We sincerely thank the Whitehall Foundation for their funding support and startup funds from The Scripps Research Institute for carrying out this work.

1. Preparation of Abdominal Ganglia, Electrophysiological Measurements, and Isolation of Single Identified Neurons from Abdominal Ganglion of Aplysia californica
<ol>
	<li>Maintain Aplysia in the laboratory aquarium with circulating artificial seawater (ASW) at 16 &#176;C under 12:12 light:dark conditions.</li>
	<li>Isolation of abdominal ganglion.
	<ol>
		<li>Anesthetize animals by injecting 380 mM MgCl2 solution for 5-10 min&#160;(equivalent to 30-35% of the animal&#39;s body weight).</li>
		<li>Identify the abdominal ganglion based on its position in the central nervous system24,28.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Protease treatment.
	<ol>
		<li>Incubate abdominal ganglion for 30 min&#160;at 34 &#176;C&#177;0.5 &#160;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.</li>
	</ol>
	</li>
	<li>Removal of ganglia sheath.
	<ol>
		<li>After the enzyme treatment, pin down the ganglia to the Sylgard Silicone base of a cell chamber (&#216;= 1 cm; vol. = 0.3 ml) and perfuse with ASW (flow rate: 150 &#956;l/min) at room temperature (18 &#176;C&#177;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 &#216;= 2 mm modified cell chamber) that can be easily illuminated from the bottom using a binocular stereomicroscope with 20X&#160;and 40X&#160;magnification.</li>
		<li>Carefully remove the sheath from the ganglion by using forceps and microscissors.</li>
	</ol>
	</li>
	<li>Simultaneous electrophysiological recording from two neurons.
	<ol>
		<li>Identify neuron of interest.</li>
		<li>Impale the neuron with a single intracellular sharp microelectrode with resistance 10-15 M&#937; filled with 3 M KCl solution.</li>
		<li>Record neuronal activity (10 kHz band pass) using an intracellular recording system.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Drug application.
	<ol>
		<li>Apply drug of choice into the cell chamber by gentle pipetting or inject directly into neurons.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Isolation of single neurons.
	<ol>
		<li>At the conclusion of the electrophysiological experiments, stop the perfusion of ASW and wash the ganglion with 100% ethanol. &#160;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.</li>
		<li>Remove single neurons under a stereomicroscope and transfer to a small Eppendorf tube containing ice cold 500 &#956;l RNA extraction reagent. Isolated single neurons can be stored in RNA extraction reagent at -80&#160;&#176;C for future analysis.</li>
	</ol>
	</li>
</ol>
2. RNA Isolation from Single Neurons and Gene Expression Analysis by Quantitative Real Time PCR (qPCR)
<ol>
	<li>Precautions to minimize RNA degradation: 
	Ribonucleases (RNases) degrade RNA and are very stable and active enzymes. Take the following steps during handling RNAs.
	<ol>
		<li>Clean the working area and instruments with RNase deactivating agents.</li>
		<li>Wear gloves at all times while handling RNA and change gloves often.</li>
		<li>Use only RNase free plastics to handle RNAs.</li>
	</ol>
	</li>
	<li>Isolating Total RNA from Single neurons:
	<ol>
		<li>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.</li>
		<li>Place the tubes on rotator for 10 min&#160;at 4 &#176;C. Spin the Trizol-Chloroform mix at 12,000 x g for 15 min&#160;at 4 &#176;C.</li>
		<li>Collect the aqueous phase and transfer to a fresh tube.</li>
		<li>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.</li>
		<li>Mix the samples well and incubate at -80 &#176;C overnight.</li>
		<li>Spin the samples at 12,000 x g for 20 min&#160;at 4 &#176;C and a small blue pellet will be visible at the bottom of the tube.</li>
		<li>Remove the supernatant and wash the pellet with 850 &#956;l of 75% ice-cold ethanol at 12,000 x g for 10 min&#160;at 4 &#176;C.</li>
		<li>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.</li>
		<li>Once dried, resuspend the RNA pellet in 10 &#956;l of RNAse free water and determine RNA concentration. 
		NOTE: Determine RNA concentration using a spectrophotometer. When not using right away, store RNA at -80 &#176;C.</li>
	</ol>
	</li>
	<li>aRNA synthesis from single neurons:
	<ol>
		<li>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 &#956;g of RNA.</li>
	</ol>
	</li>
	<li>Reverse transcription of RNA:
	<ol>
		<li>To generate cDNA from aRNA, use 1.0 &#956;g of aRNA in 20 &#956;l of a reverse transcription reaction. Several kits are commercially available for this purpose.
		 NOTE: cDNA was synthesized according to the manufacturer&#39;s protocol using the following settings on thermal cycler. 
		5 min&#160;at 25 &#176;C 
		30 min&#160;at 42 &#176;C 
		5 min&#160;at 85 &#176;C 
		Hold at 4 &#176;C</li>
		<li>After the reverse transcription step, store the cDNA at -20 &#176;C for long-term storage.</li>
	</ol>
	</li>
	<li>Primer design and standardization: 
	Several excellent software programs are available, free of charge, for designing primers for real time amplifications.
	<ol>
		<li>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&#39;-TGATTCTGACGCAAAGAAAAGA-3&#39; 
		Reverse: 5&#39;-ACCGTGAGCAGTCAGTTGTAGA-3&#39; 
		Primer set 2 
		Forward: 5&#39;-AGGGAATGTCGATGAAAGAAGA-3&#39; 
		Reverse: 5&#39;-GACACACAGGAAGTATGCCAAA-3&#39;</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Quantitative Real Time PCR (qPCR): 
	Note: Use appropriate tubes or plates that are compatible with the qPCR machine.
	<ol>
		<li>Dilute cDNA to 5x&#160;with nuclease free water.</li>
		<li>To 2 &#956;l of diluted cDNA, add 8 &#956;l of a qPCR master mix containing 2 &#956;l of H2O, 5 &#956;l of 2x&#160;SYBR Green master mix, and 1.0 &#956;l of 10 &#956;M (each) forward and reverse primer.</li>
		<li>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.</li>
		<li>Proceed to setting up the qPCR machine.</li>
	</ol>
	</li>
</ol>

Authors do not have any competing financial interests.

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&#160;and electrophysiological activity could be continuously monitored. Thus, one could study the electrophysiological effects of brief neurotransmitter exposure (exposed locally to a specific neuron or to the bath) for a long period of time. Furthermore, one could study the effects of exposure to multiple neurotransmitters (simultaneously or in tandem) on the same neuron on firing properties of itself or the follower neuron or both.
An example of gene expression measurements by qPCR is shown in Figure 4. Since the RNA content of a single neuron is low, in vitro amplification is necessary to obtain a sufficient amount of RNA for quantitating expression of multiple genes. Commercial kits are available for the in vitro transcription process. The amplification of the CREB1 transcript was quantified directly by the number of cycles required to obtain a standardized fluorescence signal, the Ct-method31. The amplified RNA from a single neuron is sufficient for the studies to determine gene expression changes in a large number of genes using microarray32 and for the entire transcriptome by RNAseq analyses33.
A limitation of this technique is that this preparation may not preserve all the synaptic connections of the neurons of the abdominal ganglia. Several of the neurons form synapses with neurons in other ganglia and in the ganglia preparation these long-distance connections will be missed. However, the methodology described here could be easily modified to include the entire CNS or could be easily integrated to semi-intact preparation13,14,16 that was described to study cellular and electrophysiological changes in gill and siphon withdrawal reflex.
Recent advances in genomics techniques now allow us to obtain a deep insight into the dynamics of expression of coding and noncoding RNAs34. The reduced preparation that is described here is ideal for studying temporal changes of gene expression in conjunction with electrophysiological measurements. For example, one could apply pharmacological agents and measure their effects on electrical properties of individual neurons or multiple neurons that are part of the same neural circuitry and isolate individual neurons at different times for RNA isolation. The effect of manipulation of a single neuron (such as electrical stimulation, or exposures to pharmacological agents) on the entire identified circuitry could be studied at the level of changes in expression of specific genes in single neurons. Thus this methodology facilitates successful integration of electrophysiological and genomic methodologies to study neural circuitry in Aplysia.
This approach could be easily extended to the study of other invertebrate models such as nudibranch mollusc Tritonia, pond snail Lymnaea, and terrestrial snail Helix whose nervous systems contain several identified neurons35-57. These models are also used extensively for the study of neural control of behaviors such as escape swimming, magnetic field orientation, synapse formation, learning and memory storage. The methodology we described is a simple integration of genomics analysis with physiological measurements of neuronal activity at single cell level. A deeper insight into molecular and physiological basis of neural control of behavior could be obtained from such studies.

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 behaviors1-14. 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 learning5,6,15-19. Decades of study on this reflex have also identified several signaling pathways that have a key role in learning and memory20-24.
Several different preparations of Aplysia were used to study cellular and molecular basis of memory storage. These include the intact animal2,3, semi-intact preparation1,7,13,14,16 and reconstitution of major components of neural circuitry25-29. 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.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="101">
			<td height="101" style="height:101px;width:120px;">Aplysia</td>
			<td style="width:123px;">National Aplysia Resource Facility, University of Miami</td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">NaCl</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">S 3014-1KG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">KCl</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">P 9333-500G</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:120px;">CaCl2&#8226;2H2O</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">C5080- 500G</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:120px;">MgCl2&#8226;6H2O</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:129px;">BP 214-501</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:120px;">NaHCO4</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">S 6297-250G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">HEPES</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">H 3375-500G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Protease</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:129px;">17105-042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Trizol</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">15596-026</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Chloroform</td>
			<td style="width:123px;">MP Biomedicals</td>
			<td style="width:129px;">2194002</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">100% Ethanol</td>
			<td style="width:123px;">ACROS</td>
			<td style="width:129px;">64-17-5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">GlycoBlue</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9515</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">3 M NaOAc, pH 5.5</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9740</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Nuclease free water</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9737</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:120px;">MessageAmp II aRNA Amplification Kit</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM1751</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">qScript cDNA SuperMix</td>
			<td style="width:123px;">Quanta Biosciences</td>
			<td style="width:129px;">95048-100</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">Power SYBR Green PCR Master Mix</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">4367659</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Forceps</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">11252-20</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Scissors</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">15000-08</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:120px;">Stainless Steel Minutien Pins&#160;</td>
			<td rowspan="2" style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">26002-10 or</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:129px;">26002-20</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Veriti Thermal Cycler</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">Veriti Thermal Cycler</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">5430R Centrifuge</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:129px;">5430R Centrifuge</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">7900HT Fast Real-Time PCR</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">7900HT Fast Real-Time PCR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:120px;">Amplifier</td>
			<td>BRAMP-01R</td>
			<td style="width:129px;">NPI Electronics</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:120px;">Digidata Converter</td>
			<td style="width:123px;">Instrutech ITC-18</td>
			<td style="width:129px;">HEKA ELEKTRONIK</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:120px;">Micro Manipulator</td>
			<td style="width:123px;">Patch Star</td>
			<td style="width:129px;">Scientifica</td>
		</tr>
	</tbody>
</table>

aplysia ganglia preparation for electrophysiological and molecular analyses of single neurons

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.

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&#160;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 potentials from R15 neuron were recorded26,31. These measurements were compared to direct (without protease treatment) recording from the intact ganglia. Concentration of the protease (0.1% protease, see step 1.3) that produced bursting potentials that were comparable to those obtained from exposed ganglion without protease were used in the subsequent experiments.
Using the protocol described, action potentials (AP) in response to Ach exposure to R15, a cholinergic neuron and L7, a motor neuron that does not respond to Ach were recorded. As expected, simultaneous electrophysiological recordings show that Ach induce depolarization in R15 whereas there was no change in firing of L7. Figure 2 shows the application of Acetylcholine (Ach) directly into the cell chamber. 1 M Ach (volume 0.3 &#956;l) was directly applied into the chamber using micropipette in order to obtain a final 1 mM concentration inside the chamber.
Ach was washed off from the bath by perfusion and recorded again from R15 neurons. Figure 3 shows waveforms suggesting that Ach effect is reversible. Analysis of the parameters AP in control, during depolarization and after wash is shown. During depolarization amplitude is decreased and duration of AP increased significantly.
Following electrophysiological measurements, R15 and L7 neurons were isolated and RNAs were prepared for qPCR analyses. A linear T7 RNA Polymerase&#8212;driven transcription using MessageAmp II aRNA Amplification Kit from Ambion was used for amplifying RNAs from single neurons. For comparison, identified neurons R2 and L11 and the whole abdominal ganglia from adult (6-9 months old) and young animals (1-2 months old) were also studied. qPCR amplification were carried out in a 7900HT Fast Real-Time PCR System under the following conditions: 95 &#176;C for 10 min, followed by 40 cycles of 95 &#176;C for 15 sec, 60 &#176;C for 1 min. There were four biological replicates in the experiment. Each biological replicate had four technical replicates in the qPCR set up. Ct values from four replicates were averaged and 1/Ct was calculated to find the absolute expression level of CREB1 in each sample (Figure 4). An estimation of the absolute expression levels of CREB1 gene in young and adult Aplysia ganglia, and in four identified single neurons was monitored by the Ct value obtained from qPCR measurements.
The starting mRNA concentration was the same in both young and adult abdominal ganglia. The same forward and reverse CREB1 primer set were used in all qPCRs, both in studies using ganglia and single neurons. The Ct values are very similar between young and adult abdominal ganglia. Similarly, Ct values were measured in the case of single neurons where the starting total mRNA concentrations were low and two rounds of amplification yielded a good amount of RNAs for cDNA synthesis. The Ct values of CREB1 amplification suggested that CREB1 expression in R15 and L7 neurons are comparable whereas R2 and L11 showed significant differences in expression when compared to L7 or R15 (Student&#39;s T test, p&#60;0.05) (Figure 4).
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51075/51075fig1highres.jpg" src="/files/ftp_upload/51075/51075fig1.jpg" /> 
Figure 1. Microscope stage and perfusion system for ganglia electrophysiology. Perfusion system and stage were made using Plexiglas and are transparent. This setup is mounted on to a metal block (not shown) to hold position. The metal block is designed such that samples can be illuminated from the bottom. A sample electrophysiological bursting pattern recorded from R15 neuron is shown.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51075/51075fig2highres.jpg" src="/files/ftp_upload/51075/51075fig2.jpg" /> 
Figure 2. Simultaneous recording from L7 and R15 neuron of abdominal ganglia.&#160;(A)&#160;Cartoon of abdominal ganglia showing the position of L7 and R15 neurons (modified from Kandel 2001, reprinted with permission from AAAS). Also shown are the positions of R2 and L11 in the abdominal ganglia. (B)&#160;Representative intracellular recordings from L7 and R15. Arrow indicates time of Ach application. One could continue these measurements for 8-10 hr.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51075/51075fig3highres.jpg" src="/files/ftp_upload/51075/51075fig3.jpg" /> 
Figure 3. Ach induced changes in action potential (AP) of R15. AP waveforms were analyzed before, during and after Ach exposure. Representative waveforms are shown. ms: millisecond, mV: millivolt.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51075/51075fig4highres.jpg" src="/files/ftp_upload/51075/51075fig4.jpg" /> 
 
Figure 4. qPCR analysis of expression of CREB1 in abdominal ganglia and single identified neurons. (A)&#160;Absolute quantification of CREB1 transcript in the young and adult abdominal ganglia of Aplysia. Total RNA was isolated using standard Trizol protocol as described in the methods. 1 &#956;g of total RNA was used to synthesize cDNA, followed by real time PCR amplification using CREB1 primers. (B)&#160;Absolute quantification of CREB1 transcript in identified single neurons of old Aplysia. Total RNA was subjected to two rounds of in vitro amplification before cDNA synthesis. 1 &#956;g of aRNA was used to synthesize cDNA, followed by real time PCR amplification using CREB1 primers.

Watch this Scientific Journal Video about Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons at JoVE.com

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

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.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

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9.0K Views.  The Scripps Research Institute, Florida. 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.

Video: Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular entanglement of the constituent polymers in the spinning dope was found to be an essential prerequisite for successful electrospinning. Rheology is a powerful tool to probe the molecular conformation and interaction of biopolymers. In this report, we demonstrate the protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan, from their dimethyl sulfoxide (DMSO)/water dispersions. Well-formed starch and pullulan fibers with average diameters in the submicron to micron range were obtained. Electrospinnability was evaluated by visual and microscopic observation of the fibers formed. By correlating the rheological properties of the dispersions to their electrospinnability, we demonstrate that molecular conformation, molecular entanglement, and shear viscosity all affect electrospinning. Rheology is not only useful in solvent system selection and process optimization, but also in understanding the mechanism of fiber formation on a molecular level.

Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan.

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...