Name: examining monosynaptic connections in drosophila using tetrodotoxin resistant sodium channels
Uploaded: 2018-02-14T14:00:00
Description: Watch this Scientific Journal Video about Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels at JoVE.com

We would like to thank Joshua Singer, Jonathan Schenk, as well as thoughtful reviewers for comments on the manuscript. We would also like to thank Ben White and Harold Zakon for discussions on the technique. Jonathan Schenk provided the data for Figure 3A. This work was supported by a Whitehall Foundation Grant and an NIH R21 to QG.

1. Prepare Flies and Identify GAL4 promoter Lines
<ol>
	<li>Select a GAL4 promoter line and cross it with flies carrying both the UAS-NaChBac transgene and an optogenetic transgene for activation. 
	NOTE: UAS-CsChrimson11 is selected due to its high conductance and the ease with which it activates NaChBac-mediated action potentials. Both UAS-NaChBac and UAS-CsChrimson are available from the Bloomington Stocks Repository.</li>
	<li>Prepare all-trans retinal as a stock solution in ethanol (35...

<ol>
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TERPS analysis compliments current techniques used for circuit cracking by enabling the detection of synapses between identified neurons. Specifically, the approach reveals monosynaptic connections by broadly silencing action potentials with TTX while restoring excitability in a select population of neurons with the TTX-insensitive sodium channel NaChBac. Synaptic release is elicited by optogenetic stimulation while postsynaptic events are monitored with whole-cell recordings. TERPS distinguishes itself from other approa...

A major goal of neuroscience is to map the connections between neurons to understand how information flows through circuits. Numerous approaches and resources have become available to test for functional connectivity between neurons in a range of model systems1,2. To generate the most accurate wiring diagrams using electrophysiology, it is important to resolve whether observed connections between two cells are direct and monosynaptic versus indirect and polysynaptic. A gold standard for making this distinction in mammalian neurons is to measure the latency between single action potentials in the presynaptic cells and the onset of excitatory postsynaptic currents (EPSCs) in the second neuron. Monosynaptic connections should have short latencies of a few milliseconds and low variability3. This approach can be complicated in invertebrate neurons because synapses may occur in their dendrites far from the somatic recording site, causing delays in detection due to long electrotonic distances between postsynaptic conductances and the recording electrode. Such delays can introduce ambiguity regarding poly- versus monosynaptic contributions. Additionally, small synaptic events may decay before reaching the somatic recordings site, and driving stronger presynaptic activity, is likely to recruit polysynaptic events.
Various techniques have been developed to test for monosynaptic connections in invertebrates. One approach uses high divalent cation solutions (Hi-Di) containing excess Mg++ and Ca++. This solution blocks polysynaptic connections by reducing release probability and increasing action potential threshold to favor detection of monosynaptic input4,5. Determining the precise ratio of Mg++ to Ca++ required, however, is not trivial and polysynaptic contributions may persist for even modest stimulation6. An alternative approach called GFP Reconstitution Across Synaptic Partners (GRASP) takes advantage of the proximity between pre- and postsynaptic membranes found at synapses to infer a monosynaptic connection 7. Here, a component of green fluorescent protein (GFP) is expressed in one neuron, and the complementary fragment of the molecule is expressed in a putative postsynaptic partner. The presence of fluorescence indicates that the two neurons are in close enough proximity to permit reconstitution of the GFP molecule and implies the existence of a synapse. GRASP can report synapses falsely, though, if two cells have closely apposed membranes, as in a nerve or fascicle. Variants of GRASP eliminate such false positives by tethering the presynaptic fragment of GFP directly to synaptobrevin, thus allowing reconstitution only at active synapses8. While GRASP and its variants have been instrumental in determining functional connectivity in the Drosophila CNS, some connections may not be made visible by GRASP if the distances between pre- and postsynaptic partners is relatively large. This is particularly pertinent in the assessment of volume transmission associated with neuromodulation9 or GABAergic inhibition10.
Here, a complementary and novel technique is demonstrated for testing direct monosynaptic connections in the Drosophila CNS. This method, called Tetrodotoxin Engineered Resistance for Probing Synapses (TERPS), works by co-expression of the tetrodotoxin (TTX)-resistant sodium channel, NaChBac, and an optogenetic activator in a presynaptic cell while recording from putative postsynaptic partners9. In the presence of TTX, all action potential-mediated activity is suppressed in cells other than those expressing NaChBac. The NaChBac channel selectively restores excitability in the targeted presynaptic cell(s), permitting light-evoked synaptic transmission. This method permits strong activation of the presynaptic neurons, such that connections can be resolved postsynaptically by somatic recording while reducing the probability of recruiting polysynaptic circuits. Importantly, this technique permits the study of volume transmission and reveals the spread of transmitter released from a single neuron throughout a circuit. In addition, TERPS can reveal the chemical nature of the connection through conventional pharmacology. TERPS is suitable for use in any model system that allows the transgenic expression of TTX-insensitive sodium channels.

<table border="0" cellpadding="0" cellspacing="0" width="946">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">UAS-csChrimson</td>
			<td style="width:219px;">Bloomington Drosophila Stock Center</td>
			<td style="width:344px;">55135</td>
			<td style="width:167px;">Used as a neural activator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">UAS-NaChBac</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>9466</td>
			<td style="width:167px;">Resotores excitibility in cells in TTX</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tetrodotoxin</td>
			<td>Tochris</td>
			<td>1078</td>
			<td style="width:167px;">Special permission may be needed to purchase TTX as it is a controlled substance</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">all trans-Retinal</td>
			<td>Sigma-Aldrichall trans-Retinal</td>
			<td>R2500</td>
			<td style="width:167px;">Require co-factor for channelrhodopsin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Weldable 321 Stainless Steel&#160;Sheet,&#160;0.002&#34;&#160;Thick,&#160;10&#34;&#160;Wide</td>
			<td>McMaster Carr</td>
			<td>3254K7</td>
			<td style="width:167px;">Used to make custom fly holder. Custom foil can be laser cut at pololu.com from the provided PDF file</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissection Microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000-C</td>
			<td style="width:167px;">Used for dissection of preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Waxer</td>
			<td>Almore</td>
			<td>Eectra Waxer 66000</td>
			<td style="width:167px;">Used during dissection to secure fly in foil</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraffin Wax</td>
			<td>Joann</td>
			<td>4917217</td>
			<td style="width:167px;">Used with waxer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Number 5 forceps</td>
			<td>Fine Science Tools</td>
			<td>#5CO</td>
			<td style="width:167px;">Used for dissection and desheathing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissection Scissors</td>
			<td>Fine Science Tools</td>
			<td>15001-08</td>
			<td style="width:167px;">Used to remove parts of the cuticle during dissection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tungsten wire</td>
			<td>A-M Systems</td>
			<td>797500</td>
			<td style="width:167px;">Use with electrolysis to make sharpened needles for dissection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Reciculating Peristaltic Pump</td>
			<td>Simply Pumps (Amazon)</td>
			<td>PM200S</td>
			<td style="width:167px;">for recirculating TTX</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Speed Controller for peristaltic pump</td>
			<td>Zitrades (Amazon)</td>
			<td style="width:344px;">N/A</td>
			<td style="width:167px;">PWM Dimming Controller For LED Lights or Ribbon, 12 Volt 8 Amp,Adjustable Brightness Light Switch Dimmer Controller DC12V 8A 96W for Led Strip Light B</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Versa-Mount&#160;Precision Compressed Air Regulator</td>
			<td>McMaster Carr</td>
			<td style="width:344px;">1804T1</td>
			<td style="width:167px;">For applying positive pressure during patching</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass capilaries</td>
			<td>World Precision Instruments</td>
			<td style="width:344px;">TW150F-3</td>
			<td style="width:167px;">For patch pipettes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Multipurpose Gauge</td>
			<td>McMaster Carr</td>
			<td>3846K431</td>
			<td style="width:167px;">Gauge for pressure regulator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrophysiology Camera</td>
			<td>Dage MTI&#160;</td>
			<td>IR-1000</td>
			<td style="width:167px;">Any camera that works in the IR range (850 nm) will work. You do not want to use red illumination as this can activate csChrimson</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IR LED</td>
			<td>Thorlabs&#160;</td>
			<td>M850F2</td>
			<td style="width:167px;">For oblique illumination</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">fiber optic for IR LED</td>
			<td>Thorlabs</td>
			<td>M89L01</td>
			<td style="width:167px;">Couples to IR LED</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective lens&#160;</td>
			<td>Olympus 40X</td>
			<td>LUMPlanFLN</td>
			<td style="width:167px;">This can be used on most microscipes and works well for visualizing fly neurons.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Amber LED</td>
			<td>Thorlabs</td>
			<td>M590L3d</td>
			<td style="width:167px;">For visualizing RFP and mCherry</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Blue LED</td>
			<td>Thorlabs</td>
			<td>M470L3d</td>
			<td style="width:167px;">For visualizing GFP</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GFP filter set</td>
			<td>Chroma</td>
			<td>49011</td>
			<td style="width:167px;">For visualizing GFP or stimulating channel2rhodopsin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Custom mCherry Filter Set</td>
			<td>Chroma</td>
			<td style="width:344px;">&#160;et580/25x and t600lpxr (from the 49306 set) but with an et610lp barrier/emission optic</td>
			<td style="width:167px;">Use only if you wish to patch identified neurons with channel2rhodopsin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dichroic to combine Amber and blue LED</td>
			<td>Thorlabs</td>
			<td style="width:344px;">DMLP550R</td>
			<td style="width:167px;">Use only patch under mCherry and excite channel2rhodopsin with blue light.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Red LED</td>
			<td>LEDSupply</td>
			<td>Cree XPE 620 - 630 nm</td>
			<td style="width:167px;">Used to drive csChrimson</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LED Driver</td>
			<td>LEDSupply</td>
			<td>3021-D-E-1000</td>
			<td style="width:167px;">Used to drive LEDs for optogenetic stimulation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Manipulator</td>
			<td>Sutter Instruments</td>
			<td>MP-225</td>
			<td style="width:167px;">Used to position pipette during recordings</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Patchclamp Amplifier</td>
			<td>A-M Systems</td>
			<td>Model 2400</td>
			<td style="width:167px;">An equivalent amplifier is suitable</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bessel Filter</td>
			<td>Warner Instruments&#160;</td>
			<td>LPF 202A</td>
			<td style="width:167px;">Auxillary filter used to filter current trace to oscilloscope during patching.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Data Acquisition System&#160;</td>
			<td>National Instruments</td>
			<td>NI PCIe-6321&#160;&#160;&#160;&#160;&#160;&#160; 781044-01</td>
			<td style="width:167px;">Used to record data from amplifier to computer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Connector Block - BNC Terminal BNC-2090A</td>
			<td>National Instruments</td>
			<td>779556-01</td>
			<td style="width:167px;">Used to connect amplifier to DAQ card.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Steel Foil&#160;</td>
			<td>McMaster Carr</td>
			<td>3254K7</td>
			<td style="width:167px;">Steel foil for custom recording chamber</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Magnets</td>
			<td>K&#38;J Magnetics</td>
			<td>D42</td>
			<td style="width:167px;">To secure recording chamber to ring stand</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;">1/16&#34; Cell Cast Acrylic Clear</td>
			<td>Pololu</td>
			<td></td>
			<td style="width:167px;">Used to make custom recording chamber. Acrylic can be laser cut at pololu.com from the provided PDF file</td>
		</tr>
	</tbody>
</table>

examining monosynaptic connections in drosophila using tetrodotoxin resistant sodium channels

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between target neurons. The method relies on co-expression of a transgenic activator with the tetrodotoxin-resistant sodium channel, NaChBac, in a specific presynaptic neuron. Connections with putative post-synaptic partners are determined by whole-cell recordings in the presence of TTX, which blocks electrical activity in neurons that do not express NaChBac. This approach can be modified to work with any activator or calcium imaging as a reporter of connections. TERPS adds to the growing set of tools available for determining connectivity within networks. However, TERPS is unique in that it also reliably reports bulk or volume transmission and spillover transmission.

TERPS is used to distinguish between mono- and polysynaptic contributions in synaptic connections between neurons. While weak stimulation of a cell may be used to test direct connections, driving greater presynaptic activity often recruits polysynaptic connections (Figure 1A). TERPS works by co-expressing the TTX-insensitive sodium channel NaChBac and an optogenetic activator, and testing connections in the presence of TTX to eliminate polysynaptic connection...

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Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

This article features a method to test the monosynaptic connections between neurons by employing tetrodotoxin and the tetrodotoxin-resistant sodium channel, NaChBac.

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between ...

The overall goal of this technique is to identify monosynaptic connections electrophysiologically in drosophila using tetrodotoxin resistant sodium channels. The main advantages of this technique is that it is straightforward and can reveal the chemical nature of synapses. It is also suitable for the study of volume or bulk transmission.Generally, in videos new to this method, we struggle because of the difficulty of the dissection. By simply understanding the method, however, the logic is easily transferable to other model systems such as mice. For the recordings, have patch pipettes for the resistance near seven megaohms.Also, have external recording saline with a pH of 7.3 or less aerated continuously with carbogen containing 95%oxygen and 5%carbon dioxide. For the subjects, have a fly stock expressing csChrimson, propagating on food containing 0.2 millimolar of all trans-Retinal. For the recordings, select one to three-day-old adults.Load them into a glass scintillation vial and immobilize it using one minute exposure to ice-cold conditions. Then, lower the light level on the dissecting scope and coarse forceps to load an anesthetized fly into the custom-made recording chamber. Secure the fly to the chamber by surrounding with wax or UV curable epoxy.Position it dorsal side up with the foil roughly halfway up the thorax. The key to obtaining a great preparation is having the hole cut in the foil to be just the right size, so the fly is not squeezed nor able to fall through. Illuminate the preparation with optical fiber lights from either side and keep them as dim as possible.Next, cover the preparation with four to six drops of recording saline. Next, sharpen the tungsten wire or filament for the dissection. Apply about 20 volts of AC current to a potassium nitrate-saturated solution and repeatedly dip the tip of the wire very quickly into the solution.After 20 dips, the tip will be sharp. Now, adjust the lights to provide just enough visibility to use the sharpened wire to expose the brain by removing the cuticle, followed by the trachea and fat bodies. To access the antennal lobes, use a bent tungsten wire to gently tuck the antenna under the foil shield.Then use sharp forceps to gently tear away the glial sheathing covering the area of interest. Now transfer the preparation to the electrophysiology rig to make recordings. At the rig, immediately start perfusing the preparation with recording saline, bubbled with carbogen.Use a gravity-fed saline solution reservoir placed above the microscope stage. To collect the waste saline, use a vacuum line and a 2 liter flask. Next, load the patch pipette with 4 microliters of internal recording solution and attach the pipette to a micromanipulator.Then, zero the amplifier's offset and set the amplifier to deliver test pulses. Then, adjust IR oblique illumination in order to obtain a clear view of the brain. Now, while applying positive pressure to the pipette, approach the cell with the pipette.When contact with the cell is made, release the positive pressure to seal the pipette to the membrane. Then, set the holding potential of the membrane to minus 60 millivolts. And the pipette should form a gigaohm seal with the cell membrane.The holding current should drop to as low sub-picoamp level. To obtain constant gigaohm seals, make sure to keep your pipette clean by keeping positive pressure and avoid hitting tissue or approaching your cells. To proceed, set the amplifier to deliver test pulses from minus 50 to minus 60 millivolts.The test pulses will reveal a large capacitative transient representing the current necessary to charge the patch pipette. To remove capacitative transience, adjust the capacity compensation knobs on the amplifier. Now, apply brief pulses of negative pressure to rupture the cell membrane and obtain the whole cell configuration.A large increase in the capacitative transience indicates success. A small increase in the holding current should also be observed. Then, set the amplifier to current clamp mode.And adjust the cell's potential to between minus 50 and minus 60 millivolts. In preparation, configure the data acquisition system as indicated in the tech's protocol. The system requires a high-powered red LED positioned beneath the preparation, positioned to illuminate the fly at 0.238 milliwatts per square millimeter.Now, activate the presynaptic neurons optogenetically, using pulsed red light in csChrimson expression. Other methods can also be used. Before applying tetrodotoxin, a synaptic connection should be visible in response to a 40 millisecond light pulse.No response means a connection is unlikely. The holding potential may be adjusted to emphasize the excitatory or inhibitory connections, accordingly. Once a connection is verified, set up the saline profusion for recirculation at the saline.Set the pump so the saline level in the bath stays stable. Then, add enough tetrodotoxin to the system for a final concentration of one micromolar. This should stop the spiking activity of the patched neuron.If not, add more tetrodotoxin to the system. Now, apply brief pulses of red light to activate what is likely to be a monosynaptic connection. Then, proceed with adding pharmacological antagonists to the recirculating saline to test the chemical nature of the synaptic connections.Transgenic expression of the tetrodotoxin in sensitive sodium channel notchback rescues excitability in neurons and results in a large plateau potential. Interneurons in the antennal lobe respond to odors. However, excitatory post-synaptic currents are not easily resolved at the soma.So, do the responses arise directly from olfactory receptor neuron input? Using tERPs reveals that the olfactory receptor neurons make direct connections with the local interneurons. Stimulation of the serotonergic neurons in the antennal lobe results in a mixture of excitation and inhibition in local interneurons.Application of methysergide, a broad 5HT-receptor antagonist, blocked a slow hyperpolarization, but had no effect on the depolarization, while mecamylamine blocks the fast depolarization. This suggests that the stimulation is cholinergic. tERPs was used to resolve extrasynaptic volume transmission that occurs with either GABA or neuromodulators.In one example, application of tetrodotoxin did not suppress an inhibitory signal in the local interneuron. This inhibition was blocked using the serotonin antagonist, methysergide, suggesting a direct signal from a specific serotonergic neuron. After watching this video, you should have a good understanding of how to obtain whole cell recordings from drosophila neurons and assess their conductivity using tERPs.While attempting this procedure, it's important to remember to be patient and practice the difficult dissection repeatedly. Once mastered, this technique can be done in about an hour if it is performed properly. Following this procedure, other methods like pharmacology can be performed in order to answer additional questions like the nature of the chemical synapse.Don't forget that working with tetrodotoxin can be extremely hazardous and precautions, such as wearing gloves and working with small volumes, should always be taken while performing these procedures.

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