This work was supported by NIH DC009613, the Human Frontiers Science Program and a Morley Care Fellowship (to JR).

1. The Recording Setup
The recording chamber is mounted on a Nikon TE2000U Eclipse inverted microscope with phase contrast optics which is fitted on an air table and electrically shielded using a Faraday cage. The Plexiglass recording chamber consists of two sections partially separated by a barrier and glued onto a silanized glass slide. One section of the chamber is used to settle the cells while the other is used for stimulus-exposure during the recording to minimize premature exposure of settled but as yet unused cells to odorant. The recording setup consists of a suction pipette (see below) mounted in a pelleted pipette holder and a ground electrode. The suction pipette is positioned using a micromanipulator. Stimulus solutions are applied using a triple-barrel glass square tube, with each square tube being 0.7 mm x 0.7 mm. The triple-barrel glass was connected to a motorized fast-step perfusion system mounted on a manipulator (Sutter SF-77B, Warner Instruments) and entered the recording chamber at an angle of ~45&deg;. The end of the triple barrel glass was beveled at an angle of again 45&deg; such that the final face (opening) of the triple-barrel glass was vertical to allow close positioning of the pipette.
The suction pipette electrode filled with standard mammalian Ringer solution and the ground electrode are connected to the 1 G&Omega; headstage of a patch clamp amplifier (PC-501A, Warner Instruments) in voltage clamp mode. The analog signal from the amplifier (low pass filtered at 5 kHz) is displayed on a digital oscilloscope to monitor the signal in real-time and also connected to an 8-pole Bessel filter (Krohn-Hite) where the signal is low pass filtered at 50 Hz. Both signals (low pass filtered at 5 kHz and 50 Hz) are fed into an A/D converter (Cambridge Electronic Design Micro 1401 mkII) which is connected to a PC for data acquisition. The data is recorded using Signal 3 acquisition software (Cambridge Electronic Design) at a sampling frequency of 10 kHz. The signal is recorded at the two different bandwidths to first record the receptor current together with the superimposed action potentials and then the receptor current alone (see Fig. 2). The perfusion system is used to switch rapidly between Ringer (control) solution and odorant stimuli or any solution of desired composition and is controlled by the PC and Signal 3 software. The solutions are contained in 60 ml syringes and the flow rate is controlled by medical- grade intravenous flow regulators. Typically, the flow rate is set to 1 ml/min.
The suction on the recording pipette is controlled in the following way: The side port of the pipette holder is connected to an oil line which in turn is connected to a height-adjustable oil reservoir. Raising or lowering the reservoir ensures that the pipette pressure and therefore Ringer flow through the tip of the pipette is minimal and slightly positive. Additional suction or pressure can be applied through a tube connected to the airspace of the reservoir with a mouthpiece at the other end to suck the cell body of an isolated ORN into the tip of the recording pipette.
The experiments are performed at 37 &deg;C in order to closely simulate the native physiological condition of the cell. The temperature is controlled by passing the solutions through a custom-made heating unit which consists of ceramic resistors through which stainless steel tubes containing the solutions are passed10. The temperature is monitored using a thermocouple thermometer (Fluke).
2. Solutions
Mammalian Ringer's solution (mM): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 0.01 EDTA, 10 HEPES, 10 Glucose. The pH was adjusted to 7.5 with NaOH. A 10 % diluted Ringer's solution was used to measure the junction current. Odorant solutions: Acetophenone and eugenol were prepared daily in Ringer's solution from 20 mM DMSO stocks.
3. Making Electrodes
<ol>
 <li>Place an unfilamented borosilicate glass capillary in a Sutter P-97 micropipette puller and pull a micropipette with a long taper.</li>
 <li>Transfer the pipette to a custom-made micropipette holder mounted on a Nikon E200 Eclipse microscope which is in turn fitted with a reticle in the eyepiece and a movable diamond knife mounted on the stage.</li>
 <li>Observe the tip of the pipette under the 20x objective and using the reticle as a guide; gently scribe the pipette at a 90&deg; angle using the custom-made diamond knife where the pipette is 10 &mu;m wide (outer diameter).</li>
 <li>Move the diamond knife further towards the tip and gradually apply pressure on the tip with the diamond knife. The pipette tip should break cleanly at the point where it was scribed.</li>
</ol>
Alternatively steps 3.1 to 3.4 can be combined and pipettes can be pulled with desired dimensions directly without having to cut the pipette tip, although it can prove harder to pull reliably pipettes with such a large tip diameter and cleanly cut edges.
<ol start="5">
 <li>Under a 40x objective, fire polish the tip of the pipette to an inner diameter of 5 &mu;m using an electrically heated filament. Once finished, the micropipette is ready to be used for recording currents from mouse ORNs. The open pipette resistance should be around 1 M&Omega; when filled with Ringer.</li>
</ol>
4. Isolation of Olfactory Receptor Neurons
<ol>
 <li>Sacrifice a mouse following institutional guidelines and regulations. Remove the head, peel away the skin overlying the skull and medially bisect the head along the midline.</li>
 <li>Place the two hemi-heads under a dissecting stereomicroscope, pull off the nasal septum and remove the olfactory turbinates. Place all tissue in a Petri dish with glucose containing mammalian Ringer solution.</li>
 <li>Peel the olfactory epithelium off the underlying cartilage from two turbinates and transfer the tissue into an Eppendorf tube containing 250 &mu;l Ringer. We use tissue from 2 turbinates to get the right density of cells for the size of our recording chamber. Store the remaining tissue at 4 &deg;C for later use.</li>
 <li>Vortex the Eppendorf tube containing the olfactory epithelium twice briefly for 1 sec at medium speed. This step leads to mechanical dissociation of the ORNs from the epithelium.</li>
 <li>Place the Ringer solution containing the dissociated ORNs in the recording chamber. Remove large pieces of tissue also present in the suspension using fine forceps.</li>
 <li>Let the ORNs settle for 20 min before beginning continuous superfusion with Ringer and proceed to the recordings.</li>
</ol>
5. Recordings
<ol>
 <li>Lower the suction electrode connected to the oil line into the recording chamber and regulate the height of the oil reservoir to establish slightly positive pressure at the pipette tip. This can be done by observing e.g. cell debris moving away from (positive pressure) or towards (negative pressure) the pipette. Try not to contaminate the tip of the pipette with cell debris.</li>
 <li>Scan the recording chamber for an isolated ORN which can be recognized by its typical bipolar morphology using a magnification of 20-40x. Move the recording electrode into close proximity of the ORN cell body. Gently start sucking so that the cell body enters into the tip of the pipette. Carefully and slowly continue to apply suction until the entire cell body is drawn into the tip of the suction pipette, leaving the dendrite and cilia exposed to the bath solution. For images of sucked ORNs and pipette shapes see7,11. Ensure that once no suction is applied, the ORN does not move in or out of the pipette. If so, adjust the height of the oil reservoir accordingly.</li>
 <li>Using the micromanipulators, move the suction pipette from the section containing the settled ORNs to the recording section of the chamber. Carefully place the suction pipette in front of the 3-barrelled tube for solution exchange. The suction pipette should be placed close enough to the opening of the 3-barrelled tube so that the ORN is exposed to a laminar flow thus avoiding mixing turbulence and to achieve fast solution exchange. The solution exchange is achieved by stepping the interface between parallel streams of flowing solution across the tip of the suction pipette. The time course of the solution exchange is typically around 20 ms, measured from the junction current evoked by stepping the cell between solutions of different ionic composition (see Fig. 2). Solutions are delivered by gravity.</li>
</ol>
6. Representative Results
<img alt="Figure 1." src="/files/ftp_upload/3862/3862fig1.jpg" /> 
Figure 1. A) Bipolar receptor neurons in the olfactory bulb. B) Signal transduction machinery in the cilia ultimately leads to excitatory current influx. The ensuing depolarization triggers action potential generation at the cell body.
<img alt="Figure 2." src="/files/ftp_upload/3862/3862fig2.jpg" /> 
Figure 2 shows the speed and reliability of solution exchange. A pipette was stepped from normal into 10 % diluted Ringer for 100 ms and the junction current resulting from the dissimilar ion concentration was recorded. Black trace is the average of 5 trials, red trace its SD. The little change in SD during the solution exchange demonstrates reliability of solution exchange from trial to trial and the lack of excess noise in either solution stream.
<img alt="Figure 3." src="/files/ftp_upload/3862/3862fig3.jpg" /> 
Figure 3 shows a eugenol-induced response from an ORN using the suction pipette technique. The ORN was exposed to 100 &mu;M eugenol for 1 second as indicated by the bar above the recording. In Fig. 3A (black trace) the receptor current was filtered at a bandwidth 0 - 50 Hz to only display the receptor current. Fig. 3B shows the same recording, now filtered at the wide bandwidth of 0 - 5000 Hz (red trace) to also display action potentials (APs, indicated by arrows). The inset shows the same trace at an expanded timescale to visualize APs more clearly at the onset of the response.
<img alt="Figure 4." src="/files/ftp_upload/3862/3862fig4.jpg" /> 
Figure 4. Dose response family of a eugenol-responsive ORN (A). Eugenol was used at concentrations ranging from 0.3 &mu;M to 100 &mu;M, the stimulus duration was 1 s and traces were filtered at 0 - 50 Hz. A progressive increase in odorant concentration led to a larger and faster response, which also terminated more slowly once odorant exposure was terminated.
ORNs can display an oscillatory response pattern during long stimulations at intermediate odorant concentrations (Fig. 4B). Here an acetephenone (3 &mu;M) responsive ORN was stimulated for 8 seconds. After a fast and transient peak response at the onset of stimulation a series of smaller, recurring responses are observed. The recording bandwidth was 0 - 50 Hz.

<ol>
	<li>Reisert, J., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on:+Information+and+coding+in+mammalian+sensory+physiology:+Response+kinetics+of+olfactory+receptor+neurons+and+the+implications+in+olfactory+coding.">Perspectives on: Information and coding in mammalian sensory physiology: Response kinetics of olfactory receptor neurons and the implications in olfactory coding.</a> J. Gen. Physiol. 138, 303-310 (2011).</li><li>Kaupp, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+signalling+in+vertebrates+and+insects:+differences+and+commonalities.">Olfactory signalling in vertebrates and insects: differences and commonalities.</a> Nat. Rev. Neurosci. 11, 188-200 (2010).</li><li>Tirindelli, R., Dibattista, M., Pifferi, S., Menini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+pheromones+to+behavior.">From pheromones to behavior.</a> Physiol. Rev. 89, 921-956 (2009).</li><li>Kleene, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrochemical+basis+of+odor+transduction+in+vertebrate+olfactory+cilia.">The electrochemical basis of odor transduction in vertebrate olfactory cilia.</a> Chem. Senses. 33, 839-859 (2008).</li><li>Baylor, D. A., Lamb, T. D., Yau, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+retinal+rods+to+single+photons.">Responses of retinal rods to single photons.</a> J. Physiol. 288, 613-634 (1979).</li><li>Wang, J., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+Suction+Recordings+from+Mouse+Cone+Photoreceptors.">Single-cell Suction Recordings from Mouse Cone Photoreceptors.</a> J. Vis. Exp. (35), e1681 (2010).</li><li>Lowe, G., Gold, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spatial+distributions+of+odorant+sensitivity+and+odorant-induced+currents+in+salamander+olfactory+receptor+cells.">The spatial distributions of odorant sensitivity and odorant-induced currents in salamander olfactory receptor cells.</a> J. Physiol. 442, 147-168 (1991).</li><li>Reisert, J., Matthews, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na+-dependent+Ca2++extrusion+governs+response+recovery+in+frog+olfactory+receptor+cells.">Na+-dependent Ca2+ extrusion governs response recovery in frog olfactory receptor cells.</a> J. Gen. Physiol. 112, 529-535 (1998).</li><li>Reisert, J., Matthews, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+the+odour-induced+response+in+frog+olfactory+receptor+cells.">Adaptation of the odour-induced response in frog olfactory receptor cells.</a> J. Physiol. 519, 801-813 (1999).</li><li>Matthews, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+compact+modular+flow+heater+for+the+superfusion+of+mammalian+cells.">A compact modular flow heater for the superfusion of mammalian cells.</a> J. Physiol. 518P, 13 (1999).</li><li>Reisert, J., Matthews, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+recording+of+receptor+current+and+intraciliary+Ca2++concentration+in+salamander+olfactory+receptor+cells.">Simultaneous recording of receptor current and intraciliary Ca2+ concentration in salamander olfactory receptor cells.</a> J. Physiol. 535, 637-645 (2001).</li></ol>

The authors have nothing to disclose.

The suction pipette technique is a electrophysiological method which is used to record the odor-induced slow receptor current and the fast biphasic action potentials from an ORN simultaneously. Since the plasma membrane of the cell is not breached, this method leaves the intracellular milieu undisturbed ensuring that the odorant responses are not altered due to changes of the cytoplasmic ion concentrations or dilution of intracellular factors. Cells can be recorded from for long durations (up to 4 h in frog and 1 h in m...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the material</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalogue / Model number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Air table</td>
 <td>equipment</td>
 <td>Newport</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Air Pump</td>
 <td>equipment</td>
 <td>Newport</td>
 <td>ACGP</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipette Puller </td>
 <td>equipment</td>
 <td>Sutter</td>
 <td>P-97</td>
 <td></td>
 </tr>
 <tr>
 <td>Borosilicate glass</td>
 <td>equipment</td>
 <td>WPI</td>
 <td>1B150-4</td>
 <td></td>
 </tr>
 <tr>
 <td>Nikon Eclipse Inverted microscope</td>
 <td>equipment</td>
 <td>Nikon</td>
 <td>TE2000U</td>
 <td>Equipped with Hg lamp, GFP filter and objectives 20X and 5X at least</td>
 </tr>
 <tr>
 <td>Amplifier PC-501A</td>
 <td>equipment</td>
 <td>Warner</td>
 <td> 64-0008</td>
 <td>Headstage 1 G&Omega;</td>
 </tr>
 <tr>
 <td>Diamond knife</td>
 <td>Equipment</td>
 <td>Custom-made</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Digitizer Mikro1401 A/D</td>
 <td>equipment</td>
 <td>Cambridge Electronic Design</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Filter unit 3382</td>
 <td>equipment</td>
 <td>Krohn Hite corporation</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Signal</td>
 <td>software</td>
 <td>Cambridge Electronic Design</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Molded Ag/AgCl Pellet</td>
 <td>equipment</td>
 <td>WPI</td>
 <td>64-1297</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipette holder </td>
 <td>equipment</td>
 <td>Warner</td>
 <td>64-0997</td>
 <td>Custom modified to fit 
 headstage</td>
 </tr>
 <tr>
 <td>Recording chamber</td>
 <td>Equipment</td>
 <td>Custom-made</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td> Micromanipulator 
 MP85-1028</td>
 <td>equipment</td>
 <td>Sutter Instrument</td>
 <td> Micromanipulator 
 MP85-1028</td>
 <td></td>
 </tr>
 <tr>
 <td>Mineral oil</td>
 <td>Solution</td>
 <td>Sigma</td>
 <td>330779-1L</td>
 <td></td>
 </tr>
 <tr>
 <td>Oscilloscope TDS 1001</td>
 <td>equipment </td>
 <td>Tektronix</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Three-barreled square glass tube</td>
 <td>Equipment</td>
 <td>Warner</td>
 <td>64-0119</td>
 <td>0.6 mm ID , 5 cm long</td>
 </tr>
 <tr>
 <td>Valve </td>
 <td>equipment</td>
 <td>The Lee Company</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Valvelink 8.2</td>
 <td>equipment</td>
 <td>Automate Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>SF-77B Perfusion fast step</td>
 <td>equipment</td>
 <td>Warner</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

odorant-induced responses recorded from olfactory receptor neurons using the suction pipette technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Watch this Scientific Journal Video about Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique at JoVE.com

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

odorant-induced-responses-recorded-from-olfactory-receptor-neurons

Research

JoVE Journal

Neuroscience

10.7K Aufrufe.  Monell Chemical Senses Center. Olfaktorische Rezeptor-Neuronen (ORN) zu konvertieren Geruchs-Signale zunächst in einem Rezeptor Strom, wiederum löst Aktionspotentiale, die bis zur zweiten Ordnung Neuronen im Riechkolben gefördert werden. Hier beschreiben wir die Saugpipette Technik gleichzeitig aufzeichnen Duftstoff-induzierte Rezeptor-Strom-und Aktionspotentiale aus Maus ORN.

Serie: Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...