1. Instrumentation 
<ol>
<li>To measure the response of olfactory sensory neurons to photolysis of caged compounds we use a flash lamp in combination with a typical patch-clamp recording system including: a patch-clamp amplifier, a recording electrode and a reference electrode connected to the head-stage of a patch-clamp amplifier, a digitizer, a computer, software for data acquisition, micro-manipulators, an epifluorescence microscope, a perfusion system, an anti-vibration table and a Faraday cage (Figure 2).</li>
<li>To generate a high-intensity flash of ultraviolet (UV) light we use a Xenon flash lamp (Rapp OptoElectronic JML-C2 Flash Unit, Figure 3A) that functions by discharging a high voltage capacitor across a short-arc flash tube that is filled with Xenon gas6-7. The light flash can be triggered manually or by the computer, using the same software that controls electrophysiological acquisition.</li>
<li>A UV short pass filter optimized for uncaging applications (270-390 nm) is inserted into an output slot of the flash lamp and a quartz fiber optic light guide is connected to the microscope (see 1.5).</li>
<li>The intensity of the light flash and therefore the extent of photolysis can be modified by changing specific parameters: voltage or capacitance6. Indeed, the light intensity is related to the energy stored in the capacitor and depends on the capacitance value and on the voltage across it, according to the standard relation 0.5 CV2. One of three capacitance values can be selected on the front panel of our flash lamp (C1=1000 &mu;F, C2=2000 &mu;F, or C3=3000 &mu;F) and the voltage can be varied between 100 to 385 Volts (Figure 3A), producing a stored energy ranging between 5 and 220 J. The flash duration varies from 0.5 to 1 ms at full half maximal width and depends on the selected capacitance value. Before selecting a different capacitance or setting the voltage to a lower value, it is necessary to discharge the capacitor, by selecting the discharge mode on the front panel of the flash lamp (Figure 3A). 
However the light energy is not simply proportional to the stored energy due to manyfold reasons related to the physics of gas discharges and to non-linear losses in the circuit as the electrical energy is increased6. To measure the energy associated to a light flash, a joulemeter, as the JM10 provided by RappOptoElectronics, can be used. Note that the energy at the exit of the quartz light guide with UV filter in place is in the range 1-15 mJ per flash, as reported in the user guide of the Rapp OptoElectronic Flash Unit. 
A photodiode assembly (Figure 3B) is provided with the flash lamp unit and can be used to estimate variations of light intensities as a function of C and V. Indeed, the output of the photodiode is proportional to the light intensity. Following the user guide instructions, the light guide is connected to the input of the photodiode assembly, while the output is connected to an oscilloscope. Figure 3C shows the peak output voltage of the photodiode in response to light flashes for the three capacitance values C1, C2, or C3, available in our flash lamp unit, versus the applied voltage. These values will change as the flash bulb becomes old. It is a good practice to regularly use the photodiode to check the deterioration of the flash bulb. 
Instead of modifying the capacitance and/or voltage on the front panel, a more precise and faster control of light intensity is obtained by placing neutral density filters into a second filter slot of the flash lamp (Figure 3A). This approach is quicker and additionally the flash duration, that depends on the selected capacitance value, is not modified. With neutral density filters ranging from 100% to 0.1 % transmission the flash intensity can be quickly modified over a large range.</li>
<li> The light flash is directed to the neuron, plated on glass-bottom dishes, through a quartz fiber optic light guide (Figure 2). The light guide is connected to the epifluorescence attachment of the inverted microscope using an adapter. A modified filter cube without excitation filter directs the UV beam to the neuron by a dichroic mirror. </li>
<li>The light is focused through a 100x oil immersion objective. The area covered by the flash is visualized by inserting the light guide into an illuminator that provides a continuous source of visible light. The image (represented in yellow in the video) is viewed on a monitor through a CCD camera connected to the microscope. The contour of the illuminated area is drawn on the monitor to localize the UV flash area for the experiments. Before delivering the UV flash, the ciliary region of the neuron is brought into the area drawn on the monitor.</li>
<li>The dimension of the spot of UV light is controlled by the objective magnification, the diameter of the light guide, and the position of the light guide at the back of the microscope. We use a 100x magnification oil immersion objective. The diameter of the circular spot is measured by directing the light from an illuminator on a micrometer slide placed on the stage of the microscope. The spot diameter in our set-up is approximately 16, 33, 50 or 105 &mu;m for a light guide with a diameter respectively of 200, 400, 600 or 1250 &mu;m.</li>
</ol>
2. Preparing solutions
Dissection
<ol>
<li>Ringer&#x2019;s solution: 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, 1 mM sodium pyruvate (pH 7.4 adjusted with 1M NaOH).</li>
<li>Stop mix solution: Ringer&#x2019;s solution with 0.1 mg/ml BSA, 0.3 mg/ml leupeptin, and 0.02 mg/ml DNaseI.</li> 
<li>Zero-divalent Ringer&#x2019;s solution: 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM EDTA, 10 mM glucose, 1 mM sodium pyruvate (pH 7.4 with 1M NaOH). Add 200 &mu;M cysteine and 2 U/ml papain for enzymatic dissociation. 
Note: Sterile filter the solutions for dissection.</li>
</ol>
Patch-clamp recording solutions
Extracellular solutions
<ol>
 <li>Extracellular Ringer&#x2019;s solution: 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, 1 mM sodium pyruvate (pH 7.4 adjusted with 1M NaOH).</li>
 <li>Low-Ca Ringer&#x2019;s solution: 140 mM NaCl, 5 mM KCl, 10 mM EGTA, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, 1 mM sodium pyruvate (pH 7.4 adjusted with 1M NaOH).</li>
</ol>
Intracellular solutions
Always prepare and use caged compound solutions in dim light to avoid degradation of caged compounds from ambient light. Protect containers from light using aluminum foil.
Caged cAMP:
<ol>
<li>Make an intracellular solution containing 145 mM KCl, 4 mM MgCl2, 0.5 mM EGTA, 10 mM HEPES, 1 mM MgATP and 0.1 mM NaGTP (pH 7.4 adjusted with 1M KOH).</li>
<li>Make a stock solution dissolving caged cAMP (BCMCM-cAMP4) in DMSO at 50 mM. Make aliquots of 5 &mu;l. Store up to one year at -20&deg;C.</li>
<li>Dissolve an aliquot of the caged cAMP stock solution in the intracellular solution to obtain a final concentration of 50 &mu;M caged cAMP. Mix using a vortex.</li>
<li>Make aliquots of 1 ml of the caged cAMP intracellular solution. Store up to two months at -20&deg;C until use.</li>
</ol>
Caged Ca:
We prepare intracellular solutions containing 3 mM DMNP-EDTA5 50% loaded with 1.5 mM Ca.
<ol>
<li>Make an intracellular solution containing 140 mM KCl (or CsCl), 10 mM HEPES (pH 7.4 adjusted with 1M KOH or CsOH).</li>
<li>Open a vial of 5 mg DMNP-EDTA. Add 52.8 &mu;l of a 0.1 M calcium chloride standard solution and 1 ml of intracellular solution. Mix using a vortex.</li>
<li>To avoid uncaging of caged Ca by ambient light, transfer the solution to a tube protected from light with aluminum foil. Add intracellular solution to reach a final volume of 3.5 ml. Mix using a vortex. Make aliquots of about 1 ml of the caged Ca intracellular solution and store up to two months at -20&deg;C until use.</li> 
</ol>
Notes: During experiments, protect caged compound solutions from light using aluminum foil and keep them on ice.
Sterile filter the intracellular solution.
3. Dissociation of mouse olfactory sensory neurons
Animals were handled in accordance with the Italian Guidelines for the Use of Laboratory Animals (Decreto Legislativo 27/01/1992, no. 116) and European Union guidelines on animal research (No. 86/609/EEC). 
<ol>
<li>Prepare three glass-bottom dishes coated with 10 mg/ml concanavalin-A and 10 mg/ml poly-L-lysine.</li>
<li>To prepare for the dissection, prepare 1 ml of zero-divalent Ringer&#x2019;s solution. Fill two 50 mm Sylgard coated Petri dishes with Ringer&#x2019;s solution and place them on ice. Also chill a papain-cysteine mix, 0.5 ml of the stop mix solution, and a syringe with a 0.22 &mu;m filter containing about 10 ml Ringer&#x2019;s solution.</li>
<li>Sacrifice a mouse by CO2 asphyxiation followed by decapitation. Remove the skin overlying the skull, cut along the jaw lines and place the head in a Sylgard coated Petri dish. </li>
<li>Place the dish under a stereomicroscope. Using a scalpel, sagitally bisect the head along the midline. Turn half of the head with the medial side up. Carefully remove the septum to expose the turbinates (see also Cygnar et al., 201013). </li>
<li>Using number 3 forceps and a scalpel remove turbinates lined with the olfactory epithelium. Transfer to a Sylgard coated Petri dish. </li>
<li> Using number 55 superfine forceps, separate the pieces of olfactory epithelium from the attached parts of turbinates. Transfer them to a tube containing 1 ml zero divalent Ringer&#x2019;s solution. Add the papain and cysteine mix. Using micro-scissors, mince the epithelia and incubate at room temperature for 3 minutes. Gently mince again with micro-scissors and wait for 5 additional minutes.</li>
<li>Using a flame-polished Pasteur pipette, transfer to a tube containing 0.5 ml of the stop mix solution.</li>
<li> Centrifuge at 300 g (1500 RPM) for 5 minutes. Discard the supernatant and add about 1 ml of filtered Ringer&#x2019;s solution. Gently triturate with a flame-polished Pasteur pipette.</li>
<li> Plate about 0.3 ml of the solution containing the cells in the center of the three glass-bottom dishes. Allow cells to settle for about one hour at +4&deg;C. Add Ringer&#x2019;s solution.</li>
</ol>
4. Recording
<ol>
<li>Prepare patch pipettes from borosilicate capillaries to obtain resistances of 3-7 M&Omega; when filled with intracellular solution.</li> 
<li>Place one glass-bottom dish containing the dissociated olfactory sensory neurons on the stage of the inverted microscope. Perfuse with Ringer&#x2019;s solution.</li>
<li>Protect a syringe containing the intracellular solution with caged compounds from light with aluminum foil and keep it refrigerated. Work in dim light to avoid degradation of caged compounds from ambient light.</li>
<li>Look for an isolated olfactory sensory neuron with intact cilia at 100x magnification. Move the stage of the microscope to position the ciliary region inside the circle drawn on the monitor to localize the previously identified UV flash area. </li>
<li>Fill a patch pipette with the intracellular solution containing the caged compound using a flexible needle. Remove bubbles from the pipette tip. Insert the pipette into the holder on the headstage of the patch-clamp amplifier. Approach the soma of the neuron using the micromanipulator and apply gentle suction to obtain a gigaohm seal. Set the voltage at -60 mV and apply additional suction to reach the whole-cell mode using standard patch-clamp techniques12.</li> 
<li>Wait for at least two minutes for the diffusion of the caged compound into the cilia.</li>
<li>On the computer start a stimulation protocol and record the response. Use a low-pass filter at 1 kHz and digitize at a sample frequency of at least 2 kHz.</li>
<li>A typical stimulation protocol clamps the voltage at a constant value and triggers the release of a UV flash from the flash lamp for photolysis of the caged compound in the ciliary region. </li>
</ol>
5. Representative results:
You should be able to produce local uncaging of caged cAMP or of caged Ca in the ciliary region of an isolated olfactory sensory neuron and record the current response in the whole-cell voltage-clamp configuration. 
Figure 4 shows a typical inward current elicited by a UV flash producing photolysis of caged cAMP, recorded at a voltage of -60 mV in the presence of an extracellular low Ca Ringer&#x2019;s solution. In this condition the inward current is due to Na entry through CNG channels. The rising phase of the current was fast and was fitted by a single exponential function with a time constant of 3.4 ms.
Figure 5A-B show the responses of another olfactory sensory neuron in low Ca and in Ringer&#x2019;s solution with 1mM Ca. The rising phase of the current at -60 mV became much slower and multiphasic (Figure 5 A-B). This is due to the action of Ca entering the cilia through CNG channels and activating a secondary Cl current10. The earlier cationic current component, due to activation of CNG, is smaller in 1mM Ca Ringer solution than in low Ca solution because of the block due to the permeating Ca ions that reduce the overall current.
Another way to reduce the increase of Ca in the cilia is to clamp the neuron at +60 mV (Figure 5 C-D). The rising phase of the response due to cAMP uncaging at +60 mV was well described by a single exponential with a time constant of 6.7 ms, indicating the presence of only one current component.
By photoreleasing Ca inside the cilia of an olfactory sensory neuron you should be able to measure a rapidly rising current. This current is carried by Cl ions. Figure 6 A shows inward currents at -50 mV induced by photorelease of caged Ca in response to UV flashes of different intensities. The rising phase of the Ca-activated Cl currents was well described by a single exponential with time constants varying between 3.8 to 5 ms (Figure 6 B).
<img src="/files/ftp_upload/3195/3195fig1.jpg" alt="Figure 1"/> Figure 1. Olfactory transduction in the cilia of olfactory sensory neurons. Odorant molecules bind to odorant receptors (OR) activating a G protein that in turns activates adenylyl cyclase (ACIII) producing an intracellular increase in cAMP. cAMP opens cyclic nucleotide-gated (CNG) channels allowing the entry of Na and Ca ions. The intracellular Ca increase activates Ca-activated Cl channels. Caged cAMP or caged Ca can be introduced in the cilia diffusing through a patch pipette. A flash of UV light produces photolysis of the caged compound (Modified, with permission, from Pifferi et al. 20062).
<img src="/files/ftp_upload/3195/3195fig2.jpg" alt="Figure 2"/> Figure 2. The patch-clamp recording and flash photolysis system. The set-up components include a patch-clamp amplifier, a computer, a digitizer, an epifluorescence microscope, a Xenon flash lamp, a CCD camera and a monitor. Blue and violet lines indicate respectively the visible and UV light path.
<img src="/files/ftp_upload/3195/3195fig3.jpg" alt="Figure 3"/> Figure 3. Xenon flash lamp. (A) Light source used for flash photolysis of caged compounds. (B) Photodiode module used to evaluate the intensity of the light flash. (C) The light guide from the flash lamp was connected to the input of the photodiode and the output was visualized onto an oscilloscope. One of the three available capacitance values (C1, C2 or C3) was selected on the front panel switch of the flash lamp and the voltage was changed turning the knob on the front panel. The output voltage from the photodiode in response to different flash intensities was plotted versus the applied voltage for each capacitance value: C1 = 1000 &mu;F, C2 = 2000 &mu;F, or C3 = 3000 &mu;F. A 600 &mu;m diameter light guide was used.
<img src="/files/ftp_upload/3195/3195fig4.jpg" alt="Figure 4"/> Figure 4. Patch-clamp recording in response to photolysis of caged cAMP in low extracellular Ca solution. (A) Whole-cell current response induced in an isolated olfactory sensory neuron by photolysis of caged cAMP localized to the cilia. A UV flash was released at the time indicated by the arrow. The holding potential was -60 mV. (B) The current rising phase was well fitted with a single exponential function (dotted line) with a time constant of 3.4 ms.
<img src="/files/ftp_upload/3195/3195fig5.jpg" alt="Figure 5"/> Figure 5. Current responses induced by photolysis of caged cAMP in low Ca and in Ringer solutions. (A) An olfactory sensory neuron was bathed in Ringer solution containing 1 mM Ca or in low Ca solution at the holding potential of -60 mV. A UV flash was released at the time indicated by the arrow. (B) Current responses plotted on an expanded timescale showed a multiphasic rising phase in Ringer, while the rising phase was well fitted with a single exponential function (dotted line) with a time constant of 3.5 ms for the response recorded in low Ca solution. (C) Currents responses from the same neuron shown in (A) bathed in Ringer&#x2019;s solution at the holding potential of -60 and +60 mV. (D) Current responses plotted on an expanded timescale displayed a multiphasic rising phase at -60 mV, whereas at +60 mV the rising phase was well fitted by a single exponential with a time constant of 6.7 ms (dotted line). 
<img src="/files/ftp_upload/3195/3195fig6.jpg" alt="Figure 6"/> Figure 6. Responses to photolysis of caged Ca. (A) Whole-cell currents induced by photolysis of caged Ca at -50 mV. UV flashes were released at the time indicated by the arrow. Flash intensities were varied with neutral density filters. (B) Expanded timescale shows the rapid increase in the current after Ca photorelease. Currents were well fitted by a single exponential function (dotted lines), with time constants of 5, 4.8, 3.8 ms. (Reproduced, with permission, from Boccaccio &amp; Menini, 200710).

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Flash fotólise de compostos enjaulado combinado com patch-clamp gravações é uma técnica útil para obter saltos rápidos e local da concentração de moléculas ativas fisiologicamente dentro e fora das células. Vários tipos de compounds1 enjaulado foram sintetizados, e esta técnica pode ser aplicada a vários tipos de células, incluindo células cultivadas expressar canais iônicos que podem ser ativadas ou moduladas por fotólise de alguns dos compostos disponíveis enjaulado 11. <p class="jove...

<table border="1"><tbody><tr><th> Equipamento </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Adaptador módulo da lâmpada de flash para microscópio </td><td> Rapp optoeletrônicos </td><td> FlashCube 70 </td><td></td></tr><tr><td> Air tabela </td><td> TMC </td><td> MICRO-g 63-534 </td><td></td></tr><tr><td> Digitizer </td><td> Axon Instruments </td><td> Digidata 1322A </td><td></td></tr><tr><td> Aquisição de Dados Software </td><td> Axon Instruments </td><td> pClamp 8 </td><td></td></tr><tr><td> Software Análise de dados </td><td> WaveMetrics </td><td> Igor </td><td></td></tr><tr><td> Espelho para módulo adaptador </td><td> Rapp optoeletrônicos </td><td> M70/100 </td><td></td></tr><tr><td> Suporte do eletrodo </td><td> Axon Instruments </td><td> 1-HL-U </td><td></td></tr><tr><td> Gaiola de Faraday </td><td> Feitos </td><td></td><td></td></tr><tr><td> Cubo de filtro </td><td> Olimpo </td><td> U-UTH </td><td> Filtro de excitação removido </td></tr><tr><td> Lâmpada de flash </td><td> Rapp optoeletrônicos </td><td> JML-C2 </td><td></td></tr><tr><td> Fórceps Dumont # 55 </td><td> Precision Instruments mundo </td><td> 14099 </td><td></td></tr><tr><td> Capilares de vidro </td><td> Precision Instruments mundo </td><td> PG10165-4 </td><td></td></tr><tr><td> Prato fundo de vidro </td><td> Precision Instruments mundo </td><td> FD35-100 </td><td></td></tr><tr><td> Iluminador </td><td> Olimpo </td><td> Destaque 3100 </td><td></td></tr><tr><td> Microscópio invertido </td><td> Olimpo </td><td> IX70 </td><td></td></tr><tr><td> Micromanipuladores </td><td> Luigs &amp; Neumann </td><td> SM eu </td><td></td></tr><tr><td> Micropipeta Puller </td><td> Narishige </td><td> PP-830 </td><td></td></tr><tr><td> Monitor </td><td> HesaVision </td><td> MTB-01 </td><td></td></tr><tr><td> Os filtros de densidade neutra </td><td> Omega Optical </td><td> varia </td><td></td></tr><tr><td> Objetivo 100X </td><td> Zeiss </td><td> Fluar 440285 </td><td> Ou Zeiss ou Olympus </td></tr><tr><td> Objetivo 100X </td><td> Olimpo </td><td> UPLFLN 100XOI2 </td><td> Ou Zeiss ou Olympus </td></tr><tr><td> Ópticos UV shortpass filtro </td><td> Rapp optoeletrônicos </td><td> SP400 </td><td></td></tr><tr><td> Amplificador de patch-clamp </td><td> Axon Instruments </td><td> Axopatch 200B </td><td></td></tr><tr><td> Foto de díodo </td><td> Rapp optoeletrônicos </td><td> PDA </td><td></td></tr><tr><td> Guia de luz de quartzo </td><td> Rapp optoeletrônicos </td><td> varia </td><td> Usamos 600 mM de diâmetro </td></tr><tr><td> Fio de prata </td><td> Precision Instruments mundo </td><td> AGT1025 </td><td></td></tr><tr><td> Prata chão pellet </td><td> Warner instrumentos </td><td> 64-1309 </td><td></td></tr><tr><td> Xenon arco da lâmpada </td><td> Rapp optoeletrônicos </td><td> XBL-JML </td><td></td></tr></tbody></table> <table border="1"><tbody><tr><th> Reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> BCMCM gaiolas cAMP </td><td> Biolog </td><td> B016 </td></tr><tr><td> Albumina de soro bovino (BSA) </td><td> Sigma </td><td> A8806 </td></tr><tr><td> CaCl2 solução padrão 0,1 M </td><td> Fluka </td><td> 21059 </td></tr><tr><td> Ca Caged: DMNP-EDTA </td><td> Invitrogen </td><td> D6814 </td></tr><tr><td> Cisteína </td><td> Sigma </td><td> C9768 </td></tr><tr><td> Concanavalina A V tipo (ConA) </td><td> Sigma </td><td> C7275 </td></tr><tr><td> CsCl </td><td> Sigma </td><td> C4036 </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D8418 </td></tr><tr><td> DNAse I </td><td> Sigma </td><td> D4527 </td></tr><tr><td> EDTA </td><td> Sigma </td><td> E9884 </td></tr><tr><td> EGTA </td><td> Sigma </td><td> E4378 </td></tr><tr><td> Glicose </td><td> Sigma </td><td> G5767 </td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3375 </td></tr><tr><td> KCl </td><td> Sigma </td><td> P3911 </td></tr><tr><td> KOH </td><td> Sigma </td><td> P1767 </td></tr><tr><td> Leupeptin </td><td> Sigma </td><td> L0649 </td></tr><tr><td> MgCl2 </td><td> Fluka </td><td> 63020 </td></tr><tr><td> Papaína </td><td> Sigma </td><td> P3125 </td></tr><tr><td> Poli-L-lisina </td><td> Sigma </td><td> P1274 </td></tr><tr><td> NaCl </td><td> Sigma </td><td> S9888 </td></tr><tr><td> NaOH </td><td> Sigma </td><td> S5881 </td></tr><tr><td> NaPyruvate </td><td> Sigma </td><td> P2256 </td></tr></tbody></table>

flash photolysis of caged compounds in the cilia of olfactory sensory  neurons

Fotólise de compostos enjaulado permite a produção de aumentos rápidos e localizados na concentração de vários compostos fisiologicamente ativos 1. Caged compostos são moléculas feitas fisiologicamente inativo por uma gaiola química que pode ser quebrado por um flash de luz ultravioleta. Aqui, nós mostramos como obter patch-clamp gravações combinado com fotólise de compostos enjaulado para o estudo da transdução olfativos em dissociada do mouse neurônios sensoriais olfativos. O processo de transdução olfativos (Figura 1) ocorre nos cílios dos neurônios sensoriais olfativos, onde odorant ligação aos receptores leva ao aumento de cAMP que abre nucleotídeo cíclico-gated (CNG) canais 2. Ca entrada através de canais CNG ativa os canais de Ca-activated Cl. Mostramos como dissociar os neurônios do epitélio olfativo do mouse 3 e como ativar canais CNG ou canais de Ca-Cl ativado por fotólise de cAMP enjaulado enjaulados Ca 4 ou 5 &lt;/ Sup&gt;. Nós usamos uma lâmpada de flash 6,7 aplicar flashes ultravioleta à região ciliar para libertar da gaiola ou cAMP Ca enquanto patch-clamp gravações são tomadas para medir a corrente em toda a configuração de tensão de células-clamp 11/08.

Watch this Scientific Journal Video about Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons at JoVE.com

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

Fotólise de compostos enjaulado permite a produção de aumentos rápidos e localizados na concentração de vários compostos fisiologicamente ativos. Aqui, nós mostramos como obter patch-clamp gravações combinado com fotólise de cAMP gaiola ou enjaulado Ca para o estudo da transdução olfativos em dissociada do mouse neurônios sensoriais olfativos.

Flash Fotólise de Compostos Caged no Cilia de Olfactory neurônios sensoriais

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

flash-photolysis-caged-compounds-cilia-olfactory-sensory

Research

JoVE Journal

Neuroscience

12.7K visualizações.  International School for Advanced Studies. Fotólise de compostos enjaulado permite a produção de aumentos rápidos e localizados na concentração de vários compostos fisiologicamente ativos. Aqui, nós mostramos como obter patch-clamp gravações combinado com fotólise de cAMP gaiola ou enjaulado Ca para o estudo da transdução olfativos em dissociada do mouse neurônios sensoriais olfativos.

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

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.

Respostas análise de rato Olfactory neurônios sensoriais Usando a gravação Electroolfactogram Air fase

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

Neurociência

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Live-imagem de translocação da PKC em células Sf9 e em neurônios sensoriais Aplysia

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Lentivírus mediada Manipulação Genética e Visualização de Olfactory neurônios sensoriais In vivo</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Intracelular de gravação, Sensorial Campo Mapping, e cultivando neurônios identificados nos Sanguessuga, Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

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.

Monte todo imunomarcação de Neurônios Receptores Olfatórios no<em&gt; Drosophila</em&gt; Antena

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

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

Monte todo Rotulagem de Cilia na Olfativo Sistema de Ratos Principal

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

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.

Perfurado gravação patch-clamp de rato Olfactory neurônios sensoriais na Intact neuroepitélio: Análise Funcional do Neurônios Expressando uma Odorant Receptor Identificados

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

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Imagens ao vivo da ependimária Cilia nas laterais ventrículos do cérebro do rato

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

Comportamentos olfativos ensaiadas pelo monitoramento do computador de<em&gt; Drosophila</em&gt; Em um olfatômetro de quatro quadrantes

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.