I thank Dr Vincent Seutin for his constant support, Christelle Gillissen and Laurent Massotte for excellent technical assistance, Drs Jean Defourny and Sandra Ormenese for advices with the confocal microscope, Dr Jacques Destin&#233; for the gift of the second Axopatch 200B amplifier, the GIGA-Imaging platform for sharing the confocal microscope and Imaris software and Dr Stephen Freeman for critically reading the manuscript. This work was supported by grants from the Belgian F.R.S. - FNRS (U.N002.13 and T.N0015.13) and published with the support of the Belgian University Foundation (Publi&#233; avec le concours de la Fondation Universitaire de Belgique).

The author declares that he has no competing financial interests.

This report describes a step-by-step protocol to implement dual somatodendritic whole-cell recordings and local dendritic recordings. It is useful for determining the influence of ion channels (i.e., Ih) on the time course of postsynaptic potentials and mapping the distribution of the ion channel (Ih) along the somatodendritic domain of nigral DA neurons, respectively. Resulting electrophysiological measurements are combined to post hoc histochemistry to recover cell morphology. The procedure was employed to investigate DA neurons located in the substantia nigra, but can be generalized for neighboring nigral GABA neurons, ventral tegmental area DA neurons or other midbrain neurons. All the steps can also be followed to examine other ion channels expressed in dendrites of nigral neurons without important modifications. Post hoc visualization is particularly pertinent for neurons with axon-bearing dendrites, such as nigral neurons 25,26, hippocampal oriens-alveus interneurons 21 or some CA1 pyramidal neurons 67. Interestingly, neurons sharing this feature seem to be more common than generally thought 67. Morphological analysis reveals also the precise position of the electrodes and axon. The detection of the latter may be optimized by the labeling of proteins expressed in the axon initial segment (voltage-gated Na+ channels or Ankyrin G) using immunohistochemistry 68,69.
The reliability of data collected with dendritic recordings and subsequent neuronal labeling invariably depends on the slice quality. Maximum effort needs therefore to be applied to preserve the viability of cells within the tissue. This is achieved with gentle handling of healthy animals, high-quality tools and reagents, sufficient oxygenation of the tissue and ice-cold temperatures throughout the preparation of slices. Stable recording conditions rely on the selection of healthy neurons. In the whole-cell mode, series resistance should initially be as low as possible and maintained constant throughout the experiment. The stability of the recordings is further dependent on high-quality manipulators devoid of drift and vibrations. These perturbations can be reduced by optimizing the pipette stability: checking the connection to pipette holder and to headstage, controlling that micromanipulator cables are slack, avoiding sudden changes in temperature or stage movement and checking the mechanism of the manipulator. For dual recordings, methylsulfate 13,15,21 has been included in the intracellular solution, but gluconate 9,14,25 can alternatively be employed. However, the main anion may alter the membrane potential 70,71 and some voltage-dependent currents 72. Intracellular solution may be supplemented with ATP, GTP and phosphocreatine to preserve the physiological functions of neurons. Additionally, adding a fluorescent dye in the pipette solution (e.g., Alexa 594 or Sulforhodamine 101 41) to visualize the dendrites during a somatic recording can be useful for instance to place a pressure application (Figure 7 in Ref. 41) or an electrical stimulating pipette. The pipette solution for cell-attached recordings contains a high K+ concentration and no Na+ to record large Ih. Noteworthy the Na+/K+ concentration ratio influences the current amplitude 10, the reversal potential of the current 11 and the gating of Ih 73. Alternatively, Ih can also be recorded using outside-outs 10. In this recording configuration however, the intracellular milieu in the proximity of the channels may be perturbed. Consequently, differences in the voltage-dependent activation of Ih is observed when comparing currents obtained using cell-attached patches and outside-outs 10. Swelling of neurons is occasionally encountered during patch-clamp recording, and often arises from distinct causes such as the low quality of the water, strong imbalance in osmolarity or pH between the intra- and the extracellular solutions 39 or errors in the composition of solutions. The quality of electrophysiological recordings has a direct incidence on the quality of the morphology of recovered neurons. High resistance somatic pipettes are used for dual recordings (6 - 10 M&#8486;, as in Refs. 6,11) and for single somatic recordings after cell-attached recordings to minimize the dilution of the intracellular milieu 14. The whole-cell somatic recording following the cell-attached recording is therefore kept short (&#60; 10 min). Outside-out patches from both the somatic and dendritic pipettes are essential for proper closing of the cell membrane and subsequent recovery of the cell morphology. In addition to the cell&#39;s structure, the neurochemical content may be determined for the recorded neurons 59,74. For instance, the intracellular protein tyrosine hydroxylase can be immunolabeled for unequivocal identification of DA neurons 13.
DA neurons are mainly concentrated in the SN pars compacta, with a much lower density present in the SN pars reticulata, where they are intermixed with a higher number of GABA neurons 75. While the cell body of DA neurons is often larger than that of GABA neurons, the visual identification of these cells with IR-videomicroscopy is uncertain and partially hindered by the opacity of the pars compacta. To circumvent these limitations, the pre-selection of DA neurons can be facilitated by the use of transgenic mice expressing a fluorescent marker in a specific population of neurons (TH 65 or DAT for DA neurons, GAD for GABA neurons) and epifluorescence illumination. Alternatively, a fluorescent dye may be included in the solution of the somatic electrode to facilitate the visualization of dendrites. Increased resolution of the fluorescent cell is brought by Nipkow spinning disk confocal 14,22,30 or two-photon microscopy 7 combined to IR-DGC 6. Several advantages are related to DGC in comparison to DIC. First, as DIC prisms are not required, the IR-DGC image can be overlaid with a fluorescence image 49,52,53,76. Second, DGC can be combined with photostimulation and optogenetics 77.
A disadvantage of slice preparation is the preservation of the integrity of neurons. DA neurons extend their dendrites in the three spatial planes 78,79 and therefore truncation of the dendritic compartment cannot be completely avoided in slices 80. The choice of the orientation of slices (coronal, horizontal or parasagittal) is a trade-off. The origin of the innervation and the experimental design must be considered to select the right orientation of slices.
Direct dendritic patching is the technique used to map the distribution of functional ion channels in the different compartments of cells. In addition, this technique offers to determine the variability in functional properties of channels 20. As a complement the location and density of ion channels can be ascertained using immunohistochemistry at the light and electronic microscopy levels 17,23. This approach also offers the possibility to determine the channel density in small caliber structures which are inaccessible to patch pipettes. However these channels might be in a distinct functional state 24 or even inactive in comparison to those recorded using patch-clamp techniques. Both techniques are therefore necessary to obtain a complete picture of the location and properties of ion channels in a specific cellular region 17. With the development of voltage-sensitive dyes, voltage imaging has been used to examine the propagation of APs and EPSPs in neurons at multiple locations 81. As an alternative to dual patch-clamp recordings, this approach can even be implemented for thin dendrites that are not accessible to patch pipettes but necessitate accurate calibration of the signal and averaging.
While DA neurons in the SN and ventral tegmental area are widely investigated via somatic recordings in the physiological and pathophysiological context, the functional properties of their dendrites remains largely unknown in both conditions. Patching from dendrites has been implemented for nigral dopamine neurons by several groups with success 13,25,26 and remains the method of choice to dissect excitable properties of these fine subcellular structures 8. Dendritic recordings provide a further opportunity to scrutinize the efficiency and plasticity of synaptic transmission and the plasticity of dendritic excitability 82,83.

Neurons receive synaptic information predominantly on their dendrites. Excitatory and inhibitory synaptic signals spread from their site of generation to the integration site where action potentials (APs) are evoked as the output signal. On their way, synaptic potentials are influenced by both the structure of dendrites and the interaction between passive and active membrane properties. The combination of these highly variable parameters widens the computational power of neurons&#160;1,2. However, the small diameter of dendrites hinders however the study of their electrical properties. The continuous development of the patch-clamp technique 3, the optics 4 and refinement of methods for slice preparation 5 during the last decades have enabled recordings from very thin (0.7 - 3 &#181;m &#216;) dendrites 6,7. These methods were, and are, still largely used to examine the excitability of dendrites in a variety of neurons 8. Direct dendritic recordings are essential to determine the distribution 9-19 and differences in the functional properties 20-22 of ion channels in distinct neuronal compartments. These data are the necessary complement of ion channel distributions detected with immunohistochemistry combined to light and electron microscopy 23,24. Dual somatodendritic recordings have been implemented to explore the propagation of action potentials 9,13-15,21,22,25-27 and spreading of synaptic potentials 13,16,18 along the somatodendritic domain of neurons, obtain detailed passive cable models 28-30 and investigate the temporal resolution of neuronal integration 31.
The substantia nigra (SN) is a region located in the midbrain involved in several functions such as the control of movement, the coding of reward and habitual behaviors. The decrease of dopamine due to the specific loss of dopaminergic (DA) neurons in the SN is associated with the motor disturbances observed in patients suffering from Parkinson&#39;s disease 32. The nigral circuit is composed of two main cell types: dopaminergic and GABAergic neurons. Interestingly, these neurons have several specific features that distinguish them from other neurons. The axon of a large proportion of DA neurons and some GABA neurons originates from a dendritic site indicating that the dendritic arbor is heterogeneous (axon-bearing and axon-lacking dendrites) 25,26,33. The morphology of these neurons contrasts therefore with the typical organization of neurons in which the information transfer follows the law of dynamic polarization emitted by Cajal: starting from dendrites, to soma and finally to axon 34. DA neurons are also known to release dopamine from their dendrites 35, generate bursting activity 36 and NMDA-receptor plasticity 37. The dissection of these phenomena is elusive without direct recordings from the site where they are initiated. To gain insights into the relationship between the precise location and functional properties of ion channels and their role in the dendritic excitability and information transfer in nigral neurons, direct dendritic recordings are the method of choice.
This report describes a detailed procedure that can be used to obtain single and dual patch-clamp recordings from dendrites of nigral neurons and the corresponding post hoc biocytin labeling. The basic principles for patching the somatic and the dendritic membrane are very similar. Practically however, recordings from dendritic sites require specific optimization in comparison to somatic recordings. Successful dendritic recordings rely on the quality of the slices, optimal adjustment of the optics, gentle approach of the patch pipette and stability of the recordings.

<table><tbody><tr><td>Double-distillated water</td><td>Millipore</td><td>Super Q</td><td>Resistivity &gt;14 M&amp;Omega; cm, ideal 18.2 M&amp;Omega; cm at 25&amp;nbsp;&amp;deg;C, filtered (0.22 &amp;micro;m), https://www.merckmillipore.com/BE/en/product/Super-Q-Plus-Water-Purification-System,MM_NF-C1760</td></tr><tr><td>Microfilter candles</td><td>Robu</td><td>Porosity 3, 6 mm or 13 mm diameter</td><td></td></tr><tr><td>Tissue slicer with vibroprobe</td><td>Leica</td><td>VT1200</td><td>cutting parameters: speed 0.07, amplitude 1.40</td></tr><tr><td>Tissue slicer</td><td>Dosaka</td><td>DTK-1000</td><td>cutting parameters: speed 4, frequency 7</td></tr><tr><td>Razor blades</td><td>Gilette</td><td>Super Silver</td><td></td></tr><tr><td>Dissection tools</td><td>Braun, Aesculap</td><td></td><td></td></tr><tr><td>Reserve chamber</td><td></td><td></td><td>Custom-made</td></tr><tr><td>PP beckers</td><td>VWR</td><td>213-1725</td><td>400 ml</td></tr><tr><td>Syringe filter</td><td>Merk Millipore</td><td>Millex - GV 0.22 &amp;micro;m</td><td></td></tr><tr><td>Cyanoacrylate glue&amp;nbsp;</td><td>UHU</td><td>&quot;Sekunden Kleber&quot;</td><td>liquid glue</td></tr><tr><td>Recording chamber&amp;nbsp;</td><td>Luigs &amp;amp; Neumann</td><td>Slice mini chamber</td><td></td></tr><tr><td>Horizontal pipette puller</td><td>Sutter Instruments</td><td>Brown-Flaming P-97</td><td></td></tr><tr><td>Pipettes</td><td>Hilgenberg</td><td>1807524</td><td>2 mm o.d./1 mm i.d. glass capilaries, ends firepolished, washed</td></tr><tr><td>Dental wax</td><td>Colt&amp;egrave;ne/Whaledent</td><td></td><td>orthodontic tray wax strips</td></tr><tr><td>Platinum grid</td><td></td><td></td><td>to anchor slices in the recording chamber, custom made with a platinum disk</td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-830</td><td>to fire-polish pipette tips</td></tr><tr><td>Potassium methyl sulfate</td><td>MP Biomedicals</td><td>215481</td><td></td></tr><tr><td>Biocytin</td><td>Molecular probes</td><td>B1592</td><td></td></tr><tr><td>Manometer</td><td>GREISINGER electronic</td><td>GDH 13 AN</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>FS</td><td></td></tr><tr><td>Dodt Gradient Contrast</td><td>Luigs &amp;amp; Neumann</td><td></td><td></td></tr><tr><td>Manipulators</td><td>Luigs &amp;amp; Neumann</td><td>LN Mini 25</td><td></td></tr><tr><td>Frame grabber</td><td>The Imaging Source</td><td>DFG/USB2pro</td><td></td></tr><tr><td>Camera</td><td>DAGE-MTI</td><td>NC-70</td><td></td></tr><tr><td>Condenser</td><td>Zeiss</td><td></td><td>high numerical aperture condenser working with oil</td></tr><tr><td>Objective</td><td>Zeiss</td><td>W Plan Apochromat 441470-9900 VIS-IR</td><td>63X magnification, long-distance, high numerical aperture 1.0 water-immersion objective</td></tr><tr><td>Fourfold-changer</td><td>Luigs &amp;amp; Neumann</td><td></td><td>between the camera and the microscope</td></tr><tr><td>Black &amp;amp; white video monitor</td><td>Sony</td><td>SSM-175CE</td><td>16-inch, 850 TV lines, analog</td></tr><tr><td>Sample Bottles, Amber Glass, with Cap</td><td>VWR</td><td>215-2409</td><td>to transfer slices from the setup to histology room</td></tr><tr><td>The Axon Guide</td><td>Molecular Devices</td><td></td><td>Book</td></tr><tr><td>Stimfit</td><td>https://github.com/neurodroid/stimfit</td><td></td><td>Analysis of electrophysiological data</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>441244</td><td>see SAFETY DATA SHEET and Dangerous Substances Directive (EU) before use</td></tr><tr><td>Well cell culture plate</td><td>Greiner-Bio-One</td><td>662160</td><td>for the staining of fixed slices</td></tr><tr><td>Triton X-100</td><td>Merk</td><td>108643</td><td>see SAFETY DATA SHEET and Dangerous Substances Directive (EU) before use</td></tr><tr><td>mouse anti-TH monoclonal primary</td><td>Immunostar</td><td>22941</td><td>working concentration: 1/1,000</td></tr><tr><td>goat anti-mouse secondary antibody - Alexa Fluor 568</td><td>Invitrogen - Thermo Fisher Scientific</td><td>A-11031</td><td>working concentration: 1/500</td></tr><tr><td>Normal goat serum</td><td>Dako</td><td>X0907</td><td></td></tr><tr><td>ProLong Diamond Antifade</td><td>Molecular Probes - Thermo Fisher Scientific</td><td>P36961</td><td></td></tr><tr><td>Fluorescein Avidin DCS</td><td>Vector Lab</td><td>A-2011</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>ImageJ</td><td>http://imagej.nih.gov/ij/</td><td></td><td></td></tr><tr><td>NeuronJ</td><td>http://www.imagescience.org/meijering/software/neuronj/</td><td></td><td>plugin for ImageJ</td></tr><tr><td>Imaris</td><td>http://www.bitplane.com</td><td></td><td>Reconstruction of biocytin filled neurons</td></tr><tr><td>Neuromantic</td><td>http://www.reading.ac.uk/neuromantic/</td><td></td><td>Reconstruction of biocytin filled neurons</td></tr><tr><td>WinWCP</td><td>http://spider.science.strath.ac.uk/sipbs/software_ses.htm</td><td></td><td>Analysis of electrophysiological data</td></tr></tbody></table>

subcellular patch-clamp recordings from the somatodendritic domain of nigral dopamine neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

The above-described protocol intends to facilitate the collection of data using dendritic patch-clamp recordings. This technique, combined with post hoc histochemistry, helps to gain insights into the mechanisms of many electrical signals originating or spreading into dendrites. Direct access to dendrites with patch pipettes is difficult, but special attention to several methodological aspects will improve the success rate of the recordings. An experimenter sufficiently experienced in stable somatic recording can expect to obtain high resistance (G&#8486;) seals and complete experiments on most of attempts. One to two high-quality dendritic recordings can be collected per dissection.
The availability of high-quality brain slices containing healthy somata and dendrites is a prerequisite for accessing these fine structures (Figure 1). The criteria for selecting these neurons are a smooth and homogeneous surface all over the cell and a soma and dendrites with low contrast when observed with optimal adjusted optics (Figure 1A and 1B). Selecting neurons too strongly contrasted generally leads to unstable recordings (Figure 1C and 1C). Therefore such neurons should be avoided.
In comparison to other neurons, nigral DA neurons display specific morphological and electrophysiological characteristics. These features usually assessed with a single somatic pipette can also be observed in dual somatic (Figure 2) and somatodendritic recordings (Figure 3). Long hyperpolarizing current injections induce a membrane potential rectification called &#39;sag&#39; (Figures 2B and 5D). The axon originates, in most cases, on a dendritic site as identified using complementary criteria 13,25,26, indicating that the dendritic compartment in these neurons is heterogeneous (Figure 3A - 3B). As a consequence, the compartment where the AP is observed first corresponds to the compartment in which the axon emerges (Figure 3C - 3D) 13,25,26. The axon is generally terminated by an axonal bleb caused by a cutting of the axon during slicing 64, but this structure is not systematically detected.
The pronounced sag observed in nigral DA neurons consecutive to the activation of Ih suggests a high expression of HCN subunits. To determine the influence of Ih on the integration of synaptic signals, synaptic-like current (EPSC) waveforms were generated and injected via the dendritic recording electrode during somatodendritic dual recordings 55 (Figure 4). The kinetics of these simulated EPSCs were based on the rise and decay time constants of real spontaneous EPSCs. The resulting aEPSP was recorded with the somatic electrode. Bath application of the Ih blocker ZD 7288 increased the duration of the aEPSP, indicating the contribution of Ih to the time course of synaptic signals (Figure 4B). ZD 7288 abolished also the membrane potential rectification confirming that the sag results from the activation of Ih (Figure 4C). The results obtained with simulated EPSPs might be correlated to experiments using electrically evoked EPSPs 65. To map the precise distribution of the channel in the somatodendritic domain of DA neurons, cell-attached recordings were implemented (Figure 5). A high-resistance seal (&#62;&#62; 1 G&#8486;) is an absolute necessity for cell-attached recordings (Figure 5B). In dendrites of DA neurons, Ih activates slowly with long hyperpolarizing voltage steps and the current steady-state is attained after hundreds of ms (Figure 5C), as previously shown 66. This observation indicates that HCN channels expressed in dendrites of DA neurons have different subunits in comparison to those in pyramidal neurons or other cell types 10,16,66. As the distribution of the channel might be nonhomogeneous in the dendritic compartment and as the dendritic compartment is itself heterogeneous, the identity of the dendrite (axon-bearing or nonaxon-bearing dendrite) is revealed by comparing the IR-DGC image with the confocal image after biocytin staining (Figure 3A and B). Recovery of cell morphology is therefore necessary for both dual somatodendritic recordings and for cell-attached recordings via subsequent somatic whole-cell recordings.
<img alt="Figure 1" src="/files/ftp_upload/54601/54601fig1.jpg" /> 
Figure 1. Visualizing the Soma and Proximal Dendrites of Nigral Dopaminergic Neurons Using Infrared Videomicroscopy. (A) IR-DGC image of a nigral DA neuron showing the soma and proximal dendrite and both somatic and dendritic pipettes. A dual somatodendritic recording was successfully performed on this healthy neuron. Observe the low contrast and the smooth surface all over the somatodendritic domain of the cell. (B) Another example of a healthy DA neuron. (C) DA neuron with a rougher and uneven surface and a stronger contrast. While a dual recording has been obtained, the stability was suboptimal, and the recording duration was short (~ 15 min) due to a gradual increase of the access resistance at both pipettes. (D) Second example of a DA neuron showing a strongly contrasted soma and proximal dendrite. In this case, a strong increase of the access resistance was observed shortly after the break in the whole-cell mode. An attempt to decrease the access resistance by negative pressure was unsuccessful. Neurons in panel C and D might have been damaged during the slicing procedure and should be avoided for experiments. (E) Simultaneous double somatic recording in a healthy DA neuron at the beginning of the recording. (F) Same neuron as in panel E with a swollen cell body after ~17 min. Note the full absence of contrast, the ball-like appearance of the soma and the presence of the large nucleus which is not apparent in healthy neurons. <a href="https://www.jove.com/files/ftp_upload/54601/54601fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54601/54601fig2.jpg" /> 
Figure 2. Simultaneous Double Somatic Recording from a DA Neuron. (A) IR-DGC image during double somatic recording from a DA neuron. (B) Hyperpolarizing and depolarizing 1 s long current injections (-160 to 80 pA, increment 80 pA) and corresponding voltage changes recorded simultaneously with both patch pipettes. Note the presence of the sag in the voltage trace with the long hyperpolarizing current step and the large after-hyperpolarization after the AP during the depolarizing current pulse. <a href="https://www.jove.com/files/ftp_upload/54601/54601fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54601/54601fig3.jpg" /> 
Figure 3. Dual S omatodendritic Recording in a DA Neuron. (A) IR-DGC image of a DA neuron. The distance between the dendritic and somatic pipette is 29 &#181;m. (B) Confocal z-projection image of the neuron in panel A labeled with avidin conjugated to fluorescein isothiocyanate (FITC). The neuron was filled with biocytin during recording. The axon originated from a proximal dendrite close to the soma as indicated by the arrow. (C) Simultaneous dual somatodendritic whole-cell voltage recording from the neuron in panel A. AP recorded in the soma (black voltage traces) and dendrite (red voltage traces) in response to a 1 s long current injection step via the somatic (left; black current trace) and alternatively dendritic pipette (right; red current trace). (D) Somatic and dendritic AP shown at expanded time scale. With either somatic or dendritic current injection, the somatic AP (black) preceded the dendritic AP (red). The delay between APs in this neuron was 120 &#181;s and 210 &#181;s with somatic and dendritic current injection, respectively. Delays were measured at AP half-maximal amplitude during the rising phase of the AP. The AP is observed first in the compartment close to which the axon emerges, confirming previous observations 25,26. In this case, the dendritic recording is made from an axon-lacking dendrite. An example of a recording from an axon-bearing dendrite is in Ref. 13 (in their Fig.&#160;1). <a href="https://www.jove.com/files/ftp_upload/54601/54601fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54601/54601fig4.jpg" /> 
Figure 4. Propagation of Artificial EPSPs Along the Somatodendritic Axis of DA Neurons. (A) IR-DGC image of a DA neuron. The distance between the dendritic and somatic pipette is 45 &#181;m. Both patch pipettes are in the whole-cell current-clamp recording mode. (B) Current recorded with the dendritic pipette in current-clamp and used to generate the artificial EPSP (top). The current is obtained by the injection of an EPSC-like waveform (&#964;rise = 0.6 ms; &#964;decay = 3 ms; amplitude = 100 pA) via the dendritic recording pipette 55. At the bottom, propagated aEPSPs recorded at the soma in control conditions (black) and after the bath application of the Ih blocker ZD7288 (30 &#181;M; red trace). Each voltage trace is the average of 40 single sweeps. Observe the large increase in EPSP duration in the presence of ZD7288 indicating the contribution of Ih to the time course of EPSPs. (C) Voltage traces recorded from the soma in control conditions (black) and after the application of 30 &#181;M ZD 7288 (red) in response to a long (30 pA; 1 s) hyperpolarizing current step. Note the absence of the membrane potential rectification (sag) after the blockage of Ih. <a href="https://www.jove.com/files/ftp_upload/54601/54601fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54601/54601fig5.jpg" /> 
Figure 5. Presence of Ih in Dendrites of DA Neuron. (A) IR-DGC image of a DA neuron and pipette on proximal dendrite. (B) Capacitive and leak currents during a 5 mV voltage command in cell-attached configuration. The seal resistance was 5 G&#8486; in this example. (C) Hyperpolarizing voltage step (90 mV; top) from a membrane potential of 0 mV induced a slowly activating Ih. The current trace was inverted and fitted with a single exponential function giving a time constant of &#964; = 632 ms. (D) Voltage responses of the whole-cell recorded soma (panel A) to 1 s hyper- and depolarizing current pulses (-160 to 120 pA, 40 pA increment). The somatic whole-cell recording was performed after the cell-attached recording. Note the absence of APs due to the extracellular application of TTX and Cd2+ to suppress spontaneous firing during cell-attached recording. These voltage-gated Na+ and Ca2+ current blockers were added to suppress action currents. Panels A - D are from the same neuron. (E) IR-DIC image of another DA neuron and a dendritic pipette. (F) Voltage-dependent currents activated by a test pulse to 0 mV from a 50 ms prepulse at -120 mV in an outside-out patch excised from the proximal dendrite of the neuron in panel E. Holding potential -80 mV. Note the time-dependent inactivation of the current. <a href="https://www.jove.com/files/ftp_upload/54601/54601fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Substance</td>
			<td>g/mol</td>
			<td>Concentration</td>
			<td>for 1 L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.443</td>
			<td>125 mM</td>
			<td>7.305 g</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.007</td>
			<td>25 mM</td>
			<td>2.100 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.551</td>
			<td>2.5 mM</td>
			<td>0.186 g</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>137.99</td>
			<td>1.25 mM</td>
			<td>0.172 g</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>198.17</td>
			<td>25 mM</td>
			<td>4.95 g</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 M (solution)</td>
			<td>1 mM</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1 M (solution)</td>
			<td>2 mM</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td colspan="4">Osmolarity : ~ 310 mOsmol/L (optimal range: 314 &#8211; 325 mOsmol/L) , pH = 7.4</td>
		</tr>
	</tbody>
</table>
Table 1: Artificial cerebrospinal fluid (ACSF).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Substance</td>
			<td>g/mol</td>
			<td>Concentration</td>
			<td>for 1 L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.443</td>
			<td>87 mM</td>
			<td>5.084 g</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.007</td>
			<td>25 mM</td>
			<td>2.1001 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>7.551</td>
			<td>2.5 mM</td>
			<td>0.18637 g</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>137.99</td>
			<td>1.25 mM</td>
			<td>0.17248 g</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 M (solution)</td>
			<td>7 mM</td>
			<td>7 mL</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>198.17</td>
			<td>10 mM</td>
			<td>1.9817 g</td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>342.29</td>
			<td>75 mM</td>
			<td>25.672 g</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1 M (solution)</td>
			<td>0.5 mM</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td colspan="4">Osmolarity : ~ 326 mOsmol/L, pH = 7.4</td>
		</tr>
	</tbody>
</table>
Table 2: Sucrose-ACSF to prepare slices.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Substance</td>
			<td>g/mol</td>
			<td>Concentration</td>
			<td>for 100 mL</td>
		</tr>
		<tr>
			<td>KMeSO4</td>
			<td>150.2</td>
			<td>120 mM</td>
			<td>1.8024 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.56</td>
			<td>20 mM</td>
			<td>0.14912 g</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 M (solution)</td>
			<td>2 mM</td>
			<td>200 &#181;l</td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>551.1</td>
			<td>2 mM</td>
			<td>0.1102 g</td>
		</tr>
		<tr>
			<td>Na2GTP</td>
			<td>523.2&#160;</td>
			<td>0.5 mM</td>
			<td>0.02661 g</td>
		</tr>
		<tr>
			<td>Na2-Phosphocreatine</td>
			<td>255.1</td>
			<td>5 mM</td>
			<td>0.1275 g</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>380.4</td>
			<td>0.1 mM</td>
			<td>3.803 mg</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>238.31</td>
			<td>10 mM</td>
			<td>0.23831 g</td>
		</tr>
		<tr>
			<td>Biocytin</td>
			<td>1 mg/mL</td>
			<td></td>
			<td>0.1 g</td>
		</tr>
		<tr>
			<td colspan="4">Osmolarity : ~ 302 mOsmol/L , pH = 7.2 adjusted with KOH</td>
		</tr>
	</tbody>
</table>
Table 3: Intracellular solution for dual recordings.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Substance</td>
			<td>g/mol</td>
			<td>Concentration</td>
			<td>for 100 mL</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.56</td>
			<td>120 mM</td>
			<td>0.8947 g</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1 M (solution)</td>
			<td>2 mM</td>
			<td>200 &#181;l</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 M (solution)</td>
			<td>1 mM</td>
			<td>100 &#181;l</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>238.31</td>
			<td>10 mM</td>
			<td>0.23831</td>
		</tr>
		<tr>
			<td>TEA-Cl</td>
			<td>165.7</td>
			<td>20 mM</td>
			<td>0.3314 g</td>
		</tr>
		<tr>
			<td>4-AP</td>
			<td>94.11</td>
			<td>5 mM</td>
			<td>0.04705 g</td>
		</tr>
		<tr>
			<td>BaCl2</td>
			<td>244.26</td>
			<td>1 mM</td>
			<td>0.02443 g</td>
		</tr>
		<tr>
			<td>CdCl2</td>
			<td>183.32</td>
			<td>0.02 mM</td>
			<td>0.3666 mg</td>
		</tr>
		<tr>
			<td>TTX</td>
			<td>1 mM</td>
			<td>200 nM</td>
			<td>20 &#181;l</td>
		</tr>
		<tr>
			<td colspan="4">Osmolarity: ~ 290 mOsmol/L, adjusted with D-glucose (Ref. 16); pH = 7.4</td>
		</tr>
	</tbody>
</table>
Table 4: Electrode solution for cell-attached recordings.

Watch this Scientific Journal Video about Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons at JoVE.com

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

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Research

JoVE Journal

Neuroscience

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of Liege. Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro. 

Video: Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of recordings are conducted on dissociated DRG neurons, which is a standard preparation for most laboratories. Neuronal properties, however, can be altered by axonal injury resulting from enzyme digestion used in acquiring dissociated neurons. Further, dissociated neuron preparations cannot fully represent the microenvironment of the DRG since loss of contact with satellite glial cells that surround the primary sensory neurons is an unavoidable consequence of this method. To overcome the limitations in using conventional dissociated DRG neurons for patch clamp recordings, in this report we describe a method to prepare intact DRGs and conduct patch clamp recordings on individual primary sensory neurons ex vivo. This approach permits the fast and straightforward preparation of intact DRGs, mimicking in vivo conditions by keeping DRG neurons associated with their surrounding satellite glial cells and basement membrane. Furthermore, the method avoids axonal injury from manipulation and enzyme digestion such as when dissociating DRGs. This ex vivo preparation can additionally be used to study the interaction between primary sensory neurons and satellite glial cells.

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of ...

Biochemistry

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 ...

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, ...

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Biology

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

Patch-Clamp Recordings from the Dendrite of a Dopaminergic Neuron in a Brain Slice

Source: Engel, D. <a href="https://app.jove.com/v/54601">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for patch-clamp recording from the dendritic membrane surface. This helps to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels in dopaminergic neurons.

Nagrania patch-clamp z dendrytu neuronu dopaminergicznego w wycinku mózgu

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Perforated Patch-Clamp Recordings of Inner Ear Sensory Neuron Cell Bodies

Source: Iyer, M. R., et al. <a href="https://app.jove.com/v/64908">Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings.</a> J. Vis. Exp. (2023).
This video demonstrates the patch-clamp technique for measuring ionic currents in inner ear sensory neuron cell bodies. It details the preparation and use of a polished patch-clamp pipette filled with an internal solution containing amphotericin B, which perforates the membrane to allow ion movement while preserving intracellular conditions. Voltage pulses are applied to trigger and record ion channel activity, providing insights into their properties and behavior.

Perforowane nagrania patch-clamp ciał komórek neuronów czuciowych ucha wewnętrznego

1. Recording
NOTE: In this procedure, patch-clamp recordings from the isolated ganglion are typically performed 12-24 h after the plating. Other labs have ...

Imaging and Optical Techniques

Whole-Cell Patch Clamp Recording in a Substantia Gelatinosa Neuron of a Spinal Cord Slice

Source: Zhu, M., et al. <a href="https://app.jove.com/v/58479">Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons.</a> J. Vis. Exp. (2019)
This video demonstrates the procedure for performing electrophysiological recordings in substantia gelatinosa (SG) neurons of spinal cord slices, using whole-cell patch-clamp techniques to analyze neuron firing patterns. This method allows the study of neural responses to stimuli in SG neurons of the spinal cord.