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 border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="137">
			<td height="137" style="height:138px;width:156px;">Double-distillated water</td>
			<td style="width:143px;">Millipore</td>
			<td style="width:137px;">Super Q</td>
			<td style="width:187px;">&#160;Resistivity &#62;14 M&#937; cm, ideal 18.2 M&#937; cm at 25&#176;C, filtered (0.22 &#181;m), https://www.merckmillipore.com/BE/en/product/Super-Q-Plus-Water-Purification-System,MM_NF-C1760</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:156px;">Microfilter candles</td>
			<td style="width:143px;">Robu</td>
			<td style="width:137px;">Porosity 3, 6 mm or 13 mm diameter</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:156px;">Tissue slicer with vibroprobe</td>
			<td style="width:143px;">Leica</td>
			<td style="width:137px;">VT1200</td>
			<td style="width:187px;">cutting parameters: speed 0.07, amplitude 1.40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:156px;">Tissue slicer</td>
			<td style="width:143px;">Dosaka</td>
			<td style="width:137px;">DTK-1000</td>
			<td style="width:187px;">cutting parameters: speed 4, frequency 7</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Razor blades</td>
			<td style="width:143px;">Gilette</td>
			<td style="width:137px;">Super Silver</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Dissection tools</td>
			<td style="width:143px;">Braun, Aesculap</td>
			<td style="width:137px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Reserve chamber</td>
			<td style="width:143px;"></td>
			<td style="width:137px;"></td>
			<td style="width:187px;">Custom-made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">PP beckers</td>
			<td style="width:143px;">VWR</td>
			<td style="width:137px;">213-1725</td>
			<td style="width:187px;">400 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Syringe filter</td>
			<td style="width:143px;">Merk Millipore</td>
			<td style="width:137px;">Millex - GV 0,22 &#181;m</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Cyanoacrylate glue&#160;</td>
			<td style="width:143px;">UHU</td>
			<td style="width:137px;">&#34;Sekunden Kleber&#34;</td>
			<td style="width:187px;">liquid glue</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Recording chamber&#160;</td>
			<td style="width:143px;">Luigs &#38; Neumann</td>
			<td style="width:137px;">Slice mini chamber</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:156px;">Horizontal pipette puller</td>
			<td style="width:143px;">Sutter Instruments</td>
			<td style="width:137px;">Brown-Flaming P-97</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:156px;">Pipettes</td>
			<td style="width:143px;">Hilgenberg</td>
			<td style="width:137px;">1807524</td>
			<td style="width:187px;">2 mm o.d./1 mm i.d. glass capilaries, ends firepolished, washed</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Dental wax</td>
			<td style="width:143px;">Colt&#232;ne/Whaledent</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">orthodontic tray wax strips</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:156px;">Platinum grid</td>
			<td style="width:143px;"></td>
			<td style="width:137px;"></td>
			<td style="width:187px;">to anchor slices in the recording chamber, custom made with a platinum disk</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Microforge</td>
			<td style="width:143px;">Narishige</td>
			<td style="width:137px;">MF-830</td>
			<td style="width:187px;">to fire-polish pipette tips</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:156px;">Potassium methyl sulfate</td>
			<td style="width:143px;">MP Biomedicals</td>
			<td style="width:137px;">215481</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:156px;">Biocytin</td>
			<td style="width:143px;">Molecular probes</td>
			<td style="width:137px;">B1592</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:156px;">Manometer</td>
			<td style="width:143px;">GREISINGER electronic</td>
			<td style="width:137px;">GDH 13 AN</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:156px;">Microscope</td>
			<td style="width:143px;">Zeiss</td>
			<td style="width:137px;">FS</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:156px;">Dodt Gradient Contrast</td>
			<td style="width:143px;">Luigs &#38; Neumann</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Manipulators</td>
			<td style="width:143px;">Luigs &#38; Neumann</td>
			<td style="width:137px;">LN Mini 25</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:156px;">Frame grabber</td>
			<td style="width:143px;">The Imaging Source</td>
			<td style="width:137px;">DFG/USB2pro</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Camera</td>
			<td style="width:143px;">DAGE-MTI</td>
			<td style="width:137px;">NC-70</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:46px;width:156px;">Condenser</td>
			<td style="width:143px;">Zeiss</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">high numerical aperture condenser working with oil</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:156px;">Objective</td>
			<td style="width:143px;">Zeiss</td>
			<td style="width:137px;">W Plan Apochromat 441470-9900 VIS-IR</td>
			<td style="width:187px;">63x magnification, long-distance, high numerical aperture 1.0 water-immersion objective&#160;</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:156px;">Fourfold-changer</td>
			<td style="width:143px;">Luigs &#38; Neumann</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">between the camera and the microscope</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:156px;">Black &#38; white video monitor</td>
			<td style="width:143px;">Sony</td>
			<td style="width:137px;">SSM-175CE</td>
			<td style="width:187px;">16-inch, 850 TV lines, analog</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:156px;">Sample Bottles, Amber Glass, with Cap</td>
			<td style="width:143px;">VWR</td>
			<td style="width:137px;">215-2409</td>
			<td style="width:187px;">to transfer slices from the setup to histology room</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">The Axon Guide</td>
			<td style="width:143px;">Molecular Devices</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">Book</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:46px;width:156px;">Stimfit</td>
			<td style="width:143px;">https://github.com/neurodroid/stimfit</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">Analysis of electrophysiological data</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:156px;">Paraformaldehyde</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:137px;">441244</td>
			<td style="width:187px;">see SAFETY DATA SHEET and Dangerous Substances Directive (EU) before use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:26px;width:156px;">Well cell culture plate</td>
			<td style="width:143px;">Greiner-Bio-One</td>
			<td style="width:137px;">662160</td>
			<td style="width:187px;">for the staining of fixed slices</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:156px;">Triton X-100</td>
			<td style="width:143px;">Merk</td>
			<td style="width:137px;">108643</td>
			<td style="width:187px;">see SAFETY DATA SHEET and Dangerous Substances Directive (EU) before use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:156px;">mouse anti-TH monoclonal primary</td>
			<td style="width:143px;">Immunostar</td>
			<td style="width:137px;">22941</td>
			<td style="width:187px;">working concentration: 1/1000</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:156px;">goat anti-mouse secondary antibody - Alexa Fluor 568</td>
			<td style="width:143px;">Invitrogen - Thermo Fisher Scientific</td>
			<td style="width:137px;">A-11031</td>
			<td style="width:187px;">working concentration: 1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Normal goat serum</td>
			<td style="width:143px;">Dako</td>
			<td style="width:137px;">X0907</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:156px;">ProLong Diamond Antifade</td>
			<td style="width:143px;">Molecular Probes - Thermo Fisher Scientific</td>
			<td style="width:137px;">P36961</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Fluorescein Avidin DCS</td>
			<td style="width:143px;">Vector Lab</td>
			<td style="width:137px;">A-2011</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:156px;">Confocal microscope</td>
			<td style="width:143px;">Olympus</td>
			<td style="width:137px;">FV1000</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:45px;width:156px;">ImageJ</td>
			<td style="width:143px;">http://imagej.nih.gov/ij/</td>
			<td style="width:137px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:156px;">NeuronJ</td>
			<td style="width:143px;">http://www.imagescience.org/meijering/software/neuronj/</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">plugin for ImageJ</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:156px;">Imaris</td>
			<td style="width:143px;">http://www.bitplane.com</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">Reconstruction of biocytin filled neurons</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:156px;">Neuromantic</td>
			<td style="width:143px;">http://www.reading.ac.uk/neuromantic/</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">Reconstruction of biocytin filled neurons</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:61px;width:156px;">WinWCP</td>
			<td style="width:143px;">http://spider.science.strath.ac.uk/sipbs/software_ses.htm</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">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 ...

subcellular-patch-clamp-recordings-from-somatodendritic-domain-nigral

Research

JoVE Journal

Neuroscience

14.8K Views.  Quartier Hôpital, 














University
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 from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.