paired patch clamp recording of the neuronal connections in acute brain slices

Paired Patch Clamp Recording of the Neuronal Connections in Acute Brain Slices

After preparing slices of the target region, allow them to incubate. During this time, pull the patch pipettes, which should have a long and slender shank. After securing a brain slice with a platinum harp in an experimental chamber, fill a recording pipette with internal solution.
Place the pipette in the preamplifier and patch a putative postsynaptic neuron in whole-cell mode. Using a searching patch pipette of 8 to 10 megaohm resistance filled with an internal solution in which potassium salts is replaced by sodium salts, apply gentle suction to the searching pipette to patch a potential presynaptic neuron in a loose-cell attached configuration.
Hold the membrane potential under the loose seal to between negative 30 and negative 60 millivolts in current clamp mode. Then, apply large current pulses of 0.2 to 2 nanoamps to elicit an action potential in the potential presynaptic cell.
Observe this action potential as a small spike lit on the voltage response. Set the stimulation frequency to 0.1 Hertz to prevent rundown of the postsynaptic response. If the postsynaptic neuron does not respond to stimulation of the tested potentially presynaptic neuron, patch a new potential presynaptic neuron in loose-seal mode.
Test up to 30 potentially presynaptic neurons in this way, continuing to use the same searching patch electrode as long as a seal with a resistance of greater than 30 megaohm can be established.
If stimulation in the loose-seal mode results in an EPSP or IPSP with a latency less than five milliseconds in the postsynaptic neuron, remove the searching pipette from the presynaptic neuron.
Using a recording patch electrode filled with biocytin containing regular internal solution with a resistance of four to eight megaohm, patch the presynaptic neuron and record in whole-cell current clamp mode. Elicit action potentials by current injection in the presynaptic neurons and record the postsynaptic response.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Paired Patch-Clamp Recording and Biocytin Filling
Depending on the type of synaptic connection, three different approaches are used to find synaptically coupled neurons. If the connection probability is low (as can be expected for most excitatory connections), proceed as follows:
<ol>
	<li>Neuronal connections with a low connectivity
	<ol>
		<li>Fill the patch pipettes with an internal solution of the composition listed in Table 1. For all recordings in the whole-cell configuration add biocytin at concentrations between 3 - 5 mg/ml to the internal (pipette) solution to obtain good staining results for the pre- and postsynaptic neurons. Let biocytin diffuse into the cell for at least 15 - 30 min (depending on the size of the neuron). 
		&#8203;NOTE: Do not add biocytin to the solution used in the &#39;searching&#39; pipettes mentioned below.</li>
		<li>Patch a putative postsynaptic neuron in the whole-cell mode. Use patch pipettes with a long and slender shank. This facilitates the maneuverability of the pipette under the objective and reduces movement artifacts that may occur when the electrode is introduced in the slice.</li>
		<li>Then, patch a potential presynaptic neuron in a cell-attached configuration using a &#39;searching&#39; patch pipette of 8 - 10 M&#8486; resistance and a small tip diameter. These pipettes establish a &#39;loose&#39; cell-attached patch. Fill the &#39;searching&#39; pipette with an internal solution in which K+ is replaced by Na+ (Table 2) to prevent neurons from depolarizing during searching for a presynaptic cell. 
		NOTE: The &#39;loose&#39; seal resistance is not in the G&#937; range but typically around 30 - 300 M&#937;.</li>
		<li>Hold the membrane potential under the &#39;loose&#39; seal to about -30 to -60 mV in the current clamp mode. Then, apply large current pulses (0.2 - 2 nA) to elicit an action potential in the potential presynaptic cell. Observe this action potential as a small spikelet on the voltage response.</li>
		<li>Set the stimulation frequency to 0.1 Hz to prevent a run-down of a postsynaptic response. Use lower stimulation frequencies if the brain tissue is very immature or the synaptic connection is not very reliable.</li>
		<li>In case the &#39;postsynaptic&#39; neuron does not respond to stimulation of the tested &#39;presynaptic&#39; neuron, patch a new neuron in &#39;loose&#39; seal mode. Do not replace the &#39;searching&#39; patch electrode as long as the seal with a resistance of &#62;30 M&#937; is established. In this way, test up to 30 potentially presynaptic neurons. 
		NOTE: To prevent damaging the neuron during the searching procedure with a loose patch clamp, only gentle suction should be applied to the searching pipette compared with that applied to the whole-cell patch pipette.</li>
		<li>If stimulation in the &#39;loose&#39; seal mode results in an EPSP/IPSP (excitatory postsynaptic potential/inhibitory postsynaptic potential) with a latency &#60;5 msec in the postsynaptic neuron, remove the &#39;searching&#39; pipette from the presynaptic neuron.</li>
		<li>Replace the &#39;searching&#39; patch pipette with a patch electrode filled with biocytin-containing, regular internal solution. Ensure this recording patch pipette has a resistance of 4 - 8 M&#937;; the larger the soma size, the lower the electrode resistance can be.</li>
		<li>Patch the presynaptic neuron and record in whole-cell, current-clamp mode. Elicit action potentials by current injection in the presynaptic neurons and record the postsynaptic response. Store the data on a computer for later offline analysis.</li>
	</ol>
	</li>
</ol>
Table 1. Low chloride pipette solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>mM</td>
			<td>g/L</td>
			<td>g/50 ml</td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>135</td>
			<td>31.622</td>
			<td>1.5811</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>4</td>
			<td>0.298</td>
			<td>0.0149</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10</td>
			<td>2.384</td>
			<td>0.1192</td>
		</tr>
		<tr>
			<td>Phosphocreatine</td>
			<td>10</td>
			<td>2.552</td>
			<td>0.1276</td>
		</tr>
		<tr>
			<td>ATP-Mg2+</td>
			<td>4</td>
			<td>2.028</td>
			<td>0.1014</td>
		</tr>
		<tr>
			<td>GTP-Na</td>
			<td>0.3</td>
			<td>0.156</td>
			<td>0.0078</td>
		</tr>
		<tr>
			<td colspan="4">Adjust pH to 7.3 with KOH</td>
		</tr>
		<tr>
			<td colspan="4">Osmolarity ~300 mOsmol/L</td>
		</tr>
		<tr>
			<td colspan="4">Add 3 - 5 mg/ml biocytin</td>
		</tr>
	</tbody>
</table>
Table 2. High Na pipette solution for searching synaptically coupled neurons.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>mM</td>
			<td>g/L</td>
			<td>g/50 ml</td>
		</tr>
		<tr>
			<td>Na-gluconate</td>
			<td>105</td>
			<td>22.92</td>
			<td>1.146</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>30</td>
			<td>1.76</td>
			<td>0.088</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10</td>
			<td>2.384</td>
			<td>0.1192</td>
		</tr>
		<tr>
			<td>Phosphocreatine</td>
			<td>10</td>
			<td>2.552</td>
			<td>0.1276</td>
		</tr>
		<tr>
			<td>ATP-Mg2+</td>
			<td>4</td>
			<td>2.028</td>
			<td>0.1014</td>
		</tr>
		<tr>
			<td>GTP-Na</td>
			<td>0.3</td>
			<td>0.156</td>
			<td>0.0078</td>
		</tr>
		<tr>
			<td colspan="4">Adjust pH to 7.3 with NaOH</td>
		</tr>
		<tr>
			<td colspan="4">Osmolarity ~300 mOsmol/L</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amplifier</td>
			<td>HEKA</td>
			<td>EPC 10 USB Triple</td>
			<td>with 2-3 preamplifiers</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>with 2 camera ports and a 4&#215; objective, a 40&#215; water-immersion objective</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>TILL Photonics</td>
			<td>VX55</td>
			<td>infrared CCD camera</td>
		</tr>
		<tr>
			<td>Workstation</td>
			<td>Luigs &#38; Neumann</td>
			<td>Infrapatch 240</td>
			<td>with a motorized x-y stage and a motorized focus axis for the microscope</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Luigs &#38; Neumann</td>
			<td>SM-5</td>
			<td>x-y-z manipulators for 2-3 preamplifiers</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>Luigs &#38; Neumann</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-vibration table</td>
			<td>Newport Spectra-Physics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Patchmaster</td>
			<td>HEKA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Microm International</td>
			<td>HM650V</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>HEKA</td>
			<td>Sutter P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurolucida system</td>
			<td>Microbrightfield</td>
			<td></td>
			<td>with Neurolucida and Neuroexplorer softwares</td>
		</tr>
	</tbody>
</table>

Source: Qi, G., et al. <a href="https://app.jove.com/v/52358">Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings.</a> J. Vis. Exp. (2015)
This video demonstrates the procedure to map synaptic connections by securing a brain slice, positioning pipettes to achieve whole-cell configurations, and stimulating presynaptic neurons to record postsynaptic responses, thereby elucidating neuronal connectivity.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Paired Patch-Clamp ...

Secure a brain slice with the target region in a recording chamber.
Position a recording pipette containing an internal solution over a postsynaptic neuron and achieve a whole-cell configuration.
Introduce a searching patch pipette near a presynaptic neuron.
This pipette is filled with a sodium-based solution to prevent neuron depolarization while searching for the presynaptic cell.
Use gentle suction to establish a loose-seal configuration with the searching pipette.
Hold the membrane potential and apply large current pulses to trigger an action potential in the presynaptic neuron.
Monitor the postsynaptic response to this stimulation.
If no response is detected, patch another presynaptic neuron and repeat stimulation.
Continue this process until a response is observed in the postsynaptic neuron.
Replace the searching pipette with a recording pipette and establish a whole-cell configuration in the presynaptic neuron.
Apply current pulses to elicit action potentials in the presynaptic neuron and record the postsynaptic response.

24 visualizações. Registro de grampo de patch emparelhado das conexões neuronais em fatias cerebrais agudas

Vídeo: Registro de grampo de patch emparelhado das conexões neuronais em fatias cerebrais agudas - Experimento

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Avaliação Funcional de neurotoxinas Biológica em Culturas em Rede de Células-Tronco derivadas do Sistema Nervoso Central Neurônios

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

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Neurociência

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

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

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

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

Examinando Monosynaptic conexões em drosófila usando os canais de sódio tetrodotoxina resistente

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

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

Paired Patch Clamp Recording of the Neuronal Connections in Acute Brain Slices

Transcrição

Paired Patch Clamp Recording of the Neuronal Connections in Acute Brain Slices