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

Samenvatting

Source: Qi, G., et al. Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings. 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.

Protocol

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:

1. Neuronal connections with a low connectivity
   1. 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).
      ​NOTE: Do not add biocytin to the solution used in the 'searching' pipettes mentioned below.
   2. 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.
   3. Then, patch a potential presynaptic neuron in a cell-attached configuration using a 'searching' patch pipette of 8 - 10 MΩ resistance and a small tip diameter. These pipettes establish a 'loose' cell-attached patch. Fill the 'searching' 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 'loose' seal resistance is not in the GΩ range but typically around 30 - 300 MΩ.
   4. Hold the membrane potential under the 'loose' 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.
   5. 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.
   6. In case the 'postsynaptic' neuron does not respond to stimulation of the tested 'presynaptic' neuron, patch a new neuron in 'loose' seal mode. Do not replace the 'searching' patch electrode as long as the seal with a resistance of >30 MΩ 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.
   7. If stimulation in the 'loose' seal mode results in an EPSP/IPSP (excitatory postsynaptic potential/inhibitory postsynaptic potential) with a latency <5 msec in the postsynaptic neuron, remove the 'searching' pipette from the presynaptic neuron.
   8. Replace the 'searching' patch pipette with a patch electrode filled with biocytin-containing, regular internal solution. Ensure this recording patch pipette has a resistance of 4 - 8 MΩ; the larger the soma size, the lower the electrode resistance can be.
   9. 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.

Table 1. Low chloride pipette solution.

	mM	g/L	g/50 ml
K-gluconate	135	31.622	1.5811
KCl	4	0.298	0.0149
HEPES	10	2.384	0.1192
Phosphocreatine	10	2.552	0.1276
ATP-Mg2+	4	2.028	0.1014
GTP-Na	0.3	0.156	0.0078
Adjust pH to 7.3 with KOH
Osmolarity ~300 mOsmol/L
Add 3 - 5 mg/ml biocytin

Table 2. High Na pipette solution for searching synaptically coupled neurons.

	mM	g/L	g/50 ml
Na-gluconate	105	22.92	1.146
NaCl	30	1.76	0.088
HEPES	10	2.384	0.1192
Phosphocreatine	10	2.552	0.1276
ATP-Mg2+	4	2.028	0.1014
GTP-Na	0.3	0.156	0.0078
Adjust pH to 7.3 with NaOH
Osmolarity ~300 mOsmol/L

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Amplifier	HEKA	EPC 10 USB Triple	with 2-3 preamplifiers
Microscope	Olympus	BX51WI	with 2 camera ports and a 4× objective, a 40× water-immersion objective
Camera	TILL Photonics	VX55	infrared CCD camera
Workstation	Luigs & Neumann	Infrapatch 240	with a motorized x-y stage and a motorized focus axis for the microscope
Micromanipulator	Luigs & Neumann	SM-5	x-y-z manipulators for 2-3 preamplifiers
Faraday cage	Luigs & Neumann
Anti-vibration table	Newport Spectra-Physics
Patchmaster	HEKA
Microtome	Microm International	HM650V
Micropipette puller	HEKA	Sutter P-97
Neurolucida system	Microbrightfield		with Neurolucida and Neuroexplorer softwares