神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

The overall goal of the following experiment is to demonstrate how synaptic connections between morphologically identified neurons are characterized both functionally and structurally. This is achieved by obtaining acute brain slices, which are optimized for studying a particular synaptic connection. Patch clamping is then used to test for synaptically coupled neurons.

For this, an individual prospective post-synaptic neuron is patched, and a second pipette is then used to stimulate surrounding neurons in order to establish the existence of a synaptic connection. If no response is seen, the stimulating electrode is moved. Once a connection is found, the presynaptic neuron is rematched in wholesale mode.

Following electrophysiological recordings from these neurons and simultaneous biotin fillings, the slice is fixed, processed for biotin and computer assisted 3D reconstructions of the neurons is performed. Ultimately, our method aims at a correlated quantitative structural and functional analysis of neuronal microcircuits. The main advantage of paired recordings from synaptically coupled neurons is that we can perform a correlated structural and functional analysis of both pre and postsynaptic neurons in neuronal micro circuits.

Even more recently, introduced techniques such as optical stimulation of neurons are so far not able to achieve this with as much detail. This method enables the study of the interactions of excitation and inhibition of morphologically identified neuron types and how the activity is modulated. Demonstrating the procedures for pet recordings will be Guan.

He who is a postdoc in my laboratory. 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 pre amplifier and patch a putative post-synaptic neuron in whole cell mode using a searching patch pipette of eight to 10 mega ohm 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 two nano amps to elicit an action potential in the potential presynaptic cell. Observe this action potential as a small spikelet 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 the resistance of greater than 30 mega ohm 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 biotin containing regular internal solution with a resistance of four to eight mega ohm. 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. Throughout the recording, biotin diffuses into the neuron to obtain adequate staining.

Allow the biotin to diffuse for at least 15 to 30 minutes to depending on the size of the neuron. For neuronal microcircuits with a high connectivity ratio, a searching electrode is not used. Rather begin by directly patching a presynaptic neuron in whole cell mode.

Then using a patch electrode filled with regular internal solution patch a potential postsynaptic neuron also in whole cell mode, both cells should be under current clamp control. Elicit an action potential in the presynaptic cell and monitor the prospective postsynaptic neuron for a postsynaptic potential. If the neuron does not show a response to presynaptic stimulation, use a new electrode to patch a new neuron again in whole cell mode.

When a connection is found, proceed with recording as demonstrated earlier without changing the patch pipette. After fixing and mounting the slices as described in the text protocol, examine the biotin labeled neurons under a light microscope. Ensure that the axonal batons and dendritic spines are clearly visible.

Using a commercial neuron tracing system, manually trace the neurons or synaptically coupled neuron pairs. Be sure to include even small axonal or dendritic collaterals. Use the traces to obtain 3D neuronal reconstructions, correct the neuronal reconstructions for shrinkage in all three spatial dimensions in the appropriate section of the tracing program.

Shrinkage correction factors for the embedding medium are approximately 1.1 for the x and y directions, and approximately 2.1 for the Z direction. A reciprocally coupled cell pair comprised of a spiny stellate and a fast spiking into neuron. In cortical layer four were filled with biotin.

During recording, the green dots indicate putative excitatory synaptic contacts between the pre-synaptic spiny neuron and the post-synaptic inter neuron. The blue dots indicate putative reciprocal inhibitory synaptic contacts. High power images of the synaptic contacts are shown here.

The green open circles show excitatory contacts and the blue open circles show inhibitory contacts. A neuro lucid reconstruction of this reciprocally connected cell pair is shown here. The inter neurons axon is shown in blue and its soma and dendrite are shown in red.

The spiny neurons axon is shown in green and its soma and dite are in white. The inset shows the somato dendritic compartments of the pre and post-synaptic neurons. Together with the putative synaptic contacts, barrel contours were identified in the low power brightfield photo micrographs made from the acute brain slice.

A representative synaptic connection with a very low connectivity ratio is shown here. A presynaptic action potential in a layer six parametal cell evoked a monos synaptic excitatory postsynaptic potential in a layer four star parametal neuron. The latency shown here of more than three milliseconds of this trans laminar connection is much longer than the approximately one millisecond latency observed in local intra laminar connections.

Two action potentials at 10 hertz elicited in the layer six parametal cell triggered post-synaptic potentials in the layer four star parametal neuron shown here. Note the short term facilitation. It is important to remember that the slashing condition need to be optimized for a given synaptic connection, and that during recording the setup needs to be absolutely stable and then the optimal recording understanding are guaranteed.

After watching this video, you should have a good understanding of how the electrophysiological measurements and the morphological reconstructions are used to get an overall picture of the structural and functional properties of neuronal micro circuits in the neocortex.