Classically Communication between the pre and postsynaptic elements involves an action potential arriving at the presynaptic terminal, which triggers by increasing calcium, the release of glutamate activating postsynaptic receptors. However, the observation that astrocytes intimately contact, synopsis and express neurotransmitter receptors has changed it view into the concept of the three partite synap. Ongoing glutamatergic transmission will therefore also activate the astrocyte and trigger by intracellular calcium rise.
The release of glio transmitters, which can then wire binding to ex extra synaptic or synaptic receptors modulate pre and post synaptic activities. Furthermore, astrocytes expressed the main uptake system for synaptically released glutamate. In addition, they clear up potassium, which is released during action, potential firing and subsequent postsynaptic activation from the pre and postsynaptic terminus via their potassium channels.
This potassium is then intracellularly transported to sites of lower extracellular potassium levels where it is released or diffuses via gap junctions to adjacent astrocytes. Thus, by controlling the ex extracellular concentration of neuroactive substances as well as by releasing clear transmitters, astrocytes play important roles in defining the strengths of synaptic activity. My laboratory investigates the role of neuroglial Interactions in cerebral physiopathology.
We have thus extensively used and perfected this technique to unravel the wall of astrocytes in the moment to moment synaptic transmission. Indeed, this technique is powerful as it allows monitoring online and simultaneously the dynamic electrophysiological responses of neurons. And as Osteocytes Evap clinic today, Ash and Jeremy Siebel members of the laboratory will demonstrate you how to perform this technique and explain you how it help us, the cing, an intimate dialogue between neurons and astrocytes.
I've investigated the impact of astroglide networks on neurons, synaptic transmission and plasticity by performing dual recordings of neuron activity and astro gly potassium and glut transport occurrence, and found that asline networking facilitates the extracellular of potassium and glutamate removal during neural activity. I have investigated the nature properties and plasticity of astroglia occurrence in relation to neurophysiology. During recording of astroglia and neuronal evok activity, allow me to identify that astrocyte response to neuronal activation, mostly by potassium currents.
This current have their own electrophysiological plasticity, which don't regulate neural acceptability and plasticity. In other words, astrocyte countdown, the very excited one.Neuros. We will present here an important method to investigate simultaneously urinal and astroglia activity in acute hippocampal slices by performing dual recordings of astrocytic membrane currents and urinal feed potentials evoked by presynaptic fiber stimulation.
Furthermore, we will perform paired recordings of single neurons and adjacent astrocytes to investigate ASTO gly responses during neuronal activity. We have chosen the hippocampus because this region exhibits a strong anatomic and functional compartmentalization and is well characterized in terms of neuro circuitry as well as synaptic Activity and plasticity. Start the experiments by preparing Hippocampal brain slices for this first prepare ice called oxygenated.
Artificial cerebral spinal fluid, deeply anesthetize your mice and quickly cut the head off and transferred into A CSF. Open the skull with three cuts now separated two hemispheres. Finally, transfer Them into oxygenated A CSF.
Let it equally braid for around five minutes. Now start with One hemisphere. The dissection of the hippocampus begin by removing the midbrain.
The detailed hippocampus Dissection is now illustrated in this figure. First position the separated hemisphere onto its cortical site to access the midbrain. With the help of two spoons, remove the midbrain.
Now the hippocampus will be visible as indicated by the dash lines. Dissect hippocampus out with a spoon starting from the fia, which is visible as a wide structure. Insert a spoon below the hippocampus and with the help of a second spoon, cut off the cortex adjacent to the hippocampus.
Transfer the hippocampus to a small culture dish with oxygenated A CSF and proceed With the second hemisphere Position both hippo on a small AGA block with the alveo side up. Now suck away DS A CSF. Glue the hippo onto your slicing chamber device.
Transfer the hippocampus Into the slicing chamber and cut 300 or 400 micrometer slices. Collect the slices in an oxygenated storage chamber and let them rest at least one hour prerecording at room temperature to perform wholesale recordings of single astrocytes. Transfer your hippocampus slice on a Polylysine coated cover slip.
Dry out the excess A CSF to allow attachment of the hippocampus slice to the poly coated cover slip and then re add A CSF on top of the slice. Transfer the slice into your recording chamber, which is constantly perfused with oxygenated A CSF at a perfusion speed of one to two milliliter per minute. To identify astrocytes mice expressing EGFP under the astroglia specific GFAP promoter can be used for patch clamp recordings.
Fill a glass pipe with a resistance of four to six mega ohms with filtered cold intracellular solution by using intracellular solution containing rodda dron, you can visualize in red the patched astrocyte in green. You see the neighboring astrocytes expressing EGFP. Under the GFAP Promoter, astrocytes can be identified By their small soma size of around 10 micrometer and their main process branching, including usually three to four branches.
A syringe connected via tubing to the pipet allows positive pressure application while descending into the tissue in the command window, trigger a test pulse of 10 millivolt and rero the holding current. Then move the pipet with the help of the Micro manipulator into the tissue to approach the cell. When we reach the cell surface, We move further until we see a dimpling visible as a light deflection indicating that we deform a little bit the cell surface.
Due to the positive pressure application, the astrocytic cell membrane will now seal tightly onto the glass piper tip opening, which can be observed By a increasing resistance when a giga seal is reached. Perform capacitance compensation go into the wholesale configuration by gentle application of negative pressure while observing the test parts on the command window. The break into the cell will be visible by current flowing Through the membrane.
Application of a Series of hyperpolarizing and depolarizing voltage steps allows the characterization of the cell membrane properties astrocytes Normally exhibit only passive current responses. Current injection in Current clamp mode does not result in action potential. Firing a further confirmation that indeed you have Patched an astrocyte addition of a Tracer such as sulfur or domine B to your patch, pipet enables the visualization of the patched astrocyte.
Furthermore, passive diffusion of the tracer during 20 minutes in the current clamp mode enables to visualize the gap junctional connected neighboring astrocytes to perform dual recordings of neuronal excitatory transmission, and a subsequently elicited astrocytic currents. We first block inhibitory transmission with precor toin to prevent hyperexcitability, which occurs in the absence of GABAergic transmission. We cut the shaer collaterals mediated connections between CS three and C one with a small iris knife.
We then position an A CSF field stimulation electrode, a field recording electrode, and the astrocytic patch clamp electrode. In the hippocampus slice, we choose an astrocyte and position the field electrode 5, 200 micrometer away from it to ensure that indeed the astrocyte integrates the activity recorded with the field electrode stimulation of the Shafer collaterals will evoke an excitatory field potential. This field potential consists of the fiber wall reflecting the number of stimulated axons and the subsequent postsynaptic depolarization to perform dual recordings of neuronal feed potentials and astroglia whole cell current responses.
First, position your field recording electrode around 50 micrometer away from the chosen astrocyte. Then move your patch electrode to the astrocytic soma and patch clamp, the chosen cell verified by its passive membrane properties that it is indeed an astrocyte. Now by stimulating Shaer Cs, we can simultaneously record neural activity and a subsequent astrocytic current, which consists in a long lasting to 50 pic oum response due to their network connectivity and the fact that one astrocyte is contacting synapses of up to 100 neurons.
These cells integrate the activity of a large number of simultaneously activated Synapses to perform dual whole cell Recordings of individual neurons and adjacent astrocytes descend with two patch electrodes into the stratum area of the hippocampus slice. Identify in the C one region a neuron of interest, and at the same depth around 20 to 60 micrometer away and astrocyte then position both pipettes around 10 micrometer above each cell while applying positive pressure. The astrocytic recording pipette should be positioned as close as possible to the targeted astrocyte to avoid further mechanical movements.
First, achieve the gga seal on the neuron. Now proceed with patching the astrocyte, which usually seals slower than neurons. To summarize the important steps to perform Dual wholesale recordings, lower first the two recording pipes into the slice.
Approach the soma of the astrocyte, then perform the gga seal on the neuron and finalize by performing the gga seal on the astrocyte neurons usually have a much higher membrane resistance than astrocytes, which is for pyramidal neurons around 200 to 500 mega having both cells. Now in voltage clamp mode T and hyperpolarizing, the membrane activates transient sodium currents in the neuron and passive potassium Responses in the astrocyte. We here show that induction Of fast excitatory postsynaptic field potentials by stimulating shaa collaterals evokes a subsequent long-lasting current response.
In the adjacent astrocyte inhibition of ionotropic glutamate receptors with remic acid reveals that the majority of the asto gly current is due to excitatory postsynaptic activation. The remaining B AIC response consists of the fast transient glutamate transport occurrence sensitive to TBOA and the small long-lasting current, most likely due to the presynaptic potassium release during action. Potential firing the ascitic current monitors very reliably the postsynaptic activity since both increased linearly while increasing the presynaptic activity.
Furthermore, astrocytic currents exhibit like neuronal responses, paired pulse facilitation. Moderate single stimulation of the shaer collaterals induces a relatively large synaptically evoked asto gly current compared to the small evoke depolarization recorded in the same cell in current clamp mode. This is due to the low membrane resistance of astrocytes.
Recording of the synaptically evoked TTO gly membrane potential dynamics is a direct measure of the local extracellular potassium levels by performing paired recordings of a neuron and an adjacent astrocyte, we observed no astrocytic current response while the neuron was firing action potentials indicating that they were not electrically coupled, excitatory postsynaptic potential evoked in the CS three region also elicit astrocytic current responses. Also, one, her stimulation strongly potentiated the neuronal response in the CS three region. It only moderately increased the astrocytic current.
After watching this video, we should Have a better understanding of how to set up and perform successful dual electrophysiological recordings of neurons and cytes. Using this technique, you can study online dynamic signaling between neurons and astrocytes for your electrophysiological response of interest. In particular, you can investigate whether new GL interactions involving iron channel functions and modulations contribute to define physiological or pathological situations.
Thank you for Watching and good luck with your experiment.
