EPFL scientists have used a cutting-edge method to stimulate neurons with light. They have successfully recorded synaptic transmission between neurons in a live animal for the first time.
Neurons, the cells of the nervous system, communicate by transmitting chemical signals to each other through junctions called synapses. This “synaptic transmission” is critical for the brain and the spinal cord to quickly process the huge amount of incoming stimuli and generate outgoing signals. However, studying synaptic transmission in living animals is very difficult, and researchers have to use artificial conditions that don’t capture the real-life environment of neurons. Now, EPFL scientists have observed and measured synaptic transmission in a live animal for the first time, using a new approach that combines genetics with the physics of light.
Aurélie Pala and Carl Petersen at EPFL’s Brain Mind Institute used a novel technique, “optogenetics”, that has been making significant inroads in the field of neuroscience in the past ten years. This method uses light to precisely control the activity of specific neurons in living, even moving, animals in real time. Such precision is critical in being able to study the hundreds of different neuron types, and understand higher brain functions such as thought, behavior, language, memory – or even mental disorders.
Activating neurons with light
Optogenetics works by inserting the gene of a light-sensitive protein into live neurons, from a single cell to an entire family of them. The genetically modified neurons then produce the light-sensitive protein, which sits on their outside, the membrane. There, it acts as an electrical channel – something like a gate. When light is shone on the neuron, the channel opens up and allows electrical ions to flow into the cell – a bit like a battery being charged by a solar cell.
The addition of electrical ions changes the voltage balance of the neuron, and if the optogenetic stimulus is sufficiently strong it generates an explosive electrical signal in the neuron. And that is the impact of optogenetics: controlling neuronal activity by switching a light on and off.
Recording neuronal transmissions
Pala used optogenetics to stimulate single neurons of anesthetized mice and see if this approach could be used to record synaptic transmissions. The neurons she targeted were located in a part of the mouse’s brain called the barrel cortex, which processes sensory information from the mouse’s whiskers.
When Pala shone blue light on the neurons that contained the light-sensitive protein, the neurons activated and fired signals. At the same time, she measured electrical signals in neighboring neurons using microelectrodes that can record small voltage changes across a neuron’s membrane.
Using these approaches, the researchers looked at how the light-sensitive neurons connected to some of their neighbors: small, connector neurons called “interneurons”. In the brain, interneurons are usually inhibitory: when they receive a signal, they make the next neuron down the line less likely to continue the transmission.
The researchers recorded and analyzed synaptic transmissions from light-sensitive neurons to interneurons. In addition, they used an advanced imaging technique (two-photon microscopy) that allowed them to look deep into the brain of the live mouse and identify the type of each interneuron they were studying. The data showed that the neuronal transmissions from the light-sensitive neurons differed depending on the type of interneuron on the receiving end.
Synaptic Transmission across Cortical States—Future Perspectives
Although on average we did not find a consistent modulation of uEPSPs in PV or Sst neurons comparing quiescent cortical states (DOWN) and active cortical states (UP), in a few cells we found that uEPSP amplitude decreased significantly during UP states. Decreases in uEPSP amplitude during UP states would be expected because the electrical driving force is different, with UP states being depolarized compared to DOWN states. In addition, the synaptic input occurring during UP states causes decreases in input resistance in some experimental preparations but not others. On the other hand, depolarization can also enhance presynaptic neurotransmitter release and activate postsynaptic voltage-gated somatic and dendritic conductances, which could boost uEPSP amplitude. The regulation of synaptic transmission across cortical states may therefore be complicated and deserves further detailed investigation. It is also possible that anesthesia directly affects synaptic transmission. In future experiments, it will therefore be important to extend these first in vivo measurements of cell-type-specific synaptic transmission to other well-defined neocortical cell types and to compare synaptic transmission across different behavioral states in awake mice.
Highlights
•Single-cell optogenetics for precise stimulation of action potentials in vivo
•In vivo whole-cell recordings from genetically defined postsynaptic GABAergic neurons
•Parvalbumin-expressing neurons receive strong, fast, and reliable excitatory input
•Somatostatin-expressing neurons receive longer-lasting, facilitating excitatory input
Summary
Intracellular recordings of membrane potential in vitro have defined fundamental properties of synaptic communication. Much less is known about the properties of synaptic connectivity and synaptic transmission in vivo. Here, we combined single-cell optogenetics with whole-cell recordings to investigate glutamatergic synaptic transmission in vivo from single identified excitatory neurons onto two genetically defined subtypes of inhibitory GABAergic neurons in layer 2 / 3 mouse barrel cortex. We found that parvalbumin-expressing (PV) GABAergic neurons received unitary glutamatergic synaptic input with higher probability than somatostatin-expressing (Sst) GABAergic neurons. Unitary excitatory postsynaptic potentials onto PV neurons were also faster and more reliable than inputs onto Sst neurons. Excitatory synapses targeting Sst neurons displayed strong short-term facilitation, while those targeting PV neurons showed little short-term dynamics. Our results largely agree with in vitro measurements. We therefore demonstrate the technical feasibility of assessing functional cell-type-specific synaptic connectivity in vivo, allowing future investigations into context-dependent modulation of synaptic transmission.
SOURCES – EPFL, Journal Cell
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