Wednesday, March 6, 2013

Amps and Neurotransmitters: The Story of Kiss and Run Exocytosis


    

            If you wanted to study the kinetics of neurotransmitter release at neuronal synapses, how would you do it? There has been a traditional answer to this question in the research community. Stick a recording electrode in the post-synaptic neuron, stimulate the pre-synaptic one with another electrode, and record the electrical response of the post-synaptic cell. However, indirect approaches like this limit our ability to detect what is really going on in pre-synaptic active zones. An innovative electrophysiological technique can address this problem. It’s known as amperometry, and this is how it works. Say you want to study the release of dopamine from neurons. Place a carbon fiber electrode in solution just over the pre-synaptic active zone. Then, generate a voltage of +700mV in the electrode. This is a high enough voltage to oxidize dopamine molecules that come into contact with the electrode, and thus generate a current in the electrode. The magnitude of this current is therefore proportional to the amount of dopamine released. This allows for direct recording of the amount of dopamine released from synapses over time in response to stimulation.  Amperometry has enabled a team of scientists at Columbia University to shed light on the nature of the synaptic release of dopamine.
            If you are already familiar with the basics of synaptic transmission, you know that depolarization of pre-synaptic cells causes the release of neurotransmitter via fusion of synaptic vesicles with the pre-synaptic membrane.  However, this fusion isn’t always complete. It is thought that often, especially with vesicles that contain small molecule neurotransmitters such as dopamine, vesicles may partially fuse with the membrane and then separate and close up again. Sometimes vesicles may repeat this partial fusion and dissociation, known as “kiss and run exocytosis”, several times in quick succession. Using amperometry, the researchers as Columbia were able to provide convincing evidence that this is the primary means of exocytosis in dopaminergic neurons.  Using neurons cultured in vitro, they recorded dopamine release in response to depolarization. Their recordings showed two distinct current patterns. Sometimes, single spikes of current were recorded, termed simple events by the researchers. Other times, they recorded repeated spikes of current, with the initial spike being comparable in amplitude to the single spike event, but the subsequent spikes always decreasing in amplitude over time. These were labeled complex events. The researchers interpreted these recordings as evidence of kiss and run exocytosis. The single spikes represented instances of single transient fusion of vesicles with the membrane. The compound spike patterns were thought to represent repeated fusion of the same vesicle with the membrane. The continual decrease in current would correspond to the slowly depleting dopamine stores in the vesicles as it continually fused and resealed, releasing more of its neurotransmitter with each fusion event.


 Figure: diagrams c and d represent current recordings of simple and complex events, respectively


            This study provides solid evidence of the prevalence of kiss and run exocytosis in dopaminergic neurons. The major limitation of these results is that these recordings were performed using cultured neurons in vitro. It will be interesting to see if these same results hold true for neurons in vivo. However, this study still lays the groundwork for uncovering the intricate details of vesicle fusion in neurons. Soon we may see if other types of neurons also use this pattern of release and to what extent, as well as what functional advantage it may convey to these neurons. 


Posted by Sean McDougall (2)

5 comments:

  1. Very interesting. So this means the neuron doesn't have to package dopamine in a completely new vesicle after releasing it... dopamine can be pumped back into the same vesicle using ATPase? Do we know how the duration of the "kiss and run" is regulated? What's stopping us from trying the same experiment in vivo?

    ReplyDelete
    Replies
    1. Yes, this allows cells to reuse the same vesicles for multiple fusion events. As far as I know, I don't think the regulatory mechanisms behind this type of transient exocytosis have been completely worked out, However, I believe both myosin II and actin coating of vesicles have been implicated in it's regulation. Seeing as this article was written some years ago, I don't know if anyone has tried to replicate the same findings in vivo. Actually, I'm not sure you could even use this same technique in vivo. It might be possible to try it using fresh tissue, instead of cultured cells. However, this could introduce some confounding variables, because it would be harder to control for dopamine being the only molecule able to generate a current in the electrode.

      Posted by Sean McDougall (2)

      Delete
  2. This is cool. Did different voltages get different events? Does kiss and run exocytosis use SNARE proteins? Because I thought all vesicle docking used SNARE proteins but that process was irreversible. Do these vesicles use a different method? Has this experiment been run for other neurotransmitters?

    Kaitlin Jones (3)

    ReplyDelete
    Replies
    1. I don't beleive increasing the voltage changed the profile of the events, since this shouldn't effect the dopamine concentration. However, it would increase the current in the electrode according to ohm's law (but you would still see the same profile proportionally). Yes, to the best of my knowedge the consensus is that these vesicles do use SNARE proteins. This was kind of an upset at the time, since it was originally believed that activation of SNAREs always caused complete fusion, and these findings seem to disprove that. Yes, amperometry experiments have been run with other neurotransmitters.

      Posted by Sean McDougall

      Delete
  3. Dopamine is one of the neurotransmitter of the CNS which has many functions: including roles in behavior and cognition, voluntary movement, motivation, punishment and reward, sleep, dreaming, mood, attention, working memory, and learning. So, would the diagrams for different functions of this NT be different? Or this is the general patterns for the kinetic of this NT?

    Setareh Sepasi (3)

    ReplyDelete