I've noticed that my general level of agitation has drastically increased ever since the Republican National Convention began in New York. I think I'm just pissed off in general that all of the top Republicans are strutting around the most Democratic city in the country acting like they own the place. So here's a message from me to all of the delegates, speakers and elected officials attending the convention: Welcome to New York city. Now get the fuck out.
Fortunately things have been going well in the lab, which has helped offset my moodiness a bit. (Prepare for more science talk, so if this bores you, you can just skip the rest of this journal entry.) Since Saturday I've been working like crazy on my electrophysiology experiments. I've spent the last year learning the techniques that I need to know to do these experiments properly, configuring the equipment that I need to use to make it work and ironing out the (sometimes significant) technical hurdles that stood between me and my experimental results. I seem to have finally cleared all of that, and the data are rolling in.
Here's the basic experimental set up: I've reconstituted the mouse inferior olive to deep cerebellar nuclear synapse in vitro in a cell culture preparation, to my knowledge the first such demonstration of a successful recapitulation of this synapse outside of a culture brain slice. I keep the IO neurons within an explant (a tiny chunk of tissue) and the DCNs are part of a mixed, dispersed population of cerebellar neurons. Most people are of the theory that a slice preparation more accurately reflects the in vivo physiology of the synapse in question than a dispersed cell culture (since it's basically just a chunk of tissue, while a dispersion means you've mechanically or enzymatically dissociated the cells and then allowed them to grow on a glass substrate.) The usefulness of this "coculture" system however is that when I study the interplay between the presynaptic neurons (the IO) and the postsynaptic neurons (the DCN) I can selectively alter the genotype of one or the other or both by culturing neurons from genetic knockout mice that our lab has developed in combination with neurons from normal, or wild-type mice. The gene in question is an isoform of a neurotrophic factor called neuregulin, which is expressed at high levels throughout adolescence and adulthood in the mouse in both IO and DCN neurons.
What I do is called whole cell patch clamp recording where I can actually monitor the electrical activity in the postsynaptic neuron in real time. By selecting DCN neurons (based on their size and shape ) that are close to IO explants with visible projections coming out from them, I'm reasonably certain that the neurons from which I'm recording are being innervated by the IO.
My hypothesis, based on a lot of different work from our lab and other labs, is that eliminating Neuregulin presynaptically will alter the synaptic characteristics of this synapse, especially with respect to modulation by the neurotransmitter acetylcholine. In the brain (unlike at muscles, where acetylcholine is the only neurotransmitter) acetylcholine mainly speeds up neurotransmission by a process called presynaptic facilitation, discovered in my lab, where basically activation of nicotinic acetylcholine receptors on the presynaptic neuron allow for additional calcium ions to enter the synaptic bouton that allows for more neurotransmitter vesicles to be released when the presynaptic neuron fires. More vesicles = more activity in the postsynaptic neuron. Hence, facilitation of transmission.
I have data showing that the IO expresses very high levels of a particular nicotinic acetylcholine receptor called the alpha7 subunit,w hich is known to be highly involved in presynaptic facilitation. So my hypothesis is that there will be acetylcholine mediated presynaptic facilitation at the IO to DCN synapse, but that neurons expressing reduced amounts of Neuregulin will be deficient in this facilitation. This can be readily tested - while recording from a DCN neuron that is innervated by an IO neuron, I use a "puffer pipette" that is positioned very close to the recording electrode that ejects a small amount of nicotine when I step on this little foot pedal. So I record from the neuron for a few minutes to establish the baseline rate of transmission (this is all done in the presence of tetrodotoxin, the reason why you aren't supposed to eat blowfish - that blocks all firing of neurons by binding to voltage gated sodium channels, so the activity that I see is only coming from a my presynaptic source and not the result of upstream activity) , puff on my nicotine, and then record for another few minutes to see the change in the rate of activity. Everybody got that? This will be on your exam.
No one has ever shown whether there is presynaptic facilitation at the IO to DCN synapse, so the first thing I did this weekend was look at cultures that were all from wild-type mice and measure presynaptic facilitation. Which I did not see. It was a bit of a let down, and I wasn't what to do, but I had cultures where the presynaptic source was from Neuregulin heterozygous animals as well, so I figured I might as well try it and on them just to complete the experiment. Here's where it gets weird. When I recorded from the DCNs I thought that I had screwed something up because I didn't see any synaptic activity. None. Which is unusual (I was blocking GABA, for those of you who would know about that sort of thing) but I checked all my parameters and everything else was normal. Then I applied nicotine. Lo and behold, about 30 seconds later I now had very robust glutamate neurotransmission at this synapse. This persisted for about 5 minutes, but then it stopped and went back to no transmission.
I have absolutely no idea what to conclude from this experiment, but the results are pretty cool, mainly because of his unexpected and bizarre they are. There's some evidence for nicotine activating silent synapses but it's scant, and nowhere near the area that I'm studying. What's next? Well, repetition, of course. No one believes anything in science until it's been done twice, and preferably three times. Or more. So I need to wait about a week before I can do this experiment again, but for the first time in a while I'm actually fairly excited about the work that I'm doing.
Fortunately things have been going well in the lab, which has helped offset my moodiness a bit. (Prepare for more science talk, so if this bores you, you can just skip the rest of this journal entry.) Since Saturday I've been working like crazy on my electrophysiology experiments. I've spent the last year learning the techniques that I need to know to do these experiments properly, configuring the equipment that I need to use to make it work and ironing out the (sometimes significant) technical hurdles that stood between me and my experimental results. I seem to have finally cleared all of that, and the data are rolling in.
Here's the basic experimental set up: I've reconstituted the mouse inferior olive to deep cerebellar nuclear synapse in vitro in a cell culture preparation, to my knowledge the first such demonstration of a successful recapitulation of this synapse outside of a culture brain slice. I keep the IO neurons within an explant (a tiny chunk of tissue) and the DCNs are part of a mixed, dispersed population of cerebellar neurons. Most people are of the theory that a slice preparation more accurately reflects the in vivo physiology of the synapse in question than a dispersed cell culture (since it's basically just a chunk of tissue, while a dispersion means you've mechanically or enzymatically dissociated the cells and then allowed them to grow on a glass substrate.) The usefulness of this "coculture" system however is that when I study the interplay between the presynaptic neurons (the IO) and the postsynaptic neurons (the DCN) I can selectively alter the genotype of one or the other or both by culturing neurons from genetic knockout mice that our lab has developed in combination with neurons from normal, or wild-type mice. The gene in question is an isoform of a neurotrophic factor called neuregulin, which is expressed at high levels throughout adolescence and adulthood in the mouse in both IO and DCN neurons.
What I do is called whole cell patch clamp recording where I can actually monitor the electrical activity in the postsynaptic neuron in real time. By selecting DCN neurons (based on their size and shape ) that are close to IO explants with visible projections coming out from them, I'm reasonably certain that the neurons from which I'm recording are being innervated by the IO.
My hypothesis, based on a lot of different work from our lab and other labs, is that eliminating Neuregulin presynaptically will alter the synaptic characteristics of this synapse, especially with respect to modulation by the neurotransmitter acetylcholine. In the brain (unlike at muscles, where acetylcholine is the only neurotransmitter) acetylcholine mainly speeds up neurotransmission by a process called presynaptic facilitation, discovered in my lab, where basically activation of nicotinic acetylcholine receptors on the presynaptic neuron allow for additional calcium ions to enter the synaptic bouton that allows for more neurotransmitter vesicles to be released when the presynaptic neuron fires. More vesicles = more activity in the postsynaptic neuron. Hence, facilitation of transmission.
I have data showing that the IO expresses very high levels of a particular nicotinic acetylcholine receptor called the alpha7 subunit,w hich is known to be highly involved in presynaptic facilitation. So my hypothesis is that there will be acetylcholine mediated presynaptic facilitation at the IO to DCN synapse, but that neurons expressing reduced amounts of Neuregulin will be deficient in this facilitation. This can be readily tested - while recording from a DCN neuron that is innervated by an IO neuron, I use a "puffer pipette" that is positioned very close to the recording electrode that ejects a small amount of nicotine when I step on this little foot pedal. So I record from the neuron for a few minutes to establish the baseline rate of transmission (this is all done in the presence of tetrodotoxin, the reason why you aren't supposed to eat blowfish - that blocks all firing of neurons by binding to voltage gated sodium channels, so the activity that I see is only coming from a my presynaptic source and not the result of upstream activity) , puff on my nicotine, and then record for another few minutes to see the change in the rate of activity. Everybody got that? This will be on your exam.
No one has ever shown whether there is presynaptic facilitation at the IO to DCN synapse, so the first thing I did this weekend was look at cultures that were all from wild-type mice and measure presynaptic facilitation. Which I did not see. It was a bit of a let down, and I wasn't what to do, but I had cultures where the presynaptic source was from Neuregulin heterozygous animals as well, so I figured I might as well try it and on them just to complete the experiment. Here's where it gets weird. When I recorded from the DCNs I thought that I had screwed something up because I didn't see any synaptic activity. None. Which is unusual (I was blocking GABA, for those of you who would know about that sort of thing) but I checked all my parameters and everything else was normal. Then I applied nicotine. Lo and behold, about 30 seconds later I now had very robust glutamate neurotransmission at this synapse. This persisted for about 5 minutes, but then it stopped and went back to no transmission.
I have absolutely no idea what to conclude from this experiment, but the results are pretty cool, mainly because of his unexpected and bizarre they are. There's some evidence for nicotine activating silent synapses but it's scant, and nowhere near the area that I'm studying. What's next? Well, repetition, of course. No one believes anything in science until it's been done twice, and preferably three times. Or more. So I need to wait about a week before I can do this experiment again, but for the first time in a while I'm actually fairly excited about the work that I'm doing.
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HAPPY BIRTHDAY!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!