Thursday, June 28, 2012

Astrocytic regulation of Up- and Downstates Last week we discussed the paper from Wang et al (From Maiken Nedergaards lab) in our journalclub. Since our lab has healthy interest in up- and downstates in Purkinje cells, it was a good choice I think.

The authors show that by stimulating the Bergmann glia via a transgenic receptor bistability is reduced and the Purkinje cells show more up- than downstates. During the stimulation, the Bergmann glia cells show a calciumtransient and the extracellular potassium concentration decreases. Interestingly, when the Bergman glia cells were hyperpolarized, Purkinje cells spent more time in the upstate. Now of course the question is "What causes what?" And that's where the paper goes off the rails for a bit. It claims a causal relation: calcium transients cause the extracellular potassium concentration decrease by uptake by Bergmann glia. This in turn causes a reduction in bistability. But a critical experiment, buffering calcium in Bergmann Glia, was not done. Also, there is no proof that the potassium is taken up by Bergmann glia cells. Still, the paper is interesting. It raises a lot of questions, but firmly establishes a role for glia in Purkinje cell modulation.

Another puzzling aspect is the exact mechanism of regulation. How does hyperpolarization of Bergmann glia induce calcium transients? Then, how does the calcium transient cause a reduction in extracellular potassium? Would the presumed uptake of potassium by Bergmann glia cells not depolarize the glia cells again? And how does this decrease in extracellular potassium influence the up- and downstates in Purkinje cells. Actually, the last question can be somewhat answered from the paper. It seems that by reducing the external concentration of potassium, the membrane potential for the upstate drops while the membrane potential for the downstate increases. Also, from the paper of Fernando Fernandez et al. it seems that potassium conductances play a large role in the generation and control of bistability.

A possibility that can be excluded is the hyperpolarization-induced release of GABA via the Best1 channel found in Bergmann glia. It seemed a very likely candidate: hyperpolarization forces anionic GABA out of the cell and the calcium transient opens the channel. However, bistability during Bergmann glia stimulation was not affected, thus excluding the possibility that GABA release plays a role here.

So, it seems that bistability in Purkinje cells can be controlled (somewhat) by Bergmann glia, it is influenced via extracellular potassium and the mechanism is hyperpolarization of the upstate, bringing the states closer together. Serotonin also does this by acting directly on Ih channels. But this is contested already by Fernandez et al. who didn't find any involvement for Ih channels in bistability. They show that potassium plays a big role in bistability, which is in line with the current study. Then, finally there is the possibility of GABA release from Bergmann glia. But that didn't play a role here....

The discussion on Purkinje cell bistability is here to stay. Not only the discussion whether it is really there during waking, but also how it works. Now we probably have to wait for someone to come up with the solution that will connect the pieces rather than introducing a new set of factors. I will stay on top of this, exciting times!

Fushun Wang, Qiwu Xu, Weishan Wang, Takahiro Takano, and Maiken Nedergaard (2012). Bergmann glia modulate cerebellar Purkinje cell bistability via Ca2+-dependent K+ uptake PNAS DOI: 10.1073/pnas.1120380109

Tuesday, June 12, 2012

Some changes...

I want to make a better blog. A blog that is easier to interpret than it is now. Clearer and easier to read.

So, there going to be some changes around here. Posts will be classified into categories, for example: "Research", "PhD life" and "Science related". This way it is easier for you, my dear readers, to navigate to the bits and pieces you find interesting. And of course, you already noticed, the layout and colorscheme have been changed.

Content will stay roughly the same of course!

Clustering VN cell types A while ago, during SfN, I wrote about an interesting poster on clustering cell types by single cell RT PCR. The paper is out and I just wanted to share some details with you (In case you're too lazy to read it yourself ;-)).
The cells in the vestibular nuclei (and in the cerebellar nuclei I can tell you) are hard, if not impossible to distinguish electrophysiologically. So, if you want to find out what different cell types are doing during behavior you're going to have a hard time. No way to distinguish the glutamatergic projection neurons from the glycinergic ones and no way of telling if you're listening to an interneuron or to a GABAergic projection neuron. But Kodama et al used expression profiles of transmitter-related genes, ion channels and marker genes based on the allen brain atlas.

To be able to compare the results to previous studies they used three mouselines characterized before: YFP-16 (excitatory neurons), GIN (somatostatin, inhibitory neurons), GlyT2 (glycine transporter 2). Only five genes for neurotransmitters and genes related to neurotransmitters were used (VGluT1/2, glycine transporter 2 and Gad1/2). These genes clustered nicely on the different mouselines. Interestingly, the clusters are not perfect, proving that you always have some sort incompleteness and bleed-through with transgenetic animals.

Now the interesting part is if you can match the expression profile of ion-channel related genes to the physiology. For example: fluorescent neurons in YFP-16 animals have narrow action potentials. And GIN neurons show less rebound firing than YFP-16 neurons do. So, you would expect differences in ion-channels mediating action potential shape and differences in T-type calcium channels and H-channels. Indeed, these differences are reflected in the expression profiles. Genes for NaV1.1 and NaV1.6 are upregulated in YFP-16 neurons as compared to GIN and GlyT2 neurons. The same goes for the hyperpolarizing currents: Kcnc1, 2 and Kcnc3 were all upregulated in YFP-16 neurons. Also the differences seen in postinhibitory rebound firing were reflected in the expression profiles. HCN and combined T-type channel expression were upregulated in YFP-16 neurons.
Now six classes of neurons can be distinguished by marker genes.
Exc1: Vglut2/ Secreted phosphoprotein 1
Exc2: Vlugt1/ Corticotropin releasing hormone
Exc3: Adcyap1
Inh1: Nav beta4/ GlyT2
Inh2: Coagulation factor C homolog
Inh3: Corticotropin-releasing factor-binding protein

By doing in-situ hybridization combined with tracer injections, the authors were able to pinpoint the roles of some of the classes. Exc1 neurons project to the motor nuclei, Exc2 neurons project to the cerebellar cortex as mossy fibers, Inh1 neurons project to the motor nuclei as well, Inh3 neurons project to the vestibular nuclei and Exc3 and Inh2 neurons could not be traced. (Nucleo-olivary?)

The tactic used here to classify neurons has some clear advantages. Even neurons that cannot be clustered (easily) on the basis of electrophysiology alone can be identified using genetic expression clustering. Also, if specific markers are known, transgenic mouselines can be generated specifically for each cluster.

There are also a few things that worry me a bit about the paper. The spike-in RNAs used to quantify the expression profiles do not show the linear relationship that you would expect (fig 1E). In other words, it is not clear whether the results from the genetic profiles are compared to the linear fit and how the outlier is handled. Another concern is that only the MVN was used and only the central part of the MVN. What about the other nuclei and the periphery of the MVN? This is especially a concern since different neuronal morphologies are not uniformly present throughout the nucleus. So, maybe there is only a subsampling of the neurons in the present study.

Some more research is needed to address these issues. Still, I think the paper is a big leap forwards for cerebellar research.

Kodama T, Guerrero S, Shin M, Moghadam S, Faulstich M, & du Lac S (2012). Neuronal Classification and Marker Gene Identification via Single-Cell Expression Profiling of Brainstem Vestibular Neurons Subserving Cerebellar Learning. The Journal of neuroscience : the official journal of the Society for Neuroscience, 32 (23), 7819-31 PMID: 22674258

Monday, June 4, 2012

Brains!!! Brains!!!

A surprising message from the CDC: 'There is no zombie apocalypse' and 'CDC does not know of a virus or condition that would reanimate the dead (or one that would present zombie-like symptoms)'.

Of course the evidence is crystal clear and shows the opposite! There are zombie killings all over the world!
San Fransisco Chronicle
Herald Sun
NY Daily News
The Onion
Clearly, CDC is getting people accustomed to the idea that zombies exist and are going to take over the world, why else all the video games and horror movies? It's a conspiracy I tell you!
There are even serious scientists researching this! Do they get the attention they deserve??? NO!

*Sane Mode Activated*
Of course there's no zombie apocalypse, but it's amazing the CDC thought it necessary to comment on this. The existence of zombies should not be a question, let alone an apocalypse. Still, zombies amaze us. Why? Is it something to do with the fact we all like control and we all like to live our lives as we choose? So, a disease that would turn you or others into man-eating brainless undead freaks people out? I guess all people are control freaks up to a certain level.
Good thing I know I've already turned half-zombie by my cat! Yes, cat-lovers, you have a good chance of being infected by Toxoplasmosis, a parasite that lodges itself in your brain and could cause behavioral changes in the host.
Fortunately the changes are mild and...... Hmpfff...... Braaaaaainnnns! Brains!!!!! Brains!!!!!

Friday, June 1, 2012

Not a landmark, just 'a thing'

Today was a bad day at the lab for me ('typical', according to Danielle). Experiments didn't work, I freaked out (again) over my results, got depressed about my chances of publishing a paper and I didn't see my PhD ending this year.

My experiments stopped working about a month ago. I've had it before; it seems to be an up and down motion of productive weeks and unproductive weeks. I think in-vivo patching is a precarious interplay between lots of factors. Get one wrong and your experiment will fail. Get them all right and you have a chance at results, provided you work hard.
I aim at patching in the cerebellar nuclei, but recently I often overshoot them and get vestibular nuclei instead (which seem to be very easy to patch for some reason). Also, the patches I get in the cerebellar nuclei are of bad quality. I get to 100-200 MOhms of seal and then they drop off. Or they just don't open nicely and I have to dump the recording because nothing can be learned from it. It's probably something to do with slight differences between mice and a slow drift of the stereotaxic location of the nuclei between generations of mice. Why the patching is so hard, I don't know. Tomorrow I will have freshly polished and flamed electrodes. I will throw out my internal solution and make some new. Hopefully this will solve some of the problems.

I freaked out over my results because I feared I might have patched a lot of vestibular nuclei cells. By inspecting my data closely, this turned out not to be the case. Thank god, I might have been forced to throw out months of work... This of course caused my slight panic attack over my chances of publishing a paper and finishing my PhD. When the attack was gone I decided to take matters into my own hands and have another try at an experiment (for the result, see above).

Fortunately I have very considerate and wise colleagues. One particular in-vitro patch clamp colleague (who always plays creationism vs evolution or religion vs atheism debates for the lab, which we all thank him for ;-)) had very helpfull insights: A PhD defence shouldn't be too much a landmark event. It doesn't define you as a scientist, it's merely a thing that you need to do to make life easier. It's something you need to pass to get to the next level, but it doesn't define your expertise or you being a scientist. It's just 'a thing'. The most important issue here is to publish thorough papers that you can always defend. So, my PhD book will be more of a 'booklet' with one or two papers and unpublished chapters. Which might end up as published work someday. Don't get me wrong, I would be very happy to get the definite proof on some of the things I've been working on, but it's just not going to stand in the way of me moving on.

So, tomorrow I'm going to talk to 'my boss' to talk about a PhD defence date for this year (2012, remind me if I missed the mark ;-)). I will continue work on my cerebellar nuclei and cerebellar cortex stuff. If it all works out, it will be submitted or published when I'm finished. If not, so be it.