Thursday, November 29, 2012

Grant application from hell

Today is the deadline for my grant, or rather: the deadline is in less than seven hours. And I am freaking out!
The grant application is done, looking great and shiny as a pdf on my computer. It has been read by several people, who all had remarks and wise advice. So, why would I freak out you ask?

The Dutch funding agency asks for an acceptance form stating that the host institute accepts the candidate and the grant if the grant is awarded. So far so good you would say, but the form has been on a desk for a week and now needs to be run by several offices in one day. Extra complicating the story is that I am at GMT+1 time, the host institute is at EST time and the deadline is midnight GMT+1. So, it is now past four in the afternoon, people at NWO will probably leave around five, which leaves me only forty-five minutes to fix things here......

Extra complicated is the fact why it is taking so long at the other side of the pond. They need to check the terms and conditions on the grant. One problem there is the Dutch code on animal experiments. The Netherlands has some strict regulations, and the universities have agreed on a code to be open about animal research. Although the document does not have legal value like a law, research funded by NWO still has to adhere to it. And here begin the problems. It states all research should adhere to Dutch animal law. Of course the document was drafted in Dutch and the possibility of non-dutch research applying to the code was not incorporated in the code. So, in principle the strange situation could arise that by agreeing to adhere to the code, Dutch law should take effect when my research is concerned, also abroad. This then causes the Dutch law openbaarheid van bestuur (Openness of Governance) to be applicable to all experiments I do abroad. Something the University probably won't agree to.

I asked NWO, they didn't know directly so I had to email them. They will get back to me today with an answer. Then, hopefully, this answer is 'good enough' for the host institute and they sign the form and email it to me. Then I can send in the grant tonight. When they are not happy, then I have to get back to NWO, if they are still there to answer the phone of course.

That's why I'm freaking out.....

Tuesday, November 13, 2012

They're moving the finishing line!!!

Dear reader,

it's been a while (again) since I last posted here. I have been caught up in the whirlwind that is called "finishing your thesis". I hoped the storm would have died down a little bit, but if anything, the gale has been growing stronger.

Last week I finished the one manuscript that was supposedly between me and a thesis. Unfortunately, it turns out non-finished manuscript #2 also needs to become a finished manuscript. So, my assumption that I would go back to more normal, less frantic, working was false. At the moment I'm in full swing getting insane electrophysiology done. I'm talking 'whole cell in-vivo at 2mm depth with a perfusion electrode nearby' crazy electrophysiology. It's an unpublished thing we developed, so I can't be too specific. But, needless to say these experiments suck b*lls when you're in a hurry finishing things.

Then there's a lot of histology that needs to be done. Some simple recovery of patched neurons and testing a new antibody that we need for immuno-EM. I heard it's always wise to learn a new technique during the last weeks or months of your thesis work (not), so I'm going to do EM now as well (Jeeeej!!!!).

On the bright side, I have been looking for good estimations of the number or density of cells in the cerebellar cortex. After some cries for help and google scholar searched I have come to the conclusion that no-one ever put all things together. So, I compiled an estimate based on a collection of literature. It all seems to point (roughly) to the same estimation: (in cells per cubic millimeter mouse cerebellum)
Purkinje cells: 20,000
Granule cells: 2.63 million
Mol. Layer Intern: 100,000
Golgi cells: 4,500
I was quite surprised with the Golgi cells being so low. Still, they have an amazing axonal branching pattern, so they can provide a large number of granule cells with inhibition. Also, the high number of interneurons in the molecular layer surprised me. So, the ration MLI:PC seem to be 2:1, quite surprising....
Where did I get this knowledge you ask?

Lange (1974) Cell and Tissue Research 153:219-26
Woodruff-Pak (2006) Neuroscience 141:233-43
Dugue (2009) Neuron 61:126-39
Sturrock (1989) Journal fur Hirnforschung 30(4):499-503
Altman (1977) Exp Brain Res 29:265-74

ps. I still love science, don't be alarmed ;-)

Thursday, October 25, 2012

Troubles of a grant virgin....

I am writing, or rather trying to write, a grant application. It is very difficult and quite different from writing a paper. (So I decided to write a blogpost instead first)
I know what is expected from me in a paper. Introduce the study, present your results in a clear way, discuss the results in the context of the scientific field. Also, when appropriate give credit to previous work (references).
But, how does that work in a grant application? There are no results, only plans. So, that means writing an introduction and then present the plans. How far should I take this? The committee is multidisciplinary, so too much details will not help, but maybe there will be some specialists there who would appreciate details. Still, I really wouldn't want to bore any members of the committee. AAAAAARGH!!!!!
Should I use references in the same way as in a paper? I guess not since the grantcommittee will not be interested in all the details. Still, it is good to show that my plans are based in reality and not too outrageously far-fetched.

One thing I picked up from a grant I could use as an example from my boss is that every figure should have a very clear, exciting and colorful message. Maybe I should include some figures from my present work, combined with some figures from papers the lab has published I'm going to work......

Decisions, decisions.....

There must be some people here who have written grants before. What are the do's and don'ts??? I would be eternally grateful ;-)

Saturday, October 6, 2012

People hang on his every word, even the prepositions...

Okay, so that was another embarrassingly long absence from blogging.

The last months have been hectic, both professionally and on a personal level. I have a postdoc (jeej!), so I have to finish my PhD in time. This means: finish my book in November, graduate in April, get to Boston in May. Two papers... two papers is the only thing in between me and my thesis. Two papers to analyse all the data for, two papers to write and then two papers to send off. I know it's ambitious, too ambitious maybe, but I don't really seem to have a choice.

Meanwhile there was a great little conference here in Amsterdam the last few days. The Cerebnet and C7 consortia had a joint meeting right here in our institute. Today was the final day with a few workshops. Together with a colleague I demonstrated in-vivo patch recordings in the cerebellar nuclei. Now I'm waiting for a PCR gel to settle and I can go home and crash. Next week, an old student will visit the lab, so that's another week out of my schedule. Two papers.....

There is one great thing though from the conference I have to share with you. The keynote lectures were done by two emiritus professors: Professor Nieuwenhuys and Professor Voogd.
Rudolph Nieuwenhuys spoke about the evolution of the brain and what that tells us about its function. He is an unbelievable speaker, with his 80+ years he still captures the audience; I never heard our colloquiumroom so quiet. A very clear lecture with clear takehome messages after every few slides.
Jan Voogd then took the audience home, back to the cerebellum. Why is the cerebellum so big in humans? Which zones got bigger? Is this only in primates or also in other mammals? Dolphins for example show a big cerebellum, but this is mainly due to skeletomuscle zones that increased in size. In primates however, the increase is in the non-skeletomuscle parts of the cerebellum.

They must be the two most interesting men in the world!

The best part actually might have been that they both refused to operate the computer, so someone else was advancing the slides for them. This took the pace out of the presentation and it all came across very calm and controlled. Maybe there is a lesson to be learned here for presentation skills!

If you want to learn more about these two absolute legends in neuro-anatomy, you can buy their book! It's a great bargain, just under $100 for about a thousand pages of wonderful diagrams and clear explanations.

Thursday, July 5, 2012

Easy Come, Easy Go

My newspaper has a section called 'Next Question' which contains intriguing questions from readers. This time the question was "How come we never 'forget' how to ride a bike?" The answer was complicated, convoluted and -at best- incomplete. The answer came from a Dutch Neuropsychology professor who explained that when learning to ride a bike you need to couple correct movements to what you see and feel on a bike.
(Not a literal translation, paraphrased)
If you learn it a little bit you are motivated to get better and better. That is why you keep on making the correct movements fitting to the perceptions. This way you produce robust connections between so-called perception cells and action cells. He makes a reference to Hebb: "Cells that fire together wire together". The more you do it, the more ingrained it becomes. Because every bike works (almost) the same, the relation between perception and action is stable. You never experience something completely different on a bike, that's why you never unlearn it.

So, you might think, that's not such a bad answer. And no, it's not. It's a bit unclear on which parts of the brain are involved. What are perception cells and action cells, but other that that it sort of makes sense. But in the last section it goes off a bit:
(again paraphrased)
Less consequent experiences are more susceptible to forgetting. That's why we have problems forgetting what we ate last week for every day. This is because we eat something different almost every day, the memories interfere and we are not able to form strong memorytraces.

Right, so riding a bike is part of declarative memory? It seems that the answer here is solely built on the principle that if a trace is enforced every time in a constant way it becomes stronger. Though true, it doesn't tell you why you never unlearn to ride a bike, even though you haven't done it for years, while you do forget what you ate yesterday!

What about those pesky Dutch spelling rules? We got drilled in school to know where to put d, t or dt. But sometimes I forget.... Why do we forget numbers so easily, but are always able to thump in our pin at an ATM machine? (Honestly, sometimes I can't remember the digits, but when I see a keyboard I can type it!). The same goes with passwords at your computer, sometimes you can't remember, but your fingers can!

The answer of course lies within the cerebellum. If you say coordinated movements, such as riding a bike, you say cerebellar involvement. On the other hand, riding a bike is also very much a sequence that you have learned, so you would expect striatal involvement. It's probably a bit of both; maybe first cerebellar coordination to get the movements right, then striatal consolidation? Why then don't you unlearn how to ride a bike, while you do forget other things? Although we do not know the cellular and molecular mechanisms, this question can be answered via a different route.

Imagine what would happen if you would learn and unlearn movements in the same vivid and thorough way as you remember your lunch. You would step onto the greens of a golfcourse and you would be able to swing the perfect ball within a few trials. Sounds perfect, doesn't it. But a quick learning implies a quick unlearning as well. You would not be able to make that perfect swing again in one go. And that's what you don't want for your movements. You would forget how to walk after sitting for an hour. So, movements are learned very slowly, so they are also unlearned or forgotten very slowly. Why don't you forget how to learn to ride a bike? Because it took long to learn.

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.

Wednesday, March 21, 2012

Art of Neuroscience - Amsterdam

So far my idea for consistent posting after my return from SfN. No messages from my side. None whatsoever, my apologies.
I have a new idea though, inspired by an email from my lovely girlfriend about efficient writing. Tips included: 'Forget the idea you're ever going to finish' and 'Forget a generalized audience, ... in writing, your audience is one single reader'. So, armed with some new energy I have to tell you about the wonderful initiative we have in Amsterdam.

Science and art have always been friends, since art can inspire scientists and science can look pretty darn good too. Everyone must have been amazed by the pictures taken with the Hubble space telescope, the pictures taken daily through microscopes in every lab on the globe and buildings no-one thought were possible, made possible by science.

To deliberately make art from science takes more effort, but can have great results. The Amsterdam 'Art of Neuroscience' competition challenges scientists to take their science from the lab, make it pretty and then present it to a general audience. The event took place at NEMO, the Amsterdam science museum, during the brain awareness week and received considerable media attention. The entries were from the fields of human fMRI, electrophysiology and imaging to name a few.

It's best not to talk (write) too much about it. Better to just direct you to the pictures!
The winner - M. Steenwijk
Honorable mentions - J. Winnubst, S. Hoyng, C.P.J. De Kock and R. Meredith

See all submissions here.