TGIF! Today seems to be one of those days that everything in the lab breaks. So I’m running back and forth and I don’t get round to anything else than troubleshooting.
One of the things that annoyed me today is that SPSS uses comma as a decimal separator instead of a period by default. This is a problem you probably wont experience in the US, because the problem arises from your local settings. In previous SPSS versions (or windows versions) it worked just fine to adjust your windows regional settings. Now I needed to adjust the local settings in SPSS as well/instead. Since I think a lot of people will have this problem, here is how you fix it:
1. In SPSS go to: File-> New-> Syntax
2. Type in the following: SET LOCALE = ‘en_US.windows-1252′
3. Run it
Easy enough right?
Back to some more trouble shooting, some screw in the vibratome is broken…
Unjustified embargo of residence permit applications for Iranian scientists coming to the NetherlandsPosted on June 18, 2012
I could use your help. Mehran is a new PhD student who recently joined our group. He arrived last week from Iran. He had a working permit but at the day he arrived, the Dutch immigration service (IND) decided to temporarily stop all processing of visa of Iranian knowledge workers (!). And so they put his visa on hold. This means that until they continue, he is not allowed to work here (and can thus not receive an income). The ridiculous thing is that the IND gives/has no information about how long this will be the case. Can you imagine what that must feel like for him? I’m ashamed to witness this act of so-called “Dutch tolerance”. To make sure that they do not forget that there are people waiting for their decision, an online petition was drafted. You can help us by signing it and spreading the word.
Good news! After one month of insecurity, Dutch immigration responded and allowed my new colleague to start his PhD. Thanks for all the support and interest. Read the reaction of the Dutch minister of foreign policy here.
We both had a typical day at the lab. I woke up at 8:30 and rushed to the journal club (9:00), I then showed some disgruntled students where they can find their enzymes in which of the many freezers, wasted some time on ordering new antibodies and then I tried to book a free slot on the confocal microscope (next Tuesday at 21:30!), I sent out a couple of emails discussing how to go about the new experiments and after lunch I set myself to do more Matlab programming to finally get that analysis program to work, (15:00) reverting to manual data analysis (again) and when all the more productive people headed home I ate a stale slice of pizza that I found in the conference room after some groups’ pizza meeting and after all that I finally got round to doing an experiment.
At the moment I’m using intrinsic signal imaging to measure how responsive the visual cortex is to one eye over the other. Pretty simple, not too exciting but essential for my experiments: relating structural synaptic changes to functional changes after inducing ocular dominance plasticity.
I must say that I cherish these times, using a simple method to collect lots of data fast. As Luther (roughly) said: the work of a women that is merely sweeping the floor is also divine as she does it with concentration and passion.
I must confess that although I’m Dutch my knowledge on calvinism is actually quite limited and I heard the phrase from philosopher Alain de Botton, supossedly quoting Luther. But the point is that it struck a cord. I love science even though sometimes practicing it is frustrating (this is a euphemism). My other half (his name is Laurens) is also a neuroscientist and quite accidentally works in the same institute. His day roughly progressed the same way as mine but didn’t result in the same blissfull data collection. As I said sometimes experiments are frustrating and unfortunately usually the good ones are. After spending 12 hours at work I dragged him home and on the bike I asked him how things had been in the lab.
What followed was a lesson I seem to have to learn over and over again (so much for one trial learning): when someone confides in you with a problem, zip it and don’t start to offer copious amounts of “good advice”.
Luckily, Laurens and I share a passion that we both like to indulge in after being frustrated with experiments which is pretending everything is possible and there are no technical limits to our experiments. It all started on our last camping trip in Amboise (France) where I picked up a little notebook which said le cahier les idées géniales (notebook for ingenious ideas). Usually, people tend to frustrate their thought processes by limiting the feasability of their plans but this childish notebook made us throw all of that overboard. And ever since, we sometimes just let ourselves go and think of crazy cool experiments.
And here’s what it resulted in:
Our lab found that adult ocular dominance plasticity induces a higher turnover of inhibitory synapses that specifically sit on dendritic spines in the most upper layers of the primary visual cortex (V1). We found that those double synapses more often receive thalamic input which led us to believe that probably the adult cortex after monocular deprivation subtly disinhibits eye specific inputs in order to effectively modulate the responsiveness to the open eye. The next question that I had was, where do the eye specific inputs come from? Thalamus, but LGN or perhaps pulvinar (lateral posterior nucleus in rodents)? There is a bit more evidence that it might be the latter and so here’s the crazy cool idea. I inject a tracer that travels from the retina to the thalamus and finally to V1, much like Ed Callaway’s technique. Only now I add channelrhodopsin and make sure I only activate the tracer in the pulvinar with a helper virus. I can then image our in utero electroporated GFP-labeled double synapses and their pulvinar input and then simply increase the strength of the input by shedding some light and of course I would have patched the labeled cell and measure ocular dominance before and after. Yeah right, so what’s your insanely crazy cool idea?
Also read Laurens’ post for more reflecting advice on surviving grad school.
I just submitted my article and so I’m left with plenty of time to waste on answering “silly” questions. For instance, take a look at this. Now take a few steps back and shake your head while looking at it again.
I was really amazed by this optical illusion when I first came across it.
How does it work and what does it tell us about the visual system?
At first glance it’s just a high spatial frequency grating, when you look really close at the picture you only see a couple of faint grey pixels, but shake your head and the purkinje cell clearly emerges.
As a proper scientist, I immediately started googling for the answer but without much success. Then I started thinking and got the entire lab to ponder over the subject. At some point someone even suggested: “Have you considered that it could be magic?”.
Here is our first attempt at a serious hypothesis:
You have your two main types of cells in the visual system: P-cells and M-cells.
P-cells are good for perception of high spatial frequencies, but they are bad at low contrast and have poor temporal resolution. Whereas the M-cells fail when it comes to fine details but they are much more sensitive to low contrast and temporal changes.
Since the black bars have such a high spatial frequency and contrast, it involves mostly the P-cells. As soon as you start shaking your head they loose track and the M-cells become more dominant.
When you take an increasing distance from the image, you start increasing the spatial frequency to a point where even P-cells start to fail to encode it properly. And as you might have noticed looking at it from an angle also makes the figure pop out, again this seems to fit since peripheral vision is dominated by M-cells over P-cells.
If our hypothesis were true, the effect should be gone when we use equiluminant colours, since M-cells are insensitive to colour. As real scientists we couldn’t help ourselves from testing it by replacing the black and white with red and green:
And… although the effect seems less, it’s still there… We’re back to the drawing board, any suggestions are welcome!
As someone correctly mentioned the above green and red image doesn’t have equiluminance and so it’s not a proper test. Here’s a true equiluminant figure with a grating on top. Can you see the hidden text?
Unfortunately, I’m missing the Thursday morning journal club, because I’m at MIT visiting labs for job interviews. But why should that stop me from discussing a hot optogenetics paper? By chance, I met the first author Michael Kohl at Boston airport last night. I knew him from the time when I was doing my internship at Ole Paulsen’s lab (now in Cambridge but back then he was still in Oxford). It’s great to unexpectedly meet people you know, especially considering, it rarely happens to me in Amsterdam, where I live.
Kohl et al. conducted a really elegant study using optogenetics to study the left-right asymmetry of spike timing-dependent plasticity (t-LTP) at synapses of CA1 pyramidals that received either input from the left or the right hemisphere.
The study of lateralization used to be more in the realm of psychologists but that changed with this study. Shinohara et al., showed that postsynaptic spines of CA1 pyramidal cells that receive CA3 projections from the right side differ from those spines that receive input from the left CA3 region. Spines receiving input from the right hemisphere tended to be bigger, had a higher GLUR1 AMPAR content and at the same time showed lower NR2B expression. This seems contradictory, given that NR2B expression favours LTP which increases spine size and AMPAR insertion in vitro. The authors suspect that these plasticity mechanisms might be very different in the in vivo situation. Another explanation could be that the bigger spines were already maximally potentiated and in response homeostatically downregulate their NR2B sununits.
Kohl used slice electrophysiology to study t-LTP in CA1 pyramidal cells by patching on to them and pairing electrical stimulation in the stratum radiatum with optogenetic stimulation of either left or right CA3 input. He found that both ipsilateral (Schaffer collaterals) as well as contralateral input (commissural fibers) coming from the right hemisphere failed to show t-LTP, whereas presynaptic input from the left CA3 showed a considerable increase in EPSP strength (~150%).
They confirmed that higher NR2B subunit expression (left) was responsible for this effect without a difference in AMPA/NMDA ratios. Given that NR2B expression is experience-dependent, it’s tempting to say that the left hippocampus shows enhanced/different activity. Interestingly, I remember hearing sometime ago that Einstein had a markedly bigger left hippocampus compared to his right. Ideas, anyone?
I’m not the lucky owner of an Ipad, but I did enter every possible raffle, lottery and prize quiz at this conference to increase my chances of finally getting one. Until then my notepad made of paper will do just fine. You might think that I am thoroughly outdated, but then you would actually be mistaken. You see, tablets are much much older than pen and paper. Around the 8th century BC, the old greeks used a wax tablet and a stylus to take notes on, whereas paper came around in the 2nd century AD.
The tabula (how the Greeks called their wax tablet) was an inexpensive way to take notes and wipe it clean by just heating up the wax, creating a tabula rasa. Neuroscientists think of something very different when they use those words. At least when I hear them I think about experience-dependent and -independent mechanisms that sculpt the developing brain.
This morning, David Fitzpatrick gave a very nice talk on just that. How much of the features encoded in our visual cortex require the circuitry to be sculpted by visual experience? Or is it mostly a fixed developmental program that runs independently of light hitting the retina?
He started with acknowledging the work of Hubel and Wiesel, who while having fun in the lab did so much groundwork on the workings of the visual cortex that afterwards nothing much was left to be discovered. Thanks guys, real great work!
Fortunately, I’m exaggerating and many questions remain. One of the questions that Fitzpatrick asked is:
Do cells in the primary visual cortex require visual input in order to become selectively responsive to certain orientations and directions?
We know from rearing animals in complete darkness that orientation selectivity develops independently of visual input. Direction selectivity on the other hand does not, since it is almost absent in dark-reared and immature ferrets and cats. This seems to be different however in rodents, Nathalie Rochefort showed very recently that mice as soon as they open their eyes have cells properly tuned to direction, albeit initially there is a bias to dorsal and anterior directions. An explanantion for this is that already at the retina there are direction selective ganglion cells, that have shown to project to the thalamus. I believe that in ferrets this has not yet been studied.
Beautiful data collected just weeks ago in the Max-Planck institute in Florida showed how cells in immature ferrets become rapidly direction selective if you train the animals by repeatedly showing them a drifting grating in a certain direction. They followed this in the time course of many hours by chronic Ca2+ imaging using the genetically decoded indicator GCaMP3. It was astonishing to watch the cells bounce back and forth between preferred direction to see them eventually organize into columns.
We’ve come to another interesting difference between mice and cats. Mice’s response properties such as orientation -, direction selectivity and ocular dominance is topographically scattered in a salt and pepper fashion, whereas cats have a very nice columnar organization. I remember distinctly when I entered the field of visual plasticity how disappointed I was to hear this. David made me realize that it just raises an extremely interesting question: What is the difference in their circuitry that gives rise to either a salt and pepper organization or columns? I always thought it was, among other things, a scaling problem. Fitzpatrick proposed that there could be differences in the respective weights of inhibitory and excitatory inputs and that different operations could play a role in determining the tuning of the cells. That sounds to me like there is still a lot for us left to discover. Hubel and Wiesel eat your heart out!
Yesterday I marked a few posters about optogenetics in my itinerary. When I arrived at the alley where they were located I was shocked to see a big mass of people pushing and fighting to see all posters there. One couldn’t fail to notice that optogenetics is hot!
Of course the technique is inexpensive and yes you can do awesome research with it. The problem is, most people don’t. There were posters about the development of new vectors (“We made a new virus that performs slightly worse than what is available”), new probes, outrageous applications and a whole lot of other things people will forget about in a year or so (“We introduced optogenetics into the common hedgehog, and guess what; it does what it’s supposed to do!”). Fortunately, Amidst all this, there were a few posters that were gems. I’ll highlight two of them:
The lab of G.J. Augustine from Duke University showed that Purkinje cells receive input from just 1-2 interneurons directly and from 3-4 interneurons indirectly via electrotonic coupling. They did this by first measuring the extent of the dendritic tree of one interneuron in the slice by patching it and stimulating the slice at several points creating a ‘map’ of the neuron. By now comparing the spatial extent of one interneuron to the area from which one Purkinje cell can be inhibited, you can estimate the number of interneurons involved in this inhibition. Furthermore, by blocking the gap junctions they estimated that ~2 interneurons provide direct inhibition and 3-4 provide inhibition via coupling. Maybe the most interesting fact was that this indirect inhibition was completely gone in coronal slices, confirming that interneurons provide inhibition within one cerebellar zone.
Another poster from the lab of M. Sur from MIT showed the difference between Parvalbumin (PV) and Somatostatin (SOM) neurons. Interneurons don’t just provide inhibition and ‘block’ spikes. They provide arithmetic operations. Apparently the PV neurons provide a divisive operation (scaling) while SOM neurons provide a subtractive operation. They found this by looking at pyramidal cell responses during the acquisition of a tuning curve. When PV neurons were stimulated during the acquisition, the pyramidal cell response amplitude was scaled down. In contrast, when SOM neurons were stimulated the tuning curve shifted downward in its entirety. Clearly, the two interneuron classes have a completely different role in shaping the pyramidal cell response.
As you can see, when the scientific question is sound, optogenetics can be a powerful tool. It’s always been the same: techniques should never be leading in research, questions should.
written by: mouseguy
Chuck Norris, err sorry I meant to say Winfried Denk, was the first to combine laser scanning microscopy with two-photon excitation, transforming it into a technique that enables the visualization of the living intact brain. More recently, he went a step further and automated serial scanning of a block of tissue with an electron microscope achieving high resolution. A technique called serial block face scanning electron microscopy (SBF-SEM).
During his lecture yesterday afternoon, the director of the Max-Planck institute in Heidelberg spoke about one of the most interesting challenges in Neuroscience: relating structure to function.
Structural neurobiology is just a new word for neuroanatomy. A circuit diagram is more than just a wiring diagram and more than just a description of the circuit elements. The circuit diagram should allow prediction of functional implications.
We both want to measure functional activity and precisely determine the underlying connectivity. For the first goal we can use two-photon microscopy, but for the second we need much higher spatial resolution. Which can be done with serial reconstruction with an electron microscope, finding back the cells and carefully tracing their dendrites and axons.
If you’re familiar with this type of work you know that it’s a hell of a job and used to be extremely slow. This venture began with mapping all 7000 connections in the 300 neuron containing C. elegans’ nervous system which took more than twelve years to accomplish. Now the field of connectomics, as it’s called, has advanced greatly due to the development of the SBF-SEM technique along with great improvements in image analysis methods pioneered by Sebastian Seung at MIT.
Winfried Denk tried to demonstrate the old image analysis software but shortly after opening the program, he said with a frustrated tone of voice:
“I’m sorry, I can’t watch it, it’s too slow.”
and moved on showing his most recent work on mapping the retina. I can tell you about it but rather you should watch this Nature video, which gives a good idea of how they studied the connectivity between star amacrine cells and direction selective ganglion cells after determining their direction selectivity with calcium imaging.
The reconstruction of neurites is still challenging. One of the problems is when experts disagree whether they are looking at a synaptic contact or just a crossing. This still happened in a lot of cases. And instead of devising a super computer, Denk came up with a more cost-effective method:
“a small army of undergraduate students”.
Fifty people put in roughly 30.000 tracing hours to accomplish the circuit diagram of a small piece of retina. And because fifty people make uncorrelated errors, it’s easy to determine the most likely underlying connectivity.
Denk’s latest project is to describe the connectome of the entire mouse brain. For that they built a whole-brain microtome and SEM system (the Denk-o-tome?). The machine is an impressive beast spanning the height of two adult people (he showed a picture which unfortunately I wasn’t able to retrieve). One of the remarks by Denk made me realize why this man is so successful, he is truly unstoppable. When he talked about the whole-brain scanning system he said:
“There’s no reason why it shouldn’t go up to 40 mega Hz.”
Here’s how long it takes to scan 0.5 ml of brain (the mouse’s) at 80 nm resolution at 40 mHz: 280 days. Not so bad, increasing the resolution to 20 nm however, will take you 50 years. One solution they are currently working on in collaboration with Zeiss, is speeding up the scanning by using a multi-beam source of 60 beams. In which case scanning the entire brain can be done in less than a year. Now the question remains where can we sign up to join Winfried Denk’s army?
Time flies at a big conference like this and with a million other things to do like blogging, socializing and writing up a paper. But I’m not complaining because I’m enjoying every second of it. Yesterday’s presidential lecture was stimulating and thought-provoking. Mu-Ming Poo presented mostly old but also his newest unpublished work. I was excited to hear him speak because it was Poo’s first papers which sparked my interest in spike timing-dependent plasticity when I was an undergraduate.
The topic was neurotrophins and their effects on axonal differentiation/initiation and guidance and finally Poo talked about the role of BDNF in synaptic plasticity. On a side note: did you notice the ages of the two people who discovered neurotrophins? They are over a hundred years old! Viktor Hamburger lived until the age of 101, Rita Levi-Montalcini (102) is still with us and Stanley Cohen who isolated BDNF turns 89 next week.
Poo showed nice pictures of axons changing course that demonstrated the chemoattractive action of BDNF on axonal growth cones. The story however, is more complicated. Axon attraction or repulsion is not only determined by these extracellular gradients but it also depends on the internal state of the neuron and can be modified by neuronal activity. It is the effect of BDNF onto cAMP and cGMP that determines axonal guidance. If cAMP and or cGMP are low the axon is repulsed and if they are high it’s attracted. The same is true for calcium levels. Poo told us that when his graduate student first told him that axons belonging to neurons with the same neurotrophic receptors could be both attracted or repulsed, he had a hard time believing it. And apparently, Poo entrusted us, telling your students that you don’t believe their data one bit is a very effective method. “They start to produce very convincing data.” Imagine Poo telling you: “If these data are true, I will get up that table and crawl on it like a dog” and then imagine him actually doing it…
What provoked me was Poo’s dictum as he called it himself. I believe it was something like this:
” Something neat found in vitro, is bound to be used somewhere in vivo.”
And yes sure if you think of any mechanism it is bound to exist. So I’m not so convinced of how useful Poo’s dictum is but I must admit, in vitro work is valuable, if asked the right questions.
Poo’s talk ended with the role of BDNF in synaptic plasticity and how bidirectional trans-synaptic transport of BDNF might achieve synapse specificity. But the most interesting part in my opinion was the preliminary data on the role of BDNF in spike timing-dependent plasticity (STDP). His postdoc Xiao-Hui Lu used glutamate uncaging and paired this with dendritic spiking. Only when she uncaged glutamate before dendritic spiking did she see an increase in BDNF secretion as measured by BDNF-GFP in a neighbouring spine. I’m not sure whether this work was done in culture, slices or in vivo. But I’ll keep an eye out for the paper.
Today’s highlight is the talk by Winfried Denk, father of the Denkoscope. Again the talk will be in hall D and it starts at 13:00.
p.s. Congratulations to Roger Nicoll for receiving the Axelrod prize, Janet Zadina for getting the Science educator award and of course Shiree Heath for winning the Brain Awareness Video Contest!
I placed myself with my laptop at the main entrance, close to the ethernet socket. It didn’t take long before colleagues and friends found me and so we’re camping out here together sharing our first experiences. The Dutch crowd is appalled by the price you pay for the food here: “Yesterday I paid 4.60! for a loaf of bread, plain bread!”. Some people worry that they can’t do experiments for a while: ” I can’t do anything for 10 days!”. Others have found alternative transport and borrowed or rented a bike. Apparently not wearing a helmet here is offensive?! How strange to experience these cultural differences: in Amsterdam wearing a helmet on a bike makes you kind of a wuss.
Yesterday, I went to the satellite of the Journal of Physiology on basket cells. I had my schedule mixed up so I was only on time for the talks by Ivan Soltesz and Ed Callaway. Judging from the list of speakers I think the symposium gave a pretty good overview on the two main types of basket cells most frequently studied, PV fast-spiking and the CCK non fast-spiking cells.
What struck me most from both of the talks was the specificity of the connectivity and the input to the two main types of basket cells and moreover, their functional differences. Ivan Soltesz showed that PV+ cells are inwardly rectifying whereas CCK+ cells are not and this difference could be contributed to expression of the CB1R. This was only found on pyramidal cells and not on granule cells of the hippocampus. Another functional difference between the two basket cell types is that CCK+ cells have a much higher IPSC amplitude than the PV+ cells, due to the expression of the Cl- channel CLC2, restricted to the somata. And ofcourse it’s the PV+ cells that mostly project somatically. It was proposed that this could be a protective mechanism, preventing the PV+ cells from exerting too much influence (Foldy et al., 2010). During the discussion, the idea of Cl microdomains was suggested, much like Ca2+ microdomains. I’m looking forward to the TINS review (in press) on the diversity of inhibition by Krook-Magnuson.
And now some posters and then the long anticipated backyard brains (cheap electrophysiology experiments in your own backyard) at 207B.