Saturday, February 28, 2009

Rotation #2 Diary: Week 8 (It's Over!)

The last week of this rotation contained many ups and downs. On the positive side, I produced the most beautiful western blot I've ever seen. My tricky protein finally behaved, and I was able to detect nice, sharp bands in most of the samples I tested. I was so excited! I showed the postdoc who's been helping me, and she was also excited. I showed my adviser, and she was pleased. Yesss! I am a western blot queen! All I had to do was repeat the experiment two more times, and I've have some quantifiable data.

Well, you know what they say about the best-laid plans of mice and scientists... I ran the gels again and the new blots looked like crap. I wasn't able to get any signal from the samples that had behaved so beautifully the previous day. I'm told that this is a common problem that people in the lab have with this protein. We hypothesize that perhaps the protein doesn't like freeze/thaw cycles, and thus it only shows up when freshly made protein lysates are used. That's pretty annoying, since I get about one mL of protein lysate from each mouse tissue, but only use 10-50 µL for each gel (thus, each tube of protein lysate should be enough for 20-100 experiments!). One potentially easy way to solve this problem, if it is a freeze/thaw thing, would be to aliquot my fresh protein lysates into 20 individual tubes, instead of one big tube, so that I'd only thaw what I need for one experiment at a time. Doing this is somewhat annoying, but if it preserves my ability to get beautiful western blots, it's worth the trouble. However, with my rotation now over, I don't have time to spend three days making another set of lysates, aliquot them, and try again. Instead, I will be creating a rotation report based on very little actual data. My methods section will be long, though -- I tried a lot of different things to make this protein cooperate with my experiments. A few of them kind of worked. I can document that.

I start my third (and probably final) rotation on March 16. No more rotation diaries until I start working there.

Thursday, February 26, 2009

Who's the Scientist?

I can't recall what chain of links initially led me to this website, but I'm so glad that I found it. It collects the drawings and descriptions of scientists made by a seventh grade class both before and after a visit to FermiLab. In addition to being absolutely adorable, these creative projects give us a sense of the popular conception of what a scientist is, and how it may differ from what real scientists are like. I was heartened to see that while many kids drew a stereotypical bald white guy as their "before" scientist, some of them knew going into this project that scientists can be, for example, women. The white lab coat was fairly universal, though. I guess kids don't know how lax we are with our PPE. (They also seemed excited to learn that real scientists wear casual clothes to work. Examining the photo of the class will show that they're wearing school uniforms, and apparently are not big fans of the restrictive dress code.)

This web discovery is especially appropriate given that I will be doing some outreach with seventh graders myself. As part of our first-year graduate seminar, Emory neuroscience students will be participating in Brain Awareness Month this March. I'm working with a partner to design a lesson for a middle school class. We'll be teaching about the neuromuscular junction, and how some native Georgia species produce toxins that affect this synapse. (Coral snakes have a curare-like toxin in their venom, which blocks acetylcholine receptors in the muscle cell. Black widow spider venom, on the other hand, induces the nonspecific release of acetylcholine. Both of them can mess you up.)

I think it would be great to have "my" seventh-graders do this exercise. I've already been matched with a local teacher for Brain Awareness Month; I may have to contact him and see if he's into it. Ideally, I'd then get to take some of the drawings home and put them on my fridge. I'll be sure to post again after my classroom visit, and let you all know how it goes!

Monday, February 23, 2009

Compound Mutants: A Better Model for Autism?

ResearchBlogging.orgDr. Mriganka Sur and colleagues at MIT's Picower Institute for Learning and Memory published a paper in Proceedings of the National Academy of Sciences earlier this month on a compound mutant mouse model of brain enlargement and abnormal social behavior. Both increased brain size and social deficits are hallmarks of autism spectrum disorders (ASDs), a very hot research topic these days. The paper shows that these mice, which have mutations in two genes called Pten and SLC6A4, exhibit some of the characteristic symptoms of ASDs. The key finding is that the combined mutations exacerbate the mices' symptoms, such that a double mutant exhibits more severe problems than a mouse with mutations in Pten alone or SCL6A4 alone. From this, the authors conclude that these two ASD candidate genes play off of each other during development, providing further evidence that while there is no single "autism gene," combined genetic risk factors seem to play a large role in the disease's etiology.

Pten and SLC6A4 were already known to play individual roles in ASDs and other neurological disorders. Pten is a regulator of a highly complex series of interlinked biochemical processes called the PI3-kinase pathway. This pathway regulates a huge number of cellular functions, including cell growth, differentiation, proliferation, and survival. Because of this, the pathway is closely linked to tumor formation -- indeed, Pten is found to be absent in many kinds of tumors. But aside from its role in cancer, Pten seems to help regulate normal brain development. One effect of Pten disregulation is macrocephaly -- fancy science language for "big heads." This molecule may be required to keep brain growth in check. Relatedly, a subset of individuals with autism and other forms of cognitive impairment are haploinsufficient for Pten, meaning that they carry one normal copy of the gene and one inactivated mutant copy. Macrocephaly is commonly associated with ASDs, and this may be one reason why. (TSC1 and TSC2, the genes that cause tuberous sclerosis, are found in the same pathway. Tuberous sclerosis, it's worth noting, is a tumor-forming disease that is also the most common comorbid disorder with autism.) People with Pten mutations display a broad range of symptoms, however -- some are far more impaired than others. For this reason, the gene has been suggested to sensitize individuals to the effects of other mutations, which in turn establish the severity of cognitive impairment in these patients.

Meanwhile, SLC6A4 is a gene that codes for a serotonin transporter. The protein made by this gene helps to regulate levels of serotonin in the extracellular space. One common biological marker for ASD is an increase in peripheral serotonin levels, so it seems that there is some association between autism and the regulation of this key neurotransmitter. In addition, some patients with ASD have been found to carry a mutant SLC6A4 allele which greatly reduces the transporter's expression. These patients, like individuals with mutant Pten, tend to have overgrown brains and some form of social impairment.

Previous research had made some associations between the PI3-kinase pathway and the serotonin pathway in vitro -- studies of protein-protein interactions and protein expression in cultured cell lines. Dr. Sur's study, however, makes the first direct link between PI3-kinase/serotonin pathway interaction and ASD symptoms in vivo. By breeding together two mutant mouse lines -- a Pten heterozygous knockout and a SLC6A4 loss-of-function line -- the researchers were able to study the combined effect of these two mutations in a living animal.

Both Pten and SLC6A4 single mutants had previously been shown to exhibit macrocephaly. When you combine the two mutations, though, the mice develop brains enlarged more severely than those of mice with individual mutations alone. The biochemical mechanisms allowing these genes to cause brain overgrowth have not been completely explained, but it seems that they are acting on different but related cues in brain development. I was unable to tell from the published figures whether the brain size increase in the compound mutants was greater than the sum of the increases of the two individual mutants, however. It's possible to imagine a situation in which the two genes were acting on completely unrelated pathways that could still cause an additive effect in the compound mutant. Just as a simplified example, we might someday learn that Pten regulates overall cell size, whereas SLC6A4 regulates the pruning of superfluous neurons during development. If this is the case, then you'd expect that combining the two mutations might lead to brain size greater than that seen in either of the single mutants (because then you'd have too many cells and the individual cells would be overgrown). A more convincing link is established if you prove that the change in brain size is somehow worse than would be predicted by the effects of the individual mutations. I wish this paper had provided more explicit measurements for the brain size experiments, to allow for this sort of comparison. Still, the study indicates that the compound mutant displays a more pronounced ASD-like phenotype than either individual mutant, which, combined with the combinatorial mutation theory of ASD, makes these mice an interesting model.

After measuring macrocephaly in these mutant mice, Dr. Sur and colleagues examined their behavior. ASDs are primarily diagnosed based on behavioral deficits, so it's important to establish whether the social behavior of an ASD mouse model is affected by the experimental manipulations being performed. The experiment used a fairly standard test of mouse sociability, in which the mice are given a choice between spending time in a chamber with an unknown "stranger" mouse (defined as the "social" chamber) or a novel inanimate object ("nonsocial" chamber). Most normal mice prefer to spend time getting to know a new mouse than playing with a new object. The Pten single mutant mice did show a deficit in this social behavior assay, spending roughly equal time in each chamber rather than showing a preference for social interaction. The SLC6A4 mutants also showed a decreased preference for the "social" chamber when compared to normal mice, although the effect was not statistically significant. Compound mutants, meanwhile, showed a social deficit more pronounced than that of either of the single mutants. This result seems to confirm the additive effect of the two mutations implied by the brain size measurements.

Other tests of social behavior, including social approach/avoidance and social recognition, also found deficits in socialization among the mutant mice. A test of prepulse inhibition, however, which measures the sensitivity of sensory and motor systems that can be disrupted in ASD patients, did not show an additive effect in the compound mutants. The researchers theorize that the two genes studied here are therefore involved in brain development relevant to social behavior, but not necessarily involved in other ASD symptoms such as sensory and motor defects. For a disease like autism, which involves symptoms in many different systems, this is not an unreasonable theory to espouse.

In the discussion section of their article, Dr. Sur and colleagues propose a few mechanisms by which the serotonin and PI3-kinase pathways could interact in ASD patients. Perhaps Pten is directly affecting the serotonin pathway by binding to serotonin receptors:
One possibility is that Pten and serotonin receptors may physically interact in a regulatory manner to influence brain development. In neurons, Pten binds the 5-HT2c [serotonin] receptor and, via its phosphatase activity, limits agonist-induced activation of this receptor and modulates the firing rate of dopaminergic neurons in the ventral tegmental area. It is interesting to note that 3 drugs that have been reported as alleviating symptoms of autism -— the atypical antipsychotics risperidone and olanzapine and the antidepressant fluoxetine -— all have antagonistic effects on the 5-HT2c [serotonin] receptor, in addition to well-known effects targeting other members of the serotonin and dopamine pathways.

Alternately, serotonin signaling might regulate the PI3-kinase pathway, as has been implied by several tissue culture experiments. Either theory could provide a mechanism by which the two pathways interact to regulate neuron growth, development, and function in a manner that might explain some ASD symptoms. They posit that the interaction between these two pathways could provide new therapeutic targets for treatment of ASD, allowing development of new drugs that ameliorate both abnormal cell growth and dysfunctional intercellular signaling.

Regardless of the exact mechanism, this study shows that a combination of genetic factors can be used to create a better animal model of ASDs. The scientists go on to discuss the fact that Pten dysfunction may increase susceptibility to spontaneous additional mutations and deleterious effects of environmental toxins in haploinsufficient patients. They propose using Pten mutations in combination with other genetic and environmental perturbations to further elucidate the role this gene may play in enhancing ASD symptoms in the presence of additional risk factors. If this line of research pans out, we may learn that while autism has no single genetic cause, there may be certain genes that greatly increase the effect of other risk factors that have yet to be fully characterized. If genes like Pten are responsible for increased sensitization to other mutations, they may provide a single target for treatment of a multifactorial and poorly-understood disease.

A press release from the Picower Institute has more information about this study.

D. T. Page, O. J. Kuti, C. Prestia, M. Sur (2009). Haploinsufficiency for Pten and Serotonin transporter cooperatively influences brain size and social behavior Proceedings of the National Academy of Sciences, 106 (6), 1989-1994 DOI: 10.1073/pnas.0804428106

Rotation #2 Diary: Week 7

Well, that was a rough week. I spent three days preparing new tissue samples according to my fine-tuned protocol. Dissecting mice, grinding up their organs, processing the resulting goo, and measuring the concentration of protein in those samples took longer than I expected. Plus, with my class schedule, I had some days where I just didn't have enough continuous lab time to do all the things I wanted to do. (I wouldn't mind staying at the lab late to get things done, but unfortunately the Emory shuttle that I use for commuting stops running at 7:00 PM. I have to impose a somewhat rigid schedule for myself, as a result.)

Because it took much of the week to prepare my samples, I'm only just now running them out on a gel. I hope they look as good as the preliminary batch. I have a meeting with my adviser this week, so we'll be going over what little new data I've generated recently and talking about how I might expand upon this project if I join her lab. I hope to have something cool to show her.

I'm now in Week 8 of the rotation, a.k.a. the last week! There's no way I'm going to finish everything I want to do. When I started this rotation, I had to submit a proposal outlining my intended project. Reflecting on that now, I was being laughably ambitious. If everything had worked perfectly from the start, I might have checked more experiments off the to-do list, but instead I spent the first six weeks trouble-shooting. Alas, science can be like that sometimes. I just hope I can scrape together a few pretty figures for my rotation report.

Tuesday, February 17, 2009

Role Models

The March edition of Scientiae is calling for submissions on role models of women making history. The prompt:
During Women's History Month we tend to look backward and acknowledge the hard work and suffering that got us where we are today. For a change, let's acknowledge history in the making and the motivations that make it possible. Who are your role models? Who first got you interested in your field, or opened new doors for you? Who inspires you on a daily basis and makes you believe in the future of science, technology, or the world?

My inspiration to become a scientist is something that's not entirely easy to nail down. I think in some sense, I was always interested in the subject. There are a lot of medical professionals on my mother's side of the family -- my grandfather was a surgeon, an uncle is a psychiatrist, and my mother and grandmother have both worked in nursing. I was raised by people who share a sense of curiosity and intellectual commitment, especially related to the study of the human body.

In school, I had a series of awesome science teachers who encouraged the development of my interest in science. My seventh grade science teacher, Ms. Bozeman, had the thrilling job of teaching life science to a gang of rowdy middle schoolers. Everyone loved her class -- we had pet snakes and tarantulas in the classroom, we got to dissect frogs, we watched uncomfortably hilarious videos about sex education. In seventh grade, I was not especially studious, although eager to learn. Ms. Bozeman did everything she could to encourage me to complete my homework and notebook assignments so that I could get the 'A' I deserved (I usually did very well on tests, but was too lazy to do other assignments). I mostly ignored these attempts, but looking back, I'm glad that my teacher was paying enough attention to me to judge my actual abilities, not just my skill at filling out worksheets. During a quick Googling spree I learned that Ms. Bozeman now has an asteroid named after her -- how rad is that?! And how rad is NASA for acknowledging the work of middle school science teachers when they name celestial bodies?!

In high school, my favorite science class was chemistry. Part of this, I think, came from my intuitive grasp of a subject that other students found to be difficult. My best friend through most of school was one of our valedictorians (yes, I went to the sort of over-achieving high school that has a nine-way tie for valedictorian), and chemistry was the only subject where I ever did better than her on exams. She still did well, but I remember how thrilling it was to have her ask me about things she didn't understand, and to teach her a thing or two for a change. Being the perfect sweetheart that she is, she never took any sort of grade discrepancies personally, and our friendship continues to this day.

But, much of the credit for my success in and love of chemistry is due to my teacher, Ms. M.J. Booher. A lot of students were scared of her, because of her no-nonsense approach to classroom discipline and generally tough attitude. But, some of us learned that she could also be great fun, and even managed to make her laugh occasionally. She demanded respect from her students, but returned it in kind. I was in her class for two years (a year of honors chemistry followed by a year of AP chemistry) and learned a lot -- so much that I was able to skip my first year of chemistry in college! That head start in science at the college level gave me time to take extra credits and ultimately earn a dual BS/MS degree in a total of four years.

When I was in her classes, Ms. Booher would often jokingly refer to herself as "a junior" -- meaning that she planned to teach for two more years, and then she would be eligible for retirement. When I looked her up to write this blog post, I saw that she actually surpassed the 30-year mark for teaching, and was still in the classroom as recently as 2006 (I graduate high school in 2002), going above and beyond the call of duty to serve her understaffed department and unappreciative school board. Here you can read an editorial on teacher salaries that she wrote in my home town's newspaper. I don't follow the local news there too closely and I'm not sure what proposal she refers to, or whether it passed, but I know that I wouldn't want to go up against my old teacher in this kind of a fight. I hope the school board listened.

Once I reached college, I had the great privilege of working with some of the finest women neuroscientists around, including Prof. Eve Marder (former President of the Society for Neuroscience) and MacArthur Fellow Prof. Gina Turrigiano. They taught my classes, they helped mentor me in the lab, and they provided examples of the incredible achievements that women in my field can attain. My undergraduate department had so many awesome female faculty, in fact, that I remember being somewhat puzzled by the whole Larry Summers fiasco about women in science that occurred during my junior year. Gender discrepancies in the sciences just weren't very apparent to me, at an institution like Brandeis.

But, of course, these discrepancies do exist. After I graduated from college and began working full-time as a research technician, I discovered the science blogosphere. Specifically, I found a bunch of blogs dedicated to women's issues in STEM, in which women bloggers shared their experiences and raged against the patriarchal machine. At that point, I was still unsure of what I wanted to do with my scientific education. Reading the stories of these women helped me to discover my own passion for science, and my determination to succeed even in a field where I might experience outright discrimination or subtler setbacks. I read everything I could find that was written by female scientists, professors, postdocs, and graduate students. I read about their successes and failures, their careers and families, their joys and concerns. Without the wealth of information provided by these people, I wonder if I would have decided to go on to graduate school. All of that reading made me feel both inspired to take the next step and prepared for the difficulties that lay ahead.

Which brings us to now: graduate school. I've been a PhD student for less than a year, but already I find myself surrounded by new role models. While my current department lacks some of the female superstardom of Brandeis's neuroscience faculty, I have met great women professors here who have aided in my scientific training as well as provided personal support through groups like Emory Women in Neuroscience. I'm also continually inspired by my peers: my cohort is overwhelmingly female, and these young women remind me each day that I'm not the only one reaching toward lofty scientific goals. I also enjoy the support of older students -- second-years, third-years, and beyond -- who have handed down their wisdom on classes, rotations, and school/life balance.

As I reflect on all of these people who have helped me get where I am today, I'm anxious to give something back to other women like me. Although I'm relatively inexperienced, I, too, have support to offer. This semester, I'll be working on a Brain Awareness Month project in the Atlanta public school system, designed to expose K-12 students to neuroscience taught by real scientists. It's not the same as teaching high school for 30+ years, but it's a chance to reach out to young kids and hopefully show them something new and exciting. Then, starting next year, I'll be serving as a TA in at least one class. We have the option to choose graduate or undergraduate classes, but I think I'd like the opportunity to work with undergrads. It may be naive to think that a TA can have a major impact, but I'd like to put myself out there and do what I can to teach these students about science, and about how to pursue scientific careers themselves. Meanwhile, I'll continue to interact with my current amazing cohort as we help each other through each grueling exam. I'm also privileged to interact with the next generation of prospective graduate students in my program, answering their questions over email and participating in interviews and recruitment events.

I have no delusions that doing any of these things makes me some kind of a hero, but I think this is a good start, at least. The whole idea of "history in the making" means I don't know exactly how everything is going to go, yet. All I can do is my best, when it comes to giving back -- with thanks to those who helped me make it this far.

(Written for .)

Rotation #2 Diary: Week 6

I only spent three days of week 6 in the lab, since I had some rearranged classes on Thursday and a full day of prospective student activities on Friday. I've also missed a day of week 7, due to a bad cold/flu thing that hit me on Sunday and gave me a high fever for two straight days.

So, in week 6, one of my experiments actually worked! I think we've fine-tuned the protocol for making tissue lysates in order to actually detect my protein of interest. That only took 3/4 of my rotation time... heh. Now to repeat it and make sure that it really works. If so, I can move on to testing human brain samples!

As this rotation winds down, I have to start thinking about my next (and probably last) one. I've got one lab in mind, and just emailed the PI to see if she still has space for a rotation student. If she can't take me, I may have to go back to the drawing board... but, there are so many great faculty here at Emory, I suffer more from having too many choices than from a lack of good options.

Monday, February 16, 2009

Mystery Receptor's Ligand is an Endogenous Hallucinogen

ResearchBlogging.orgDr. Arnold Ruoho's lab at the University of Wisconsin has just published a paper in Science linking the endogenous hallucinogen N,N-Dimethyltryptamine (DMT) with the "orphan" (no known endogenous ligand) sigma-1 receptor.

The sigma-1 receptor was previously known to be a regulator of voltage-gated sodium, potassium, and calcium ion channels, found throughout the mammalian nervous system and periphery. While this receptor was linked with many binding partners (including cocaine, haloperidol, and fenpropimorph), its endogenous ligand was not known. DMT occurs naturally in lung and brain tissue, and had been found in human urine, blood, and cerebrospinal fluid. In some cultures, DMT is extracted from plants and used as a ceremonial hallucinogen, as in the South American sacramental tea, ayahuasca. Because it chemically resembles the known ligands of the sigma-1 receptor (they all contain an N,N-dimethylated amine) and occurs endogenously, Dr. Ruoho's group decided to investigate the binding of DMT with sigma-1 receptors.

To test DMT's affinity for the sigma-1 receptor, the researchers measured its ability to competitively bind the receptor when challenged with other sigma-1 ligands (cocaine and fenpropimorph). Rat liver homogenates containing sigma-1 receptor were allowed to bind DMT or a similar amine (tryptamine, N-methyltryptamine) before being exposed to radioactively labeled cocaine or fenpropimorph derivatives. By measuring the amount of radioactive ligand bound to the receptors after this experiment, the scientists were able to discern how well their pre-treatment with DMT and other amines had filled the receptors' binding sites. DMT was found to be the best inhibitor of cocaine and fenpropimorph binding in the rat liver homogenates, indicating that it binds strongly to sigma-1 receptors and does not allow binding of the radio-labeled drugs in this assay.

After proving that DMT and the sigma-1 receptor can bind together, Dr. Ruoho's group needed to show that DMT binding can affect the sigma-1 receptor's function as an ion channel regulator. To test this, they performed electrophyisology studies in cultured human embryonic kidney (HEK293) cells artificially expressing a cardiac voltage-gated sodium channel (hNav1.5), as well as COS-7 cells expressing the same channel, and mouse cardiac muscle cells (which contain this type of ion channel naturally). The cell culture experiments showed that in all cases, treatment with DMT decreases the activity of these ion channels, although the degree of inhibition varied between cell types (perhaps because the different cell types contain different levels of sigma-1 receptors). The mouse cardiac myocyte experiment was especially important, because it showed that normal mouse tissue responds to DMT treatment in a measurable way, indicating that further experiments in mice, including genetically modified animals, were possible.

Dr. Ruoho and his colleagues acquired a sigma-1 receptor knockout mouse for their next series of experiments. They repeated the cardiac myocyte test using tissue derived from their knockout animals and showed that while DMT decreases sodium channel current by about 29% in normal cells, the knockout cells' sodium current only decreased by about 7%. This provides further evidence that the sigma-1 receptor plays a crucial role in DMT's effect on ion channel regulation.

After completing this series of in vitro experiments, the researchers decided to test the effects of DMT in vivo. In mice, treatment with DMT and other sigma-1 receptor ligands induces hypermobility, especially if the animals are first given a monoamine oxidase inhibitor (MAOI; inhibits the naturally-occuring enzyme that breaks down DMT and other monoamines in the body). Dr. Ruoho and his team tested the behavioral effects of MAOI + DMT in wild-type and sigma-1 receptor knockout mice and found that only wild-type animals displayed characteristic hypermobility after treatment. They used methamphetamine as a positive control, showing that a drug that works through a non-sigma-1 receptor system could still induce hypermobility in the knockouts.

In their conclusions, the scientists state that "These studies thus suggest that this natural hallucinogen could exert its action by binding to sigma-1 receptors, which are abundant in the brain. This discovery may also extend to N,N-dimethylated neurotransmitters such as the psychoactive serotonin derivative N,N-dimethylserotonin (bufotenine), which has been found at elevated concentrations in the urine of schizophrenic patients. The finding that DMT and sigma-1 receptors act as a ligand-receptor pair provides a long-awaited connection that will enable researchers to elucidate the biological functions of both of these molecules."

This study suggests that our bodies use low levels of a hallucinogenic compound to regulate normal physiological processes. I find this especially interesting, since DMT is classified as a Schedule I drug in the United States. This means that the government has decreed a naturally-occuring brain chemical to have "no currently accepted medical use" with "a lack of accepted safety for use of the drug under medical supervision." This classification implies that all of us are criminals for possessing this drug in our brains. If further studies indicate that DMT might have therapeutic promise for neurological or psychiatric diseases, one would hope that the DEA (and similar bodies around the world; DMT is a Class A drug in the UK and similarly prohibited in many other countries) would re-evaluate its status as a controlled substance. Given the way things have gone with acceptance of medicinal marijuana, though, I can't say I feel too hopeful.

Further discussion of this paper can be found at the Royal Society of Chemistry and at Psychedelic Research.

D. Fontanilla, M. Johannessen, A. R. Hajipour, N. V. Cozzi, M. B. Jackson, A. E. Ruoho (2009). The Hallucinogen N,N-Dimethyltryptamine (DMT) Is an Endogenous Sigma-1 Receptor Regulator Science, 323 (5916), 934-937 DOI: 10.1126/science.1166127

Sunday, February 15, 2009

Blogging Tools

This week, I discovered a couple of helpful tools that have improved my blogging capabilities. Hopefully they'll lead to higher quality posts in the future. (Some might say we'd need a higher quality blogger to ensure that, but fie upon them!)

The first one may be a no-brainer for most of you, but I've started using Google Reader to receive tables of contents from Nature, Science, Cell, Nature Neuroscience, Neuron, and The Journal of Neuroscience. Every week I am exposed to dozens, if not hundreds, of intriguing articles. The idea is that I will eventually get around to blogging about some of them, although there's no way to find time for them all.

I've also switched from using Blogger's web-based WYSIWYG blog post editor to MarsEdit, a piece of shareware that works with many different blogging sites. It's so much better -- Blogger's web utility seemed to randomly reformat my text anytime I tried to edit a post, and it wouldn't remember things like my preferred font. It also didn't work well with copy and paste. So, I'm happy to have an easier way to update my blog, and one that will hopefully lead to fewer inexplicable formatting problems (like the weird spacing in some parts of my previous post).

Tuesday, February 10, 2009

Enrichment Matters, Even Across Generations

ResearchBlogging.orgNewScientist has written an article about a new paper from Dr. Larry Feig's group at Tufts University School of Medicine. The paper expands on Feig's previous work(1, 2), which showed that exposure to an enriched environment increases long-term potentiation (LTP) -- a cellular process critical for learning and memory -- in the brains of both normal juvenile mice and mice with genetic LTP deficiencies. (The same effects were not seen in adults, indicating that a critical period exists for this effect.) The most recent study describes how the effects of an enriched environment can be passed on from mother to offspring in both wild-type and mutant mice. If a female mouse is exposed to an enriched environment while she is a juvenile, her offspring from later pregnancies show enhanced LTP and improved performance on memory-based tasks. This is true even though the offspring themselves are never exposed to any environmental enrichment, and the female is removed from the enriched environment before she becomes pregnant. The researchers posit that environmental enrichment leads to some form of heritable epigenetic modification in the mother mice that can be passed on to their offspring.

In the Feig group's previous papers, we learned that mutations in a family of genes called Ras-GRFs can disrupt LTP (as well as long-term depression, or LTD, the converse cellular mechanism to LTP) in the brains of mice. These genes code for proteins that regulate certain other proteins called kinases, which regulate a wide variety of cellular processes by controlling signaling via phosphorylation of different amino acid residues. These biochemical pathways and mechanisms can get extremely complicated and require vast diagrams to explain in full, but for the purposes of this discussion we can just say that Ras-GRF1 is involved with a protein called a p38 Map kinase, and Ras-GRF2 is involved with Erk Map kinase. Together, these proteins and many others help to regulate synaptic plasticity in the brain by helping to potentiate or depress the connections between neurons (that is, by creating LTP or LTD). 

Dr. Feig and his colleagues conducted further studies to see whether environmental manipulations might affect the activity of the Ras-GRF pathway in normal and Ras-GRF knockout mice. It was known that environmental enrichment can play a major role in adult neurogenesis, as well as protection against or recovery from certain neurological diseases. You can read more about this in many, many articles, but one interesting paper on the topic comes from Dr. Li-Huei Tsai's lab(3). The Tsai paper describes how environmental enrichment can help mice recover learning and memory function even after massive neuronal loss similar to that seen in Alzheimer's disease. It also associates environmental enrichment with a process called histone acetylation, a form of genetic remodeling that has been implicated in heritable epigenetic modification.

When Dr. Feig exposed his mice to an enriched environment, defined in this study as "an enriched cage (45 x 30 x 25 cm) containing plastic play tubes, cardboard boxes, running wheel, various pet toys, and nesting material that were all changed or rearranged every other day to provide novel stimulation," he observed activation of a normally latent p38 Map kinase signaling cascade. You may remember p38 Map kinase -- it's one of the signaling pathways that works with Ras-GRF proteins to create LTP and LTD. Indeed, it turns out that activation of this signaling cascade, brought on by environmental enrichment, can compensate for the loss of Ras-GRF in knockout mice. Ras-GRF knockout mice that were exposed to an enriched environment have levels of LTP comparable to those of normal mice. Ras-GRF knockouts that received no environmental enrichment had significantly impaired LTP. 

So far, fairly straightforward research. The scientists found a biochemical pathway associated with LTP. They observed that genetic manipulations to disrupt that pathway can disrupt LTP. They also observed that environmental manipulations activating a related pathway can restore normal LTP to mutant animals. Now it gets interesting, because they noted that the effects of environmental enrichment on LTP in both normal and mutant mice persist for several months. The group decided to see whether mice that reproduced during the two-month window after environmental enrichment would pass on any benefit to their offspring.

It turns out that they do. The mutant offspring of enriched mutant mice have normal LTP function. They also show enhanced memory formation ability compared to non-enriched mutants in a simple fear conditioning test. (The mice learn to fear a chamber where they have previously received a series of mild electrical shocks.) This effect is independent of paternal enrichment -- that is, it doesn't matter whether or not the fathers are exposed to an enriched environment; only the mother's experience matters. The effect is also independent of maternal care: mutant offspring of enriched mothers who were raised by non-enriched mutant foster mothers still exhibit improved LTP and fear conditioning. 

The researchers then tested whether these effects could be passed on to a subsequent generaton -- the grandchildren of the original enriched mice. They found that it could not. The authors offered: "One possible explanation for the limited transmission of this [environmental enrichment] effect is that the phenotype ends at a younger age in the offspring of enriched mice than in their parents, such that it is no longer present and thus not transferable to offspring by the time the F1 generation are old enough to reproduce." Still, this study provides evidence that a mother mouse can somehow respond to changes in her environment in a way that not only alters her capacity for synaptic plasticity but improves the learning and memory function in her young. Why might this happen?

Dr. Feig and his colleagues make a great point in the discussion section of the article. They say: "The enriched environment used as an experimental paradigm in these studies may actually be more natural than a conventional laboratory environment that may border on sensory deprivation. Thus, the transgenerational inheritance of this new LTP-inducing signaling pathway may be a mechanism that has evolved to protect one's offspring from deleterious effects of sensory deprivation, which may be particularly potent in the young and exacerbated by the presence of mutations in signaling molecules like GRF proteins that contribute to synaptic plasticity." In other words, the "control" mice in studies on environmental enrichment might be the ones with abnormal brain function. Laboratory mice spend their entire lives in a shoebox-sized cage where they have nothing to play with and little space for exercise. It may be that the pathway activated by "enrichment" in Dr. Feig's experiments is active in normal, wild mice, and that the mice living in a control environment have lost their ability to induce LTP as effectively. Perhaps in the wild, mother mice with access to the outside world pass on this activated p38 Map kinase to their offspring in a transient manner to protect their brains while they are living in the relatively deprived environment of the nest. The age at which the heritable effect of environmental enrichment wears off is, coincidentally, the age at which baby mice are weaned and leave their nests. 

There are many future avenues of research to be pursued based on this work. The next logical step would be to try to determine exactly what is happening in mother mice to allow them to pass on this effect to their offspring -- What physiological changes are occurring upon exposure to enrichment? What genes are being epigenetically modified, by what mechanisms, in response to this environmental stimulus? Since environmental enrichment likely affects many different things, it may be difficult to tease out the exact mechanism by which increased sensory or social stimulation activates a p38 Map kinase cascade, but it couldn't hurt to try. I'm also interested to learn more about why this enrichment-dependent effect on LTP only works in juvenile animals. Can this critical period for p38 Map kinase activation be extended or shifted by further experimental manipulation? Would a similar effect be seen in enriched versus deprived animals of other species, perhaps with a critical period that maps onto some important developmental stage across species? 

Regardless of where Dr. Feig's group goes from here, their work has brought a hint of the Lamarckian to a month of celebrating Darwin. Epigenetics is a growing field, and I'm interested to learn more about the intersection between environmental factors, epigenetic modification, and neurological processes in healthy and dysfunctional brains. 

J. A. Arai, S. Li, D. M. Hartley, L. A. Feig (2009). Transgenerational Rescue of a Genetic Defect in Long-Term Potentiation and Memory Formation by Juvenile Enrichment Journal of Neuroscience, 29 (5), 1496-1502 DOI: 10.1523/JNEUROSCI.5057-08.2009

References

1. Li S, Tian X, Hartley DM, Feig LA. The environment versus genetics in controlling the contribution of MAP kinases to synaptic plasticity. Curr Biol 16:2303–2313. (2006) doi: 10.1016/j.cub.2006.10.028 

2. Li S, Tian X, Hartley DM, Feig LA. Distinct roles for Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) and Ras-GRF2 in the induction of long-term potentiation and long-term depression. J Neurosci 26:1721–1729. (2006) doi: 10.1523/JNEUROSCI.3990-05.2006

3. Fischer A, Sananbenesi F, Wang X, Dobbin M, Tsai LH. Recovery of learning and memory is associated with chromatin remodelling. Nature 447:178–182. (2007) doi: 10.1038/nature05772

Rotation #2 Diary: Week 5

Another week of my rotation has come and gone. Indeed, I'm halfway through with Week 6 as I write this Week 5 diary (I won't be in the lab on Friday; my department is interviewing potential new graduate students and I"ll be helping with the assorted interview and recruitment activities). 

Last week I repeated experiments that I'd done before, in an attempt to get better, cleaner-looking western blot data. I did see some improvement, but so far haven't produced any figure-quality blots. This week I got some new mouse protein lysates prepared in a variety of different ways to try to optimize the protein yield, and I'm repeating the experiment yet again. Hopefully, the experiments this week will be more fruitful. The lab has secured some human brain tissue samples for me, so as soon as I optimize my technique in rodent lysates, I'll be able to look for my protein of interest in the human brain. That'll be pretty cool. If I make it that far.

I also learned that my lab meeting presentation for this rotation has been pushed back until two weeks after the rotation officially ends. That's probably a good thing -- I'll have some time to work on my rotation report and the presentation without worrying about lab work. I'm planning to take a couple of weeks off between this rotation and my next one (conveniently, my spring break falls in there...) so for a while I'll have nothing to do but attend classes and study while I'm writing up my results. And updating my blog, of course...

Friday, February 6, 2009

I Love Brains

I love YellowIbis.com. Not only do they have great science t-shirts that really "get it," but they're dedicated to customer service, too. Someone from their staff saw me praising their products elsewhere on the web, but lamenting the fact that grad students can't afford to buy as many new shirts as they'd like. In their infinite mercy, they offered me a small, but thoughtful, discount. I still can't spend too much of my income on fun clothes, but after they got in touch with me I decided to order some shirts anyway, just to support this cool company that's making a great effort to work with their customers. So, I ordered the I Love Brains shirt.
I'm ready to do some neuroscience this morning! (Before I've even had my first cup of tea, so excuse the slightly crazed look in my eyes.)

I also purchased the popular "This is what a scientist looks like" shirt (which was featured at ScienceWomen, and went over well at my Women in Neuroscience meeting) and the Skull and Pipets shirt. Hooray for scientific sartorial spendor! 

Wednesday, February 4, 2009

Connections

I recently finished watching James Burke's excellent 1978 documentary series Connections. The entire series (10 episodes, an hour each) is available from Netflix, although annoyingly you only get two episodes per DVD. The first episode, "The Trigger Effect," is also available on Google Video

The series is dedicated to exploring the history of science and technology. Burke chooses one invention to focus on in each episode, then delves back in time to explain the numerous advancements that had to occur before we could get to the telephone, the jet engine, the atomic bomb, and other world-changing creations. I was a little worried about watching a technology-centric series from 30 years ago, especially when we got to the episode about computers (picture a room full of servers the size of refrigerators...), but was pleased to discover that it has aged very well. The bulk of each episode is devoted to historical events, which don't feel at all dated (unlike Burke's wardrobe!). 

After spending a few idle evenings watching this series, I learned that: alarm clocks were invented by monks who needed a reliable way to say their prayers on time, computer punch cards arose from the technology used to create jacquard fabrics on a loom, the electric light bulb was preceded by "limelight" -- literally heating a piece of limestone until it became incandescent, air conditioning was originally intended to cure malaria, and you can do a hell of a lot with various coal byproducts. I especially enjoyed the way Burke pointed out all of the failures and accidents in the history of each technological advancement, telling us about the people who tried to build a better steam engine but sucked at it (showing that the idea was around before the actual machines were perfected -- who should get the credit, then?), or the person who invented artificial dyes while trying to make a synthetic form of quinine. I think these sorts of discoveries make a great case for continuing to fund basic science research -- you never know how someone's failed experiment or accidental breakthrough might become important. A sense of Burke's style of storytelling can be found at his Knowledge Web project -- see the "Mystery Tours" for some examples of the connections between inventions and events.

Definitely an enjoyable show! Check it out if you're in the market for some good educational programming.

Sunday, February 1, 2009

Rotation #2 Diary: Week 4

Well, I've been working on this rotation for about a month, now. I still don't really have any great data. After repeating some experiments and trying every troubleshooting method we could think of, it seems that sometimes things work, sometimes they don't, and the things that do work give different results each time. Ah, science... you can be a cruel mistress.

At the end of the week I had a meeting with my rotation adviser. She meets with her lab members individually, once a month or so, to check in with them and get progress reports on their projects. So, I scanned my films and emailed them to her for us to look at during our meeting. And we did look at them... briefly. We talked about what was working (very little) and what wasn't working (almost everything I've tried), and she gave me some directions to focus on during the second half of my rotation. Then, my adviser did something very smart: she stopped talking about my rotation project and switched gears to other, more exciting projects that I could work on if I join her lab.

It's not that I don't like my rotation project. I do like using molecular biology techniques, and I've learned some useful new skills here. I also think that the protein I'm studying is interesting given what's known about it, and I wouldn't mind continuing to study that protein in the future. But, my adviser was listening during my previous meetings with her, when I mentioned how I like to conduct research at multiple levels, including in vivo. So, she told me about a new line of transgenic mice the lab has just acquired, and the kinds of studies I could do using that mouse model if I was interested in that kind of project. I've got some papers to read for more information, but it sounds like interesting work. If I did join the lab, I think it would be really useful to balance a more molecular project like what I'm doing for the rotation with an in vivo project like that mouse model. Working with mice can be slow, since you have to worry about breeding them, genotyping them, and aging them out if your phenotype is age-dependent. Having something to do during down times with the mice would keep my productivity up. Although, working on two projects might not add up to a cohesive dissertation, I suppose. That's something to discuss further, if I do decide to join this lab. 

So, while I'm not thrilled that my western blots haven't been working out, I am glad that my adviser is thinking about other things that I could be doing, and that she has been paying attention to what I want to study. She was also responsive to my concerns with doing this kind of project, i.e. working with mice can get really annoying when you're managing your own colony, doing all of the genotyping, and so on. This lab doesn't have any technicians, so I'd be responsible for doing all of that grunt work. But, this particular mouse line uses coat color as a readout of the genotype, so at least I wouldn't be bogged down in tail preps and PCRs if I worked with these animals.

Of course, this professor is also acting in her own interests. Right now they don't have many people using in vivo techniques, so I'd be a useful addition to the lab in that respect. And she's a little low on students right now, since several of her lab members recently graduated, so she needs to bring more people in to keep producing data. Even so, it is flattering to be recruited in this way. It also makes my lab decision more complicated, with new research topics to consider, but I'm glad to know what my options are.