Friday, May 29, 2009

More Travel Grants Available!

As some of you may know, I recently received a $500 travel grant from NextBio, makers of a free web-based "curated, correlated database of public data, integrated literature, clinical trials, and scientific news." The grant can be used to attend the scientific conference of my choice, which is awesome.

Well, they're at it again! If you're a graduate student or medical student interested in applying for a travel grant, check out the NextBio grants website. To apply, you must submit a one-page essay describing how you've used NextBio tools in your research. The deadline for applications is June 30, 2009, and recipients of the award will be notified by July 15. It looks like the company has decided to award grants like this on a continuing basis, which is great news for poor grad students everywhere.

TRP Channel Variations Determine What's "Too Cold" In the fairy tale of Goldilocks and the Three Bears, Goldilocks encounters three bowls of porridge in the bear household. Papa Bear's porridge is too hot; Mama Bear's porridge is too cold; Baby Bear's porridge is just right. Why would Papa Bear and Mama Bear choose to heat their porridge to non-optimal temperatures? A new study by Benjamin Myers et al. published in PLoS ONE suggests that their perception of "just right" depends on their TRP channels.

Okay, it's unlikely that three members of an anthropomorphic bear family would possess significantly different temperature-sensitive ion channels. But the paper shows that warm- and cold-blooded species living in different environments have evolved ion channels with different temperature thresholds to help them maintain a body temperature that is "just right."

Transient receptor potential (TRP) ion channels are responsible for many different sensory functions. The first TRP channel was discovered in the fruit fly, where it plays a role in visual signaling. Many other TRP channels have since been found. Though these channels share a similar structure, they contribute to many different sensory systems, including touch, pain, taste, and smell. A class of TRP channels also responds specifically to temperature, allowing nerve endings in the skin to sense heat and cold. Interestingly, the channels also detect certain chemicals that create a hot or cool sensation even without a change in temperature. In 1997, we learned that the heat-sensitive TRPV1 channel also responds to capsaicin, the molecule that makes chili peppers taste "hot" (Caterina et al.). In 2002, other research showed that TRPM8, a cold-sensing ion channel, can be activated by the minty freshness of menthol (McKemy et al.; Peier et al.). But although we have learned a lot about the function of temperature-sensitive channels in mammals, not much work has been done in other model organisms.

Myers et al. examined the TRPM8 channel in a commonly used cold-blooded model organism, the South African clawed frog (Xenopus laevis). They wanted to see whether this species, with a core body temperature and preferred environmental temperature much lower than that of mammals, would have a different range of temperature sensitivity in its TRPM8 channels. They hypothesized that these animals would have channels tuned to temperatures appropriate for their ecological niche, meaning that they would require a colder temperature to become active than the TRPM8 channels of a warm-blooded mammal or bird.

To test this theory, the researchers dissected out the dorsal root ganglia (DRG) of several frogs and used calcium imaging to measure the cells' responses to different temperatures. The DRG is the portion of the spinal cord that contains the cell bodies of sensory neurons, including the cells that produce temperature-sensitive nerve fibers. Thus, the cold-sensitive cells in the DRG express the TRPM8 channel. Calcium imaging involves use of a fluorescent dye that produces light when exposed to calcium inside a neuron. Because calcium influx is related to neuronal activity, we can use the fluorescent intensity to determine which neurons respond, and how strongly, to a given stimulus.

About 23.7% of the X. laevis DRG neurons responded to menthol, the chemical activator of TRPM8. Similar percentages of menthol-sensitive neurons are seen in mammalian sensory ganglia. Frog neurons differ from those of mammals, however, when the neurons are stimulated with cold temperatures instead of menthol. While rat neurons have a thermal activation threshold of 25.4°C, frog neurons have a much colder threshold of 9.6°C. Thus, a frog TRPM8-positive sensory neuron requires a much colder temperature to become active and produce a "cold" sensation.

Myers et al. wanted to be sure that this change in temperature threshold was caused by differences in the TRPM8 channel and not some other difference between rat and frog sensory neurons. Therefore, they expressed frog, chicken, and rat TRPM8 in oocytes (egg cells which do not normally express any ion channels; this is a common experiment used for studying ion channel physiology in an isolated system). Voltage-clamp recordings were used to measure the changes in oocyte membrane potential caused by activation and opening of the TRPM8 channels. The oocytes expressing X. laevis TRPM8, as well as TRPM8 from the related frog species X. tropicalis, displayed a much lower activation temperature than the oocytes expressing chicken or rat TRPM8. This experiment also showed a slightly higher activation temperature for chicken TRPM8 than rat TRPM8, which is consistent with the elevated body temperature of birds compared to mammals.

The differences in the temperature-sensitive properties of TRPM8 channels between species occur because of slight changes in the ion channel's structure through evolution. X. laevis TRPM8 differs from rat TRPM8 in about 25% of its amino acids. The differences in protein structure allow the ion channels to exhibit slightly different responses to temperature, even though they are similar enough to retain their general TRP structure and cold sensitivity.

The researchers summarize their findings as follows:
Within visual and chemosensory systems, stimulus detectors (receptors) undergo great functional diversification as organisms evolve to inhabit a wide range of ecological niches. Our findings demonstrate that genes encoding somatosensory receptors display the same capacity for adaptation to species' environmental conditions. Specifically, we have shown that a cold receptor can be tuned to respond within a temperature range most relevant to the normal resting temperature of the primary afferent nerve terminal, whether determined by an internally regulated core body temperature or the environmental milieu.

They go on to add that it is not clear whether the X. laevis and X. tropicalis TRPM8 channels are specifically tuned to the niches of these two species, or whether they represent a universal amphibian cold-sensitive channel that does not vary between frogs inhabiting different environments. As we sequence the complete genomes of more organisms, it will become possible to search for genes homologous to TRPM8 in other cold-blooded species and compare them to these frog channels. The authors add, "It may also be interesting to examine species that experience substantial variations (short- or long-term) in environmental temperature, as there may be corresponding changes in TRPM8 expression and/or function that allow for optimal temperature detection under such circumstances." Some cold-blooded species become dormant during the winter months, and it would be informative to see whether their temperature-sensitive neurons exhibit different properties during dormant and active seasons.

This research further elucidates how evolution has shaped individual species to thrive in different environments, down to the smallest molecular details. Frogs, rats, and chickens, like Goldilocks, sense heat and cold while searching for a temperature that is "just right." But what's good for one species might not suit another. Their specialized ion channels allow them to appropriately respond to thermal stimuli -- even the ones that don't like porridge.


Caterina M.J., Schumacher M.A., Tominaga M., Rosen T.A., Levine J.D., et al. (1997) The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389 (6653): 816–24

McKemy D.D., Neuhausser W.M., Julius D. (2002) Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416: 52–58.

Myers, B., Sigal, Y., & Julius, D. (2009) Evolution of Thermal Response Properties in a Cold-Activated TRP Channel PLoS ONE, 4 (5) DOI: 10.1371/journal.pone.0005741

Peier A.M., Moqrich A., Hergarden A.C., Reeve A.J., Andersson D.A., et al. (2002) A TRP channel that senses cold stimuli and menthol. Cell 108: 705–715.

Wednesday, May 27, 2009

Synaptic Vesicles Are Not All Created Equal

ResearchBlogging.orgA pair of articles in Nature Neuroscience this month provide new insights into the mechanisms underlying spontaneous and evoked release of synaptic vesicles. Spontaneous release of a single vesicle (a "mini" release event) at a synaptic site was first observed over 50 years ago. A mini is considered to represent a single "quantum" of neurotransmitter, therefore the quantal theory of neurotransmitter release states that all synaptic responses will reflect some multiple of the response to a mini (depending on the number of neurotransmitter vesicles released). This provides a convenient way to study neurotransmitter release, and many experiments have been conducted under the assumption that minis are a reliable shorthand for evoked synaptic responses. Scientists have debated the mechanism behind spontaneous release, however. Are minis produced by the same vesicles as those that produce evoked neurotransmission (that is, release of neurotransmitter in response to an action potential)? A paper by Naila Ben Fredj and Juan Burrone address this question, showing that two non-overlapping pools of synaptic vesicles exist in rat hippocampal neurons for spontaneous and evoked release. Are minis truly random, or are they responding to signals that we don't fully understand? Jun Xu et al. show that the same calcium-sensing molecule, synaptotagmin-1 (Syt1), is responsible for regulating spontaneous and evoked release of synaptic vesicles, and that Syt1 further regulates some yet-unknown sensor responsible for other mini release events. Both of these papers reveal new complexities behind spontaneous vesicle release that challenge some of our fundamental assumptions.

Synaptic vesicle release depends upon molecules called SNAREs, which are present on the vesicle (v-SNAREs) and the target membrane (t-SNAREs). Complementary SNAREs allow vesicles to fuse with the target membrane and release their contents (neurotransmitters) into the synaptic space. The prevailing theory of release states that these fusion events occur spontaneously at a very low rate, producing minis. An action potential causes an influx of calcium into the presynaptic cell, which activates a calcium sensor and greatly increases the probability of vesicle fusion, creating a large, synchronized release of neurotransmitter from many vesicles.

The first paper I will discuss, by Fredj and Burrone, addresses the assumption that spontaneously-released vesicles are the same as evoked vesicles. If minis are caused by the random fusion of synaptic vesicles with the cell membrane, then we would expect spontaneously-released vesicles to resemble all other vesicles, with the only difference being that they accidentally fused without receiving a calcium signal. Previous experiments have indicated that this is probably not the case (Sara et. al, 2005), but Ben Fredj and Burrone developed a new technique for labeling presynaptic vesicles that supports the notion of a separate pool of spontaneously-released vesicles. Vesicles that have released their contents are recycled by the cell, allowing them to be refilled with neurotransmitter and used again later.

The researchers created a tagged version of an internal vesicle protein called VAMP2, which they called biosyn. Fluorescently-labeled streptavidin will permanently bind to biosyn. If streptavidin is present in the synaptic space, it will label the biosyn that is exposed when vesicles fuse to the cell membrane. This allowed the researchers to visualize active, fused vesicles in a living synapse under different conditions. After verifying that biosyn is a reliable measure of spontaneous and evoked fusion events, and that the tagged protein does not interfere with normal synaptic processes, they went on to test whether spontaneous and evoked release events use the same populations of vesicles.

After testing biosyn labeling of spontaneous and evoked vesicle release, Fredj and Burron noticed that they saw only about half as much biosyn signal when examining spontaneous release, compared to evoked release. This occurred even though the vesicle populations appeared to be saturated (that is, the biosyn signal reached a maximum level after a sustained period of release, indicating that all of the available vesicles had already fused and been labeled with fluorescent streptavidin). This could mean that spontaneously-released vesicles represent a sub-population of vesicles that can undergo evoked release, or it could mean that the two types of release draw from distinct vesicle pools, with fewer spontaneous vesicle than evoked vesicles.

To distinguish between these two possible explanations, the scientists sequentially labeled evoked and spontaneous vesicles within the same cell using two different colors of fluorescent streptavidin. They describe that experiment as follows:
Synapses were first stimulated with a saturating stimulus of two 90-s depolarizations in the presence of strep488 [green streptavidin], which strongly labeled the entire recycling pool. A further depolarizing stimulus in strep594 [red streptatividin] resulted in no further labeling (or very small amounts of labeling), as all biosyn binding sites were occupied by strep488, confirming that our depolarizing stimulus mobilized all possible vesicles. On the other hand, after labeling the recycling pool with strep488, a further 15-min exposure to strep594 in conditions that would only allow spontaneous fusion events resulted in a substantial amount of labeling.

The data show that two different populations of vesicles are exposed to the fluorescent streptavidin probes under different release conditions. This prompted them to ask, "If the recycling pool of vesicles cannot account for spontaneous release, then where do these vesicles come from?" They propose that the spontaneous vesicles may come from the so-called "resting" pool. This is the name given to a previously-identified pool of vesicles that are not mobilized by neuronal activity. Further experiments by Fredj and Burrone provide evidence that this resting pool does provide the vesicles for spontaneous release.

A News and Views summary of the paper gives us more to think about:
The big question now becomes what other differences might exist between these vesicles besides the pools that they come from. Are they released from different locations in the presynaptic terminal, as suggested by a recent study? Does their protein and/or lipid composition differ? We can take some comfort in Fredj and Burrone's observation that the sizes of the evoked and spontaneous pools were highly correlated in individual axon terminals, consistent with previous studies. Further experiments will be required to identify all of the similarities and differences between these two forms of vesicle fusion and validate the continued use of spontaneous release to characterize evoked transmission.

Another paper from the same issue of the journal, by Jun Xu et al., indicates that while spontaneously-released vesicles may be drawn from a separate pool, they are using the same calcium sensor as evoked release (contrary to the belief that minis are, by definition, calcium-insensitive). They studied spontaneous (mini) and evoked inhibitory post-synaptic currents in cultured cortical neurons. While removing extracellular calcium from the culture medium depressed evoked currents more strongly than minis, the application of the membrane-permeable calcium chelator BAPTA blocked over 95% of minis. Meanwhile, applying caffeine (which increases intracellular calcium availability) increased the number of minis observed. This implies that while evoked vesicle release depends strongly on extracellular calcium influx, even minis are not calcium-independent -- some calcium must be present to trigger a spontaneous release event.

Evoked neurotransmitter release is known to be regulated by the calcium sensor synaptotagmin. Somewhat paradoxically, genetic deletion of synaptotagmin-1 (Syt1) causes an increase in the number of minis, leading to the theory that this protein both allows evoked release and prevents spontaneous release through some sort of clamping mechanism. But as the authors note, "The clamping hypothesis ... argues against the notion that spontaneous release may be biologically meaningful, as it is difficult to imagine how an accidental byproduct of evoked release could control a physiological process. Moreover, the clamping hypothesis fails to explain why at least some mini release is Ca2+ dependent."

Xu et al. decided to better elucidate the role of Syt1 in spontaneous vesicle release. By studying minis in neurons lacking Syt1, they found that the upregulated minis in those cells were still calcium-dependent (they, too, could be blocked by BAPTA). They showed that the synapses with no Syt1 seemed to exhibit greater calcium affinity than wild-type synapses. This indicates that Syt1 is not acting merely as a clamp to block spontaneous release in normal cells, but that some other, more sensitive calcium sensor is able to produce spontaneous -- but not evoked -- vesicle release in the absence of Syt1. This was true for both excitatory and inhibitory synapses.

One explanation for this result would be that Syt1 serves as the primary calcium sensor for spontaneous release, but that an unknown sensor, normally clamped by Syt1, can lead to spontaneous release in Syt1's absence. To test this, Xu et al. generated neurons expressing mutant varieties of Syt1 with different calcium affinities. If Syt1 is indeed responsible for both evoked and spontaneous release, one would predict that changing the calcium affinity of Syt1 would create a commensurate change in the magnitude of both spontaneous and evoked release. Indeed, this is what they found. The result held true when they tested the different Syt1 mutations in brain slices as well as in cultured neurons.

The authors leave us with this conclusion:
Evoked and spontaneous neurotransmitter release are generally considered to represent distinct types of release that are differentially regulated. Their distinct natures are evidenced by the fact that spontaneous release is maintained in the presence of the sodium-channel inhibitor TTX, which abolishes action potentials and evoked release. We found, however, that despite their differential regulation, these two types of release are mechanistically identical in that they both are triggered by Ca2+ binding to Syt1. The major evidence for this conclusion rests on the three Syt1 knockin mutations that we used. We previously demonstrated that these mutations either increase Ca2+-dependent binding of Syt1 to SNARE complexes or decrease the apparent Ca2+ affinity of Syt1 binding to phospholipids. In a direct comparison of all three knockin mutations, we found that they cause a corresponding change in evoked release and a precisely equivalent change in spontaneous release. ... Moreover, our results support previous suggestions that spontaneous release is physiologically important. Ca2+ regulation generally implies a physiologically controlled function; thus, the finding that spontaneous release is controlled by Ca2+ binding to Syt1 implies a physiological role... Many neurotransmitters and neuromodulators act by increasing or decreasing presynaptical Ca2+ concentrations, suggesting that these agents may control synaptic circuits, at least in part, by regulating Syt1-dependent spontaneous release without triggering action potentials.

Of course, this article also leaves us with some burning questions. What is this second calcium sensor that is clamped by Syt1? Why is it so sensitive? What important physiological roles are being played by these highly-regulated mini release events? Clearly, more research is needed into these areas.

In conclusion, we've learned from this month's Nature Neuroscience that spontaneous release events are not the same as evoked neurotransmitter release. Although these two types of synaptic vesicles use the same major calcium sensor (and are both calcium-dependent, contrary to popular belief!), there exist separate pools of spontaneous and evoked vesicles that respond differently to intracellular calcium fluctuations, and never the twain shall meet. It will be interesting to see where we go from here, teasing apart the distinct roles that these two types of vesicles play in neurotransmission.


Fredj, N., & Burrone, J. (2009). A resting pool of vesicles is responsible for spontaneous vesicle fusion at the synapse Nature Neuroscience, 12 (6), 751-758 DOI: 10.1038/nn.2317

Xu, J., Pang, Z., Shin, O., & Südhof, T. (2009). Synaptotagmin-1 functions as a Ca2+ sensor for spontaneous release Nature Neuroscience, 12 (6), 759-766 DOI: 10.1038/nn.2320

Sara, Y., Virmani, T., Deák, F., Liu, X., & Kavalali, E. (2005) An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission. Neuron, 45 (4), 563-573 DOI: 10.1016/j.neuron.2004.12.056

Tuesday, May 26, 2009

Scientiae: Moving Forward

How are you moving forward in life? Are you close to your degree, tenure, sabbatical, or summer holiday? Is that paper almost ready to go out the door? Is your baby almost potty trained or are you training for a marathon? What keeps you moving forward in your science, work, and life? Is it the drive to cure a disease, make the world a more sustainable piece, or discover something that no one else knows? Is it the promise of exciting data at the end of a long assay? Is it the thought of people calling you Dr.? Is it your daughter's smile when she wakes up in the morning, or the enthusiastic tail wagging of your dog? When things get tough, how do you motivate yourself to move forward?

The "moving forward" theme is an appropriate one for this time of year. After a two-year break while working as a research assistant, I once again find myself in step with the academic calendar, when May marks the end of the year. It's a bit unsettling to think that school has been the major focus of my life for so many years. When I was working full time, I felt a bit of resentment when I didn't get a summer vacation -- but then, I didn't really take summer vacations in college (I spent each summer taking classes, doing research, or both), and I'm not getting too much of a break this year, either.

Even so, I've finished the first year of my PhD work. That is such an exciting accomplishment. I haven't taken my written qual yet, but based on my grades in my classes I'd say I'm doing well. For someone who spent many years as a squeaking-by slacker, and made some rather embarrassing grades in college, this is a big deal. I spent much of my academic career feeling smart, but not particularly successful. Now I'm actually feeling a little hint of pride in my accomplishments, which is a good feeling, although I worry about things going to my head. When I receive independent confirmation of my success, like an A in a course or some praise from a professor, my self-esteem actually seems grounded in reality.

Related to that self-esteem is the idea that I might actually be of use to other people. In the lab, I'm starting to come up with my own ideas for experiments. In other academic settings, I've been elected as a student representative on my program's executive committee, I'm working with a cool group of students from other departments on an interdisciplinary journal club, and I'm looking forward to TAing my first class in the fall. All of these things make me feel like I'm part of a team, doing important work that might even have some impact on the outside world. My ideas might contribute to a publication, a policy, an inspiration. And, in my personal life, I recently heard from my aunt that my teenaged cousin declared a science "major" (her high school has students on different themed academic tracks). I'm sure I can't claim to be the sole determining factor in that choice, but I like to think that I've been a good role model for her, and that I can be there to offer her advice as she continues her education.

Overall, I'm in a good place right now. I have new challenges to face in the next year, and I continue to fret over failed experiments, family crises, and the legions of bugs that have settled in my kitchen, but I draw strength from some great support systems. Quals and roaches had better watch their backs.

Written for .

Monday, May 11, 2009

Rotation #3 Diary: Week 8

I'm now in the last week of my third rotation. Soon I will be choosing the lab where I'll spend the rest of my graduate school career. Thankfully, I'm in the happy position of choosing between several good labs. I haven't completely made up my mind, but I am leaning in one direction. I hope to sort everything out and join a lab by the beginning of June. If any former or current grad students would care to offer me some advice on making this decision, I'd be happy to get your input!

Last week I presented my rotation work at lab meeting. I made a little PowerPoint presentation that went over my progress on a couple of projects, complete with cute cartoon mice and screenshots from the gene sequencing software I've been using. Then, much to my horror, I deleted the entire thing 30 minutes before the meeting. I know I should have backed it up, and I had intended to. My original plan was to fine-tune the presentation the night before, which would have involved transferring it from my laptop to my desktop either by email or by flash drive. Unfortunately, I spent the night before the meeting at the emergency room, being treated for a bad allergic reaction to a bee sting. (It really wasn't my week...) So, I got no work done, and never transferred the file between computers, and thus had only one copy. Which I deleted. As Charlie Brown would say, "AAUGH!"

Fortunately, my PI and labmates were understanding of the mishap, although they did scold me for not making a backup copy. (I know. I know!) I gave lab meeting as a "chalk talk" instead, drawing diagrams on the board when I needed to, but mostly just talking and glancing at some hastily-scribbled notes to remind me of the details. Despite the awkwardness, this forced me to really explain the reasoning behind what I did and summarize the relevant results, rather than being like, "Look! I ran a gel! And here it is!" Hopefully the lab doesn't think I'm too much of an airhead, despite my technical difficulties.

More lab foibles: We transformed some bacteria last week. After spending a while cloning and sub-cloning, we finally had some DNA plasmids that we wanted to make in large quantities. This is done by tricking bacteria into taking up the DNA, i.e. transforming them, and then growing cultures of the bacteria so we can extract plasmid DNA from them once they've been fruitful and multiplied. I'd done transformations before using heat shock (putting the bacteria into a hot water bath for a minute or two), but this protocol called for electroporation (zapping the bacteria with electricity). No one in the lab had used the electroporation machine before, and our first run did not go too well. In fact, a very impressive spark was produced, along with a crackling sound and a strong aroma of barbecued competent cells. After daring everyone in the lab to smell the tube, we adjusted the settings on the machine and it seemed to be okay after that. No more fireworks occurred, and my transformed bacteria grew on some ampicillin plates, so it worked for at least some of them. (The plasmid we put into the bacteria contains an ampicillin resistance gene, so we know that successfully transformed bacteria will grow even when treated with that antibiotic. The untransformed bacteria die when you put them in ampicillin.) Now I've selected transformed bacterial colonies and grown enough of them to make DNA preps. We'll have to do some more analysis to see which colonies have the plasmids we want -- some of them might have recombined in weird ways, which isn't desirable. Progress marches on, with tiny little steps. Someday we'll make these into a lentivirus, honest.

I also had my last class of the semester last week. (We had our final session of my graduate seminar at a pub. It was quite grueling.) This August, I'll be taking my written qualifying exam. Until I pass that, I don't want to jinx myself by updating my Blogger profile from "first-year" to "second-year." But, I am very happy to be one year closer to a PhD. Next year I'll be working on dissertation research, TA assignments, grant-writing, and serving as a student committee representative for the Graduates in Neuroscience organization. I'm excited!

Vaccine Dinner Club: Simultaneous Administration of Vaccines

Wednesday night, I attended a meeting of the Vaccine Dinner Club at the Emory University School of Medicine. This group of clinicians, researchers, policy makers, and other interested parties meets monthly in Atlanta to discuss issues related to vaccines. This is sort of tangential to my own research, but the May meeting theme (Simultaneous Administration of Vaccines: How Many is Too Many?) is a hot topic right now, so I was interested in hearing what the speakers had to say. Plus, they serve food and drinks. If you're in Atlanta and think this sounds like a good deal, I encourage you to click the link and learn more about the club -- it's free to join and to attend the nosh-filled meetings.

I must admit that I am a bit hesitant to make this post, because so many people have such strong opinions about immunizations these days, but I think it's important to share the kinds of things that experts talk about when they get together. This contributes to public education on the subject of vaccines, and it also lets people know that their concerns are being heard, and that medical professionals are trying to understand and alleviate them.

This month we heard from Andrew Kroger, MD, MPH, and Melinda Wharton, MD, MPH, both from the Center for Disease Control's National Center for Immunization and Respiratory Diseases. I did my best to takes notes during both talks, and I'll try to hit the key points in this post, but you should know that I'm not a doctor or a vaccine expert, so my interpretation of these lectures may be inaccurate. If I write something in error, please blame me and not Dr. Kroger or Dr. Wharton.

Dr. Kroger, a medical health educator, gave the first talk. He discussed the current vaccination recommendations established by the CDC, which cover a total of 17 different vaccines administered to children, adolescents, and adults. Because we now have the ability to protect against more diseases with vaccinations, people are getting more shots. Simultaneous administration of vaccines (which is defined as getting two or more shots during the same doctor's visit; not to be confused with combination vaccines, which consist of multiple antigens in a single syringe) is a strategy used by health care providers to ensure that people receive all of the recommended immunizations within the appropriate time frame. Dr. Kroger cited several studies indicating that giving two vaccines in one office visit is just as effective as giving them separately. In fact, it can sometimes be more effective. If vaccines are not administered simultaneously, they should be spaced out by at least one month. Giving two vaccines within the same month can lead to reduced efficacy of the second vaccine. Giving both shots at once, however, induces an immune response that is just as effective as the response to individual vaccines that have been spaced further apart. The only vaccines for which simultaneous administration is not recommended are varicella and smallpox. This is because doctors need to be able differentiate between the two diseases in the event of an adverse reaction. That is, if the shot gives you the pox (this is extremely uncommon, but possible), doctors want to be sure they know which pox you have, so they can treat it properly.

The CDC has been recommending simultaneous administration of vaccines for many years, but has added eight new vaccines to the recommended schedule since 1994. Dr. Kroger investigated simultaneous administration of these newer vaccines to see whether the historical data held true for them as well. You can find a wealth of information on this subject at the FDA's vaccine website. Dr. Kroger spent quite a while summarizing studies on new vaccines, and concluded that while we haven't rigorously tested every possible combination of shots (there are a lot of them), none of the studies conducted thus far present a contraindication for simultaneous administration.

Dr. Kroger went on to a bit of basic immunology, explaining why it's possible for our immune system to handle simultaneous vaccines. It comes down to a bit of math. Seroprotection (the goal of vaccination) is defined by antibody levels of 10 ng/mL of blood. To achieve this within a week of vaccination, only one B cell clone is needed per immune epitope for each mL of blood in the body. A typical vaccine contains about 100 epitopes for each disease that it protects against, so we need 100 B cells/mL of blood to achieve seroprotection for a disease within one week of vaccination. One mL of blood normally contains about 10 million B cells, so a healthy immune system should easily be able to handle several vaccines at once while still responding to other antigens that the body encounters.

These data left me convinced that simultaneous vaccines are effective. But are they safe? Fewer studies have been done on safety than efficacy, and there are data that indicate certain vaccine combinations can lead to a higher risk of side effects than individual administrations. Dr. Wharton, the second speaker, discussed these issues during her talk. While Googling for some of the studies she mentioned, I found this PowerPoint presentation, which is similar to the one she gave at the Vaccine Dinner Club. I suggest downloading it and looking through the slides if you're interested in this subject, as she covers more material relevant to common concerns about vaccine safety than I could possibly summarize here. The take-home message seems to be that while simultaneous vaccines may increase the incidence of side effects, these side effects are already rare, such that any additional risk caused by simultaneous administration doesn't make a huge difference. (Studies cited in Dr. Wharton's talk involved the combination of MMR and varicella vaccines. One study tracked over 531,000 children, divided into cohorts who received single, simultaneous, or combination vaccines. Only about 1% of the children across all groups were brought back to their doctor for fever symptoms after vaccination. The incidence of more severe complications, like febrile seizures, was much lower -- about 0.1%, with similar rates for sequential and simultaneous vaccines.) Parents who wish to take every possible precaution can ask their pediatrician about spacing vaccines out over several months, but this can present practical concerns for people who must take time off from work, arrange care for other children, and pay office fees during each trip to the doctor. If these issues create enough of a stumbling block, children might not be brought back for subsequent vaccines in a timely manner. And it's crucial to have children vaccinated within the recommended age range to protect them as early as possible from potentially fatal diseases. Delaying vaccination over concerns about simultaneous administration also delays protection against those diseases, so the relative risks must be balanced. While it is important to acknowledge that some children suffer unpredictable complications from vaccines, the optimal way to understand and prevent those complications is to come up with better screens for risk factors, not to simply stop vaccinating.

I thought Dr. Wharton brought up some excellent points during her discussion of risk communication with patients and parents on the subject of vaccines. Concerned parents will often reject expert opinions in favor of advice from individuals that they feel to be more caring and compassionate, even if that advice is misguided or inaccurate. It's important for healthcare providers to communicate effectively with parents, letting them know that not only do scientific studies support the importance, efficacy, and safety of vaccines for all children, but that adhering to a vaccination schedule is the best way for them to protect their child. Doctors with children of their own should be candid about their decision to vaccinate, and all physicians should make it clear that providing preventative medical care is the role of every loving mother or father. One survey of parents found that the most common concern with simultaneous vaccination comes not from spurious connections drawn between vaccines and conditions like autism, but from the additional pain and stress that children experience when receiving multiple shots. A kinder bedside manner can help with this problem, although it may be impossible to keep a child completely calm when he sees a needle coming.

The internet is teeming with discussions of vaccine safety and the dangers of the anti-vaccination movement, of course. For further reading on the subject, you can check out this article in the New England Journal of Medicine on vaccine refusal, Chris Mooney's summary of the vaccine/autism controversy in the June issue of Discover Magazine, and med student blogger Whitecoat Tales' Hard Conversations: Vaccines and Autism series.

Saturday, May 2, 2009

Rotation #3 Diary: Week 7

I think I lost count of rotation weeks somewhere back there... for consistency, I'll call this Week 7, but I actually have two more weeks left. Admission to PhD program in neuroscience? Check. Counting to single-digit numbers? Fail.

The sub-cloning project continues. I had one restriction enzyme digest that didn't work, so we chose a new enzyme for those samples and seemed to get it on the second try. I cut and blunted and cut and gel-purified, in between studying for my final exam and finishing my group project. These experiments lend themselves well to a crazed finals week, as I could set up a lot of reactions in an hour of lab time and just let them go overnight. Running the gels takes a while, though, as I'm working with some pretty big chunks of DNA and they are slow to separate, even on a low-density agarose gel. Outside the lab, I was proud of myself for coming up with a study schedule and sticking to it. I outlined all of the lectures, made flash cards, and got together with classmates to review material, without last-minute cramming. After taking the test on Thursday, I felt pretty good about it (although one professor did ask some really picky questions... we'll see).

I'm still waiting on the sequencing results from the mystery mice. Actually, I haven't even sent the samples to the sequencing facility yet. For some reason it's taking a long time to get purchases approved by the person managing the grant that funds these experiments, so every time I want to get something sequenced, there's a turnaround time of sometimes three days before I get the purchase order. By the time we got the funds approved for this batch, it was Friday, and no one at the sequencing facility would be there to receive our samples on Saturday, so they'll sit in the freezer this weekend and go out on Monday. One solution to this issue would be to put in a request for a really huge purchase order and reuse it many times over a couple of months, but you're technically not supposed to do this. You're just supposed to wait. Red tape is everywhere, even in the lab. Oh well.

Practiced some more stereotaxic surgeries this week. The concept seems pretty simple: anesthetize the animal, stabilize the head in the stereotax, do the procedure (in this case, we expose the skull, drill a tiny hole in it, and insert a needle into the brain to deliver our treatment), close the animal up, and let it recover (including medication for any post-operative pain). In practice, it's hard to get the head position right and adjust the arm that holds the needle/syringe into the right spot. I was constantly worried that I would do something wrong and hurt the mouse, although it was under anesthesia and felt no pain. We didn't do any survival surgeries; we sacrificed the animals immediately after injection and dissected them to check that the injection site was in the right place. Although this doesn't seem very nice to the mice, it's important to optimize the procedure before conducting the real experiments, to ensure consistent and meaningful results. This cuts down on animal use, overall. The dissection looked pretty good, so this weekend a postdoc from the lab will be doing the real experiment with some Cre virus. Once the animals recover and the virus has a chance to do its thing, we could see some interesting results. Probably this will take more time than I have left in my rotation, but I feel invested enough in it that I want to know what happens.

Although I'm just a trainee, I felt useful this week. A labmate presented some data a while ago and mentioned a weird result that might indicate a flaw with either the transgenic mouse line or with the fundamental assumption behind the experiment. When this happens, we tend to assume a technical problem first -- the mouse isn't what you think he is. Checking on this is difficult, though, because we don't have good antibodies for the protein this mouse is supposed to lack, and it's a conditional knockout, so some of its cells do have the gene and some don't (making it hard to just isolate mRNA and look for gene expression at that level). In situ hybridizations to look at the gene expression would work, but it can be hard to look at individual cells this way. Anyway, I read some papers for my last rotation where a group used laser microdissection to isolate individual cells from a brain slice, purified RNA, and then did RT-PCR to figure out if the cells were expressing certain genes of interest. I looked around and saw that Emory has a core facility that will do laser microdissection for a reasonable rate, or train lab members in the technique so they can do it themselves. We can already do RT-PCR in the lab, too. So I suggested that we try that, rather than in situs, and my labmate seemed really into the idea. I think he was already exploring this possibility with our PI, but they didn't know about the core facility. So I felt all smart and helpful for mentioning it. It's been cool to realize just how much I'm learning in grad school, and that I'm coming up with good ideas for experiments on my own. My education is working!

Now that finals are done, I have a lot of free time in my schedule for these last few rotation weeks. Then I'll be presenting my results in lab meeting, writing up a report, and choosing which lab I want to join. I might take a little summer vacation first, though...