The Effect of Spinal Cord Severing in Zebrafish
Blue Expo

ABSTRACT
Previous research indicates that nervous and cardiac tissue regeneration occurs in zebrafish because they lack some inhibitory characteristics found in mammals. The purpose of this project was to observe spinal cord regeneration in zebrafish of various stages of development. Zebrafish spinal cords were severed and the surviving groups were observed for visible signs of neural regeneration at that site. Revascularization was visible in zebrafish from later trials but no neural regeneration was observed in this experiment.

INTRODUCTION
The purpose of this lab was to observe the effects of spinal cord injury inflicted upon young, developing zebrafish and determine if any of spinal cord regeneration occurs. The hypothesis was that the spinal cords of the zebrafish would regenerate to some degree after being experimentally severed. Previous research has found that zebrafish neural and cardiac tissue regeneration does occur given the correct procedures and conditions. In a study on zebrafish heart regeneration by Poss, Wilson, and Keating, roughly 20% of an adult zebrafish heart is surgically removed and heart tissue regenerates through cardiomyocyte proliferation. After a period of sixty days, the regenerated portion is histologically indistinguishable from the original cardiac tissue. (Poss, Wilson, and Keating, 2002)

Another study performed by Fetcho and colleages found that some Mauthner axons in zebrafish in the presence of the second messenger, cyclic adenosine monophosphate (cAMP), begin regenerating to a small extent within two days of the zebrafish spinal cord being severed. (Bhatt, Otto, Depoister, and Fetcho 2004) To test the hypothesis that neural tissue regeneration occurs in zebrafish, they were physically immobilized and their spinal cords were carefully severed. Survival rates were recorded for each severing trial. Zebrafish surviving longer than one day were observed microscopically for evidence of neural regeneration.

METHODS

Spinal Cord Severing Procedure
The zebrafish used were from 3-12 days old were pipetted out of the 250mL beaker that they were hatched in and transferred to test tubes in 1-2 drops of water. A microwave was used to melt agar to a warm liquid, 3-4 drops of which was then added to the zebrafish test tubes and gently swirled. The zebrafish were then drawn up in 1-2 drops of the agar/water mixture and transferred onto a slide. Once on the slide, the agar cooled slightly forming a layer around the zebrafish with gel-like consistency. The purpose of the agar in this experiment was to physically restricted the movement of the zebrafish without knocking them out or harming them. With the zebrafish immobilized on a slide, spinal cord severing could be performed under the stereoscope with a steady hand using an X-acto knife blade. The slide could be rotated under the scope to achieve the best angle of approach for the dominant hand holding the blade. The goal of this step was to sever the spinal cord that is ventral to the thin black line of pigment along the back of the zebrafish in one careful and steady movement, taking care not to harm the underlying transparent notochord and adversely affect development. Early in the experiment a thin layer of water was not added to the slide on top of the agar layer containing the zebrafish during the spinal cord severing but later in the experiment a thin layer of calcium water (14g Ca/ 100mL water) was added to limit the exposure of the young zebrafish to air. Post-procedural zebrafish were pipetted using a small amount of water into a small plastic dish containing a thin layer of water. Zebrafish early in the experiment (trials 1-4) were put in normal fish water and zebrafish late in the experiment (trials 5-7) were put in calcium water for a half hour after spinal cord severing and then transferred to normal fish water. Increased calcium ion concentration water was used because calcium ions are involved in the coupling of filopodia to the actin filaments during growth cone movement. Filopodia extend or retract in response to chemotropic molecules binding to the membrane receptor of a neurite. (Matthews, 2001) Although zebrafish cannot be left in calcium water for an extended period of time because it does interesting things to their development. Post-procedural Zebrafish were fed small amounts of food after age four days when they begin feeding.

Observation and Data Collection
Post-procedural zebrafish were observed for survival rates and level of activity. Placing a pipet in the water near the zebrafish and observing whether or not they attempt to swim away is one indication of survival of the spinal cord severing. If zebrafish did not attempt to swim away then they could be observed in the plastic dish of water under the stereoscope at high power for indications of survival. The heart pulsating or sometimes light reflecting on the moving blood cells of the vascular system and revealing blood flow could be used to indicate survival. Zebrafish not surviving the spinal cord severing procedure would not have any of these indications of survival and signs of decay were obvious within one day. The hyperosmotic body fluids of the freshwater zebrafish cause continuous water gain. When zebrafish are deceased the gills and kidneys do not maintain the necessary ion/water balance with the surrounding environment causing excess water to enter the cells and often cell lysis. (Ricklefs2007, Marieb 2004) Zebrafish surviving more than one day after spinal cord severing were monitored for normality of swimming movements and microscopically for tissue regeneration.

RESULTS
Early in the experiment survival rates after 48 hours of post-procedural zebrafish were zero so the methods of capturing the zebrafish, transferring them to and from the slide, and spinal cord severing were examined for flaw. The discovery was made that not adding a thin layer of water on the slide over the agar that immobilized the zebrafish allows increased air exposure and can damage the yolk of the developing zebrafish. Survival rates after 48 hrs. increased from zero to 60-67% after the methods were changed to add a thin layer of calcium water over the agar during the spinal cord severing.

Trial 1
Date of Trial: 11/6/07
Number of Zebrafish in Trial: 15
Age of Zebrafish During Trial: 4 days
Zebrafish surviving initially: 2
Zebrafish surviving after 24 hrs: 2
Zebrafish surviving after 48 hrs: 0
Survival Rate after 48 hrs: 0%
Longest post-procedural survival (days): 1 day

Trial 2
Date of Trial: 11/11/07
Number of Zebrafish in Trial: 15
Age of Zebrafish During Trial: 6 days
Zebrafish surviving initially: 2
Zebrafish surviving after 24 hrs: 0
Zebrafish surviving after 48 hrs: 0
Survival Rate after 48 hrs: 0%
Longest post-procedural survival (days): 0 days

Trial 3
Date of Trial: 11/14/07
Number of Zebrafish in Trial: 20
Age of Zebrafish During Trial: 9 days
Zebrafish surviving initially: 11
Zebrafish surviving after 24 hrs: 0
Zebrafish surviving after 48 hrs: 0
Survival Rate after 48 hrs: 0%
Longest post-procedural survival (days): 0 days

Trial 4
Date of Trial: 11/17/07
Number of Zebrafish in Trial: 15
Age of Zebrafish During Trial: 12 days
Zebrafish surviving initially: 2
Zebrafish surviving after 24 hrs: 2
Zebrafish surviving after 48 hrs: 0
Survival Rate after 48 hrs: 0%
Longest post-procedural survival (days): 1 day

Trial 5
Date of Trial: 12/2/07
Number of Zebrafish in Trial: 15
Age of Zebrafish During Trial: 4 days
Zebrafish surviving initially: 11
Zebrafish surviving after 24 hrs: 9
Zebrafish surviving after 48 hrs: 9
Survival Rate after 48 hrs: 60%
Longest post-procedural survival (days): 6 days

Trial 6
Date of Trial: 12/4/07
Number of Zebrafish in Trial: 15
Age of Zebrafish During Trial: 6 days
Zebrafish surviving initially: 13
Zebrafish surviving after 24 hrs: 12
Zebrafish surviving after 48 hrs: 10
Survival Rate after 48 hrs: 67%
Longest post-procedural survival (days): 10 days

Trial 7
Date of Trial: 12/6/07
Number of Zebrafish in Trial: 15
Age of Zebrafish During Trial: 8 days
Zebrafish surviving initially: 11
Zebrafish surviving after 24 hrs: 11
Zebrafish surviving after 48 hrs: 9
Survival Rate after 48 hrs: 60 %
Longest post-procedural survival (days): 8 days

DISCUSSION
The hypothesis of this project was that spinal cord neurons of the Zebrafish would regenerate to some degree after being experimentally severed. Previous research has found that neural regeneration occurs in some zebrafish central nervous system (CNS) neurons provided the chemical environment in the tissue is facilitating for neural growth. Human neural regeneration in the CNS does not occur because of chemical inhibitors in these tissues and characteristics of the nervous tissue that disable it from successfully regenerating. The affects of cyclic adenosine monophosphate (cAMP) on regeneration in 5-7 day old zebrafish of the Mauthner cell, a myelinated neuron that functions in escape behavior, has also been studied. Fetcho and colleagues found that with the addition of cAMP, about a third of Mauthner axons in zebrafish display some degree of regeneration within two days after being severed along with part of the spinal cord. These partially regenerating Mauthner axons typically deviate away from the spinal cord injury rather than growing through indicating that if spinal cord regeneration occurs the severed region will not appear the same histologically as before the procedure. (Bhatt, Otto, Depoister, and Fetcho 2004) There was no observed zebrafish spinal cord regeneration throughout this project, however, there was observed revascularization at the sight of spinal cord severing in two zebrafish from trial 6 and one zebrafish from trial 7. The blood flow, visible under the microscope, travelled ventrally toward the notochord around the severed region . Although it is unlikely, especially since cAMP was not used in this experiment, undetected spinal cord regeneration may have occurred in zebrafish from the later trials in which water with increased calcium ion concentration was used.

Severing of the notochord was avoided because extirpation of these cells adversely affects zebrafish development. The research done by Greenspoon and colleagues, in which the notochord of zebrafish embryos were ablated before axon generation using lasers, resulted in development errors of growth cones. They discovered that the accuracy of growth cones at the ventral midline of the spinal cord is dependent on both the floor plate (a group of cells at the ventral midline) and the notochord. Growth cones remain accurate either without the floor plate or the notochord but not without both. (Greenspoon, Patel, Hashmi, Bernhardt, and Kuwada 1995) Given that exacto blades are far less accurate than lasers, caution was taken during spinal cord severing not to make incisions too deep. This would likely damage the nerve tissue that growth cones depend on during development. Zebrafish surviving more than one day after spinal cord severing often had decreased tail functionality in swimming.

Diminished immune systems or disease may have been a factor affecting zebrafish survival in this experiment. The X-acto knife was washed before each trial but was not sterilized so the zebrafish could possibly have been infected with a disease during the spinal cord severing procedure. Deceased fish were removed from containers upon their discovery. Aside from the beaker that the zebrafish hatched in, the number of fish per container throughout the experiment did not exceed fifteen fish so epidemic disease is not probable.

The limited precision of the X-acto blade under the stereoscope at high power during the spinal cord severing procedure and the deplorable cooperation of the zebrafish often resulted in deeper incisions and greater injury than intended. This project did not yield neural regeneration, however, zebrafish tissue regeneration remains a beneficial area of research. Zebrafish heart regeneration research found that if roughly 20% of an adult zebrafish heart is surgically removed, heart tissue regenerates through cardiomyocyte proliferation and after a period of sixty days, the regenerated portion is histologically indistinguishable from the original cardiac tissue (Poss, Wilson, Keating). A subsequent study lead by Keating has found that signaling of the PDGF gene induces DNA synthesis in the cells and is required for cardiomyocyte proliferation during heart regeneration. (Gross 2006) Further understanding the underlying processes in both neural and cardiac zebrafish tissue regeneration could lead to knowledge of how to stimulate these processes in mammals.

LITERATURE CITED

1) Kenneth D. Poss, Lindsay G. Wilson, Mark T. Keating. 2002. Heart Regeneration in Zebrafish. Science. 13 December 2002: Vol. 298. no. 5601, pp. 2188 – 2190

2) Ricklefs, R.E. 2007. The Economy of Nature 5th ed. W.H. Freeman and CO. New York,
NY. pp. 180-198. 3) Marieb, Elaine N. 2004. Human Anatomy & Physiology 6th ed. Pearson Benjamin Cummings. San Francisco, CA. pp

4) Bhatt, H., Otto, S.J., Depoister, B., Fetcho, J.R. Cyclic AMP-Induced Repair of Zebrafish Spinal Circuits. Science. 9 July 2004: Vol. 305. no. 5681, pp. 254 – 258

5) S Greenspoon, CK Patel, S Hashmi, RR Bernhardt and JY Kuwada. 1995. The notochord and floor plate guide growth cones in the zebrafish spinal cord. Journal of Neuroscience, Vol 15, 5956-5965

6) Gross L. Regenerating Zebrafish Hearts Reveal the Molecular Agents of Repair. 2006. PloS Biol 4(8): e281 doi:10.1371/journal.pbio.0040281

7) Matthews, G. Neurobiology. Molecules, Cells, and Systems. Second Edition. Blackwell Science. 2001

Here’s another interesting question from our most recent neurobiology exam. With some luck PZ won’t get irritated that I keep recycling my work. This paper was a bit of a brain thumper but also very interesting after deciphering what it’s talking about.

3) Summarize this paper and describe both the neural circuit and the genes underlying this particular rhythm.
Stoleru D, Peng Y, Agosta J, Rosbash M (2004). Coupled oscillators control morning
and evening locomotor behavior of Drosophila. Nature 431:862-868

The roughly one hundred bilaterally arranged circadian clock neurons in adult fly brains occur in six groups: dorsal neurons (DN1, DN2, DN3), dorsal lateral neurons (LNdS), and the PDF neuropeptide expressing small and large ventral lateral neurons (LNvS). Extirpation via proapoptotic genes was used to assess that D.melanogaster lacking LNvS in natural light/dark conditions displayed little change however in continuously dark environments yield arrhythmicity. Time intervals in the light/dark experiment were determined using Zeitgeber time in which lights on (sunrise) corresponds with ZT0 and lights off (sunset) corresponds with ZT12. Although the two LNvS cell groups have an imperative role in rhythmic gene expression, neurons expressing circadian photoreceptor cyrptochrome (cry) genes were also found to assist rhythmicity in natural light/dark conditions.

Green fluorescent protein reporter was used to stain the six clock neuron groups, determining that the cry-GAL4 driver, which facilitates cry gene expression, is present in all dorsal and ventral lateral neurons (LNdS and LNvS) and in two dorsal neurons (DN1). The proapoptotic gene hid was used to excise the cry gene in LNdS and LNvS generating cry-GAL4;UAS-hid flies that were arrhythmic in both natural light/dark and continuously dark environments. The dorsal neuron groups (DN1, DN2, DN3) are mostly unaffected by hid expression meaning that because cry-GAL4;UAS-hid flies are arrhythmic in both environments, these neuron groups are incapable of maintaining circadian rhythms independently.

Crossing D.melanogaster exhibiting the Pdf-GAL80 gene, which represses GAL4-mediated transcriptional activity, with flies exhibiting green fluorescent protein in circadian neuron groups via Pdf-GAL4 and cry-GAL4 drivers yielded flies without green fluorescent protein. This means that the cry and Pdf promotors, segments of DNA that control gene expression, coupled with GAL80 genes override and prevent their corresponding GAL4 drivers from transcribing. With the crossing of cry-GAL4 driver and Pdf-GAL80 repressor, and other mixed crosses, green fluorescent protein was observed only in some circadian neuron groups.

Crossing Pdf-GAL80 with cry-GAL4;UAS-hid allowed researchers to determine the effects of extirpating PDF+, CRY+PDF-, and CRY+ neurons on D.melonogaster circadian rhythm. They found that flies extirpated of CRY+ neurons were phenotypically arrhythmic and flies extirpated of PDF+ neurons had diminished morning lights-on anticipation with normal evening lights-off anticipation. Flies extirpated of CRY+PDF- had diminished evening lights-off anticipation and normal morning lights-on anticipation. These flies maintained a circadian rhythm in continuous darkness indistinguishable from wild type flies based unimodally on the morning oscillator. The phenotypes of these three strains of D.melonogaster are not affected by whether the environment is light and dark or continuously dark meaning that the oscillators are not driven by light. Using deductive logic, the researchers concluded that PDF+ neurons correspond to lights-on behavior and oscillate independently of CRY+PDF- neurons, which correspond to lights-off behavior.

Green fluorescent protein techniques for visualizing neurons confirm that the CRY+PDF- and PDF+ oscillators are coupled through PDF- axonal processes that protrude from the LNd neuron group into the LNv region. Immunostaining visualization techniques reveal that PDF neuropeptide travels in the opposite direction that the PDF- axons extend, that is, from the LNv to LNd neuron group. PDF neuropeptides function to coordinate the lights-on anticipation behaviors in the morning with the independently oscillating lights-off anticipation behaviors in the evening.

References:
Stoleru D, Peng Y, Agosta J, Rosbash M (2004). Coupled oscillators control morning
and evening locomotor behavior of Drosophila. Nature 431:862-868

This week is the second to last week of the semester before finals and everything is coming down to the wire, including my neurobio lab project. PZ was so kind as to come in and help me out this past Sunday morning; the morning after the blizzard had quieted leaving everything covered in various quantities of snow. In going over my methods we found that I wasn’t adding a drop or two of water on top of the auger layer with the immobilized zebrafish. The reason this is important is that so after the spinal cord severing is accomplished, the auger layer is separated allowing water to surround the fish immediately and preventing air exposure. The fish can then be pipetted up and put into a dish of water for observations. PZ also suggested using water with an increased concentration of calcium (14g/100mL) to facilitate better fish recovery. The fish should not be left however in the calcium water for an extended period of time because it can adversely affect development.

Repeating my methods and taking into practice the slight changes that PZ recommended, I found that after one day, four of eleven fish were still alive! After slicing up more than sixty fish with a 100% mortality rate after one day and wondering what on earth I could have been doing wrong, I was ecstatic. It’s unfortunate that this success has come so late in the game and the writeup for this project won’t show much for results other than how not to butcher zebrafish. I have learned quite a bit though about the interesting techniques I’ve been using and also about the differences in zebrafish at various stages of development. So with that, back to the lab I go to continue working with zebrafish.

The seed of this mornings discussion in neurobiology was “Time, Love, Memory” by Jonathan Wiener. As has been the norm in past weeks we met in the on campus cafe bringing along with us four insightful questions each to keep the discussion rolling along throughout the hour. Wiener describes later in his book (p192) the three necessary components of living clocks. Living clocks are the basis of circadian rhythms and must have an input pathway so that the clock can be reset by the sunrise and sunset. A good example of why this is important is that humans actually have a twenty-five hour clock that resets itself everyday to correlate to the actual day length of twenty-four hours (23 hours, 56 minutes, and 4.1 seconds for any physicists reading this). People who are blind or people who are not exposed to the sun at all will exhibit a twenty-five hour clock, out of synchronization with the earth’s rotation.

So my question was about animals that live near the poles. How do polar bears or lynxes reset their clocks in the arctic summer when the sun doesn’t set? Some thoughts were that perhaps the living clocks are reset by magnetism but quickly realized that there is no shift of magnetism that corresponds to the length of a day. Another thought was that if it’s always light out, does it matter when the polar bear sleeps? The polar bear could have a period of activity, followed by a period of decreasing activity, and then rest and sleep. Lynxes often hunt at night and rest during the day but if it’s always light out does their clock remain synchronized with the earth’s rotation? PZ mentioned there isn’t much research pertaining to this but If anyone knows of any interesting papers that would enlighten this topic post them up.

References: Jonathan Wiener. “Time, Love, Memory.” Vintage Books, A Division of Random House, Inc. New York. 1999.

As I wrote about before, my semester lab project for neurobiology has to do with regeneration. The idea is to damage the spinal cord and observe wonderful regeneration. This proposal was based on some articles I read about regeneration of zebrafish hearts, fins, tails, etc. Unfortunately I haven’t had much luck so far.

Last week, armed with an exacto knife, I performed my first round of spinal cord butchering on fifteen zebrafish that were only a few days old. The zebrafish are captured with a glass pipet and then immobilized using auger that’s just warm enough to be in liquid phase. They are then mounted on a slide under the stereoscope. I quickly discovered many faults in my methods, one of which being that I captured the fish in too many drops of water so when the auger was added in a test tube, the fish weren’t completely immobilized. I would then carefully approach a fish with the exacto knife on the slide under the scope and it would turn into a bucking bronco. Eventually I perfected the art of capturing the few day old zebrafish with the pipet and putting them in test tubes in only one drop of water, thus partially solving the immobilization issue.

The second problem I encountered is that there is nothing exact about an exacto knife under a stereoscope. Accomplishing this spinal cord severing is much like peeling an orange with a baseball bat in that it’s extremely difficult without making a mess. Even with the fish immobilized the tail doesn’t really stay put when pressure is applied with the seemingly crowbar sized exacto knife. The key to this dilemma, although I have yet to master it, is probably making the layer of auger on the slide as thin as humanly possible so that there isn’t as much room for movement.

All the zebrafish from my first attempt died. Two of them were alive for a day or so but barely. I did another round of zebrafish butchering with fifteen more fish yesterday (yes I enjoy spending my Sunday afternoon in the neurolab) and from what I could tell, all but two of them died immediately. I’ll have to see if the elite two are still swimming around today but if not, I’ll try yet another round of fifteen and see how it goes. If anyone has any ideas for instruments or methods that could improve my success, feel free to insert a comment.

Synesthesia is going to be the discussion topic for our upcoming neuroslam in two weeks. Synesthesia is the rare ability of a select few individuals to see numbers as colors or as in the article that I’m preparing to discuss (Hubbard 1996), experience varying degrees of light and dark as melodic intervals. The observed pattern is that individuals experience lower pitches or descending melodic intervals in correlation with darker stimuli and higher pitches or ascending melodic intervals in correlation with lighter stimuli. The important detail about synesthesia is that individuals experience it involuntarily whereas most individuals without synesthesia can choose to consider a set of stimuli using a secondary sense that they normally wouldn’t.

One of the experiments discussed in this article was conducted with undergraduate students coaxed into participating with the offer of some credit for an intro to psychology course. The students were placed in front of an Apple RGB color monitor and grey squares of differing light intensities were presented in conjunction with a perfect fifth for four seconds. Eight perfect fifths were used, each beginning on a different tonic and thus each having a unique frequency. The squares were presented on either a white or black background. The students then rated how similar the square and the interval were on a scale with one as the least and nine as the most. One of the questions considered with this experiment is the effect of contrast between the background and the grey squares on perception.

A second experiment was set up similar to the first experiment except that students (who had not participated in the first experiment) were presented with an interval at one of the selected frequencies and asked to choose among several light intensities of grey which correlated best. Correlating one perfect fifth to a light intensity that was presented with multiple light intensity options successfully diminished (no pun intended) the effects of background contrast on perception.

I thought of some questions when I was reading this article and then afterward studying for music theory. Are there individuals who experience synesthesia such that they correlate varying degrees and intensities of lightness with more complex types of music intervals? Do minor or diminished intervals correlate to a different light intensity than major or augmented intervals? What about different intervals of the same quality? Do ascending minor sixths correlate to a different light intensity than ascending minor thirds? If an individual with true synesthesia enters a concert hall do they experience sensory overload? (just kidding) I’m sure one of us neurobio students will post about neuroslam in a couple weeks to fill everyone in on our discussions but until then there is a lot of good reading on the subject.

References:

Timothy L. Hubbard. “Synesthesia-like mappings of lighness, pitch, and melodic interval.” American Journal of Psychology. 1996. v109n2: p219

The beginning of this week was fall break at our college campus. We had the weekend off as well as Monday and Tuesday. Since I had been planning to return home to northern Minnesota for the first time since moving down to west central Minnesota in August, I decided to take Thursday and Friday off also. The few days I spent away from this desolate prairie wasteland and back among the conifers and lakes were phenomenally enjoyable.

This is my first year of college away from home and a long way from home it is. I remember the first few weeks I was down here, only vaguely though, a lot of adapting has taken place since then. The three-day dragged out orientation process for freshmen and transfer students at the end of August was intensely boring. I had been informed by mail that my presence was required but it would have been great had I known what it entailed and that I very well could have gotten away with not attending (this sounds negative, I know, but I’m sure some of you can relate). I remember the immense amount of time and effort it took to meet new people and figure out who to make friends with. Luckily the homework load had not picked up yet and there was plenty of free time to devote to this. Did I mention I had never bought my own groceries before coming to college? The first night I went to buy food I had no idea what I needed to sustain myself in a semi-healthy manner. The first five items I put in my basket had something to do with hotdogs. I’ve since learned a few things about grocery shopping and cooking. Although the first weeks were uncomfortable and sometimes frustrating, it was well worth the effort to take them on.

The drive home was long and rainy. There were a few deer that crossed as I approached them but staying attentive kept my vehicle intact. My lily plant sat quietly on the front passenger floor and when I inadvertently opened the glove box on its head I caught myself apologizing. Talking to a plant seemed odd to me so I ignored it and its personal temperature preference for the rest of the trip.

When I finally got home just before midnight, I sat in my living room with a bowl of soup, quietly but excitedly looking at everything I hadn’t seen for a while. The pictures hanging on the wall, the bookshelf filled with various things, the scent of my dad’s cooking brought forgotten memories rushing back to me. The plant atop the bookshelf with vines that had hung down half way in August had now reached the floor. My dad, who was sitting in his recliner on the opposite side of the room with his own bowl of soup, talked about things that had happened while I was gone, the new addition on the back of the garage, why our silverware was different, and how my aunts and uncles are doing.

The weekend flew by quickly and before I knew it I was sitting in neurobio again yesterday morning (we have wednesday discussions in the cafe now so PZ can get coffee). The time I spent among the seemingly infinite number of trees and lakes in northern Minnesota was enough to keep me going until I can go back again. For now it’s just good to know that everything I call home and everyone who means the most to me are still up there, safe and sound.

I noticed that PZ posted one of our take-home exam questions on Pharyngula and so I decided to make an entry with my answer (I okayed this with PZ first although he did warn me of the certainty of harsh reader criticism). The question referred us to a recent article in Nature about TRPV1 ion channels and asked us to describe TRPV1 ion channels and the testing that was done on them.

The transient receptor potential cation channel (TRPV1), also referred to as vanilloid receptor subtype 1, is a ligand-gated cation channel (2). This means that the channel contains organic molecules that can form covalent bonds with positive ions and is thus operated chemically. TRPV1 ion channels are non-specific and can be found on TRPV1 nociceptor (pain sensing) neurons in the central nervous system and peripheral nervous system. It may be related to thermal hyperalgesia (abnormally increased sense of pain) in both regions (2). The TRVP1 ion channel The opening in the TRPV1 ion channel was determined experimentally to be large enough to pass a 452 Da (1 Dalton = 1.657×10-24 g) dye molecule through (1). The TRPV1 ion channel, when opened by the proper agnostic, can allow anesthetic molecules to be introduced into nociceptic neurons, making it an important channel some regional anesthesics.

Most anesthetics are hydrophobic, cell membrane permeable, and function by blocking sodium ion channels on the inside of the cell. This blocks sensory nerves as well as motor and autonomic nerves (1). The main idea behind Binshtok, Bean, and Woolf’s experiment was to formulate an anesthetic that blocks the pain of sensory nerves but not motor and autonomic functions. They sited multiple sources stating that when QX-314, a charged derivative of lidocane, is introduced to the inside of a nerve cell it can inhibit sodium channels and produce analgesia. QX-314 was experimentally found to have no significant effect on nociceptor neurons externally. QX-314 is impermeable to nerve cell membranes but with a mass of 263Da is small enough to fit through TRPV1 ion channels. Capsaicin is a TRPV1 agonist, meaning it has an affinity for TRPV1 channel receptors and can affect them physiologically, in this case causing them to open.

Binshtok, Bean, and Woolf observed the membrane potential changes of rat dorsal root ganglia of various diameters exposed to QX-314 (an anesthetic), capsaicin (a TRPV1 agonist), and a mixture of the two. The voltage clamp method was used to determine whether or not the neuronic sodium nerve channels were inhibited and also which nerves channels were inhibited on. The voltage clamp method involves two wires placed in the axoplasm of a nerve cell. The first wire measures potential across the membrane and the second wire propagates electrical current (3). The voltage across the membrane is controlled while the ionic current is measured. Using this method, they found that QX-314 and capsaicin applied together could block the generation of action potentials (1). This effect can be attributed to neuronic sodium channel inhibition. If sodium channels are blocked then the depolarization phase of the action potential cannot take place and a wave of depolarization, a nervous signal, cannot be propagated along the neuron (3). The voltage clamp method used in this experiment involved blocking potassium and calcium ion currents so that the sodium ion current could be recorded by itself.

Binshtok, Bean, and Woolf concluded that neither QX-314 nor capsaicin produced significant effects on nociceptor neurons when applied individually but almost entirely block nociceptor sodium channel function when applied together. The brilliant idea behind all of this is that, if QX-314, an anesthetic, is introduced along with capsaicin, a TRPV1 agnostic, it will only travel through the TRPV1 ion channels of nociceptor neurons. Sodium channels in nociceptor neurons will be blocked while other neurons that lack TRPV1 will remain unaffected. The end result is an anesthetic that blocks painful sensation but does not compromise autonomic and motor nerve function.

References:
1. Alexander M. Binshtok, Bruce P. Bean, & Clifford J. Woolf. Inhibition of nociceptors by TRPV1-mediated entry of impermeant sodium channel blockers. Nature. 4 October 2007. Vol449 pp607-610.

2. M.Cui, P.Honore, C.Zhong, D.Gauvin, J.Mikusa, G.Hernandez, P.Chandran, A.Gomtsyan, B.Brown, E.K.Bayburt, K.Marsh, B.Bianchi, H.McDonald, W.Niforatos, T.R.Neelands, R.B.Moreland, M.W.Decker, C.H.Lee, J.P.Sullivan, C.R.Faltynek. TRPV1 receptors in the CNS play a key role in broad-spectrum analgesia of TRPV1 antagonists. 13 September 2006. Neuroscience Research. Global Pharmaceutical Research and Development. Abbott Laboratories.

3. Elaine N. Marieb. Human Anatomy and Physiology. Sixth Edition. Pearson, Benjamin, Cummings. 2004.

A two hour presentation was given at a local church last night by creation scientist whom I won’t name. This presentation overall lacked direction and seemed to jump from one topic to another without really stopping to make a point. About a third of the presentation was about dinosaur diversity, talking briefly about neat features that a variety of dinosaurs have. Various weather phenomena that could have caused the flood described in Genesis were vaguely presented without any solid background or logic. Fossils were also discussed, again without really any rhyme or reason.

There were two highlights thorughout the evening. The first was when the presenter enthusiastically exclaimed, “I do believe that there were fire-breathing dragons!” From behind me a women shouted an equally enthusiastic, “Amen!” The second highlight of the presentation was the time allotted for questions at the end when PZ Myers, who had been sitting quietly in the front row throughout the entire hour and a half presentation, raised his hand and fired one off. For some reason, this reminded me of the nationally televised Bush vs. Kerry campaign debates of 2004. Whenever Bush was asked a question, he seemed to stutter ignorantly all over his podium for a few moments and then say some elaborate nonsense that didn’t really provide an answer.

To me, trying to scientifically explain an interpretation of the Bible, an interpretation that may not even be accurate, completely misses the meaning of having faith. Some of my fellow neurobio students agreed with me that science and the Bible should not have to be in opposition. It’s a shame that some creation scientists deliberately ignore valid research in areas such as glacial geology and evolutionary ecology to formulate what they consider to be a scriptural explanation of how the Earth came about. The Bible does not define the chemical and genetic specifics of the origin of this planet and the life existing on it. So is creation science attempting to make the Bible say something it doesn’t? Perhaps people have been set in their interpretations for so long, that it’s too difficult to accept that current research in science (that may not jibe with these long held interpretations) does not have to disagree with the Bible.

In discussing Soul Made Flesh this past Wednesday morning in PZ’s neurobiology class, I brought up what I thought to be an interesting, though somewhat tangential, point. Zimmer mentioned Anne Conway and how ambitious she was in her studies despite not being allowed to attend a university. The fact that females were not given the same opportunities throughout history is something I remember learning about in grade school. But where did the ideology that females are inferior to males begin?

One of my fellow students argued that because females give birth they were probably not expected to hunt and gather food while they were pregnant. I thought about this and although I don’t know for sure, in early civilizations females probably tended fields and gathered crop until while pregnant until they were no longer physically able to, returning to the fields as soon as they recovered from the stress of giving birth. Males, meanwhile, tended to be stronger and did not have to give birth to maintain their population.

Another thought that I had on this topic was that male aggression and anger tendencies probably have something to with the ambition to control their domain. Considering male influence in government, it would be interesting to see the effects of a female United States president. There have been several queens as well as kings in European countries over the last thousand years. Is there a difference in how a country operates that is dependent on the gender of its leader?

Things seem to be much different today than they were a hundred years ago. Females driving, voting, becoming doctors, and all these things that would have been unheard of. Are males falling behind and if they do will females dominate males? Is society moving toward a codominance of gender? There is plenty of debate on this topic and I’m sure it won’t be resolved anytime soon.

References:
Zimmer, Carl. 2004. Soul Made Flesh. Free Press, New York, NY.