The Effect of Spinal Cord Severing in Zebrafish

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

Sex for sale

‘Tis the season for ridiculous commercialism: I’ve been seeing these unbelievable commercials that feature some smug guy surprising his wife by giving her a luxury car (with a bow on top) as a present, or popping open a box with a big honkin’ diamond in it. The women always clap their hands with glee and lean in for a hot passionate kiss. I see these and I wonder…just how stupid do advertisers think men are?

I can tell you exactly what would happen if I spent a month’s salary or more on jewelry (or worse, a year’s income on a car). My wife would look aghast, and waver between calling the hospital for an immediate psychiatric consult and kicking me in the groin. I would spend that much on inessential frippery? Without consulting her? There sure wouldn’t be any sexual arousal, unless these commercial makers easily confuse that sinking feeling in the pit of the stomach at the thought of budget-busting debt with “sexy.”

Desirable women are too smart to be bought with flashy gee-gaws. My wife and I are talking about getting ourselves a snow-blower for Christmas…now that is romantic.

Don’t delude yourself — you can’t buy monogamy.

Circadian Clock Neurons

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

People behaving badly

We shouldn’t leave the Moslems hanging with all the blame for bad behavior — so here’s some more deplorable activities that have show up in my mailbox in the last few hours.