Abstract Introduction

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1 Decreased colocalization of synapsin I and Munc13 within presynaptic axon terminals of the earthworm neuromuscular junction when stimulated could help determine how the two proteins interact during neurotransmitter release. Michael P. Tekin and William L. Coleman Department of Biological and Allied Health Sciences Bloomsburg University, 400 E. 2 nd St. Bloomsburg PA Abstract Synapsins are a group of proteins classically thought to regulate synaptic vesicle cycling well upstream of vesicle exocytosis. Several previous studies however have suggested that synapsin I may have multiple regulatory roles, including functions closer in time to the actual release event. This led to the hypothesis that synapsin I may interact with the major vesicle priming protein Munc13. Synapsin I / Munc13 interaction was investigated using double immunofluorescence staining at earthworm (Lumbricus terrestris) neuromuscular junctions. Earthworms were anesthetized by soaking in 95% ethanol until cessation of motor activity and pinned ventral side down to a dissecting tray. The dorsal integument was bisected longitudinally and the ventral nerve cord and longitudinal muscle fibers were exposed by removing any overlaying organs. Sections of tissue were then transferred and pinned to a Sylgard filled dish. Throughout dissection, tissue was moistened with a saline solution that mimics normal extracellular fluid. Five tissue samples were left in the unexcited state and one tissue sample was chemically stimulated. Tissue was then fixed with 2% paraformaldehyde and processed using standard methods for fluorescence immunohistochemistry. Fluorescent images were taken using an inverted epifluorescence microscope, and analyzed using freely available ImageJ software with the colocalization plug-in JACoP to obtain the Pearson coefficient. The Pearson coefficient indicated strong colocalization at rest (average R = 0.883±0.005). This high degree of colocalization suggests that synapsin I and Munc13 may interact at rest. Moreover, the sample of stimulated tissue was compared to one sample of unstimulated tissue. The average Pearson coefficient was significantly lower in the stimulated tissue then the unstimulated tissue supporting Synapsin I/ Munc13 interaction regulate synaptic transmission (percent difference = 46.3%, T=0.999, df =8, P= ). Future studies will investigate what caused this observed decrease of colocalization during stimulation and if similar trends occur in the interaction of munc13 and other forms of synapsin I. Introduction When an action potential reaches the presynaptic terminal the voltage-gated, calcium channels open and synaptic vesicles migrate to end of the presynaptic terminal pools (Südhof 2004). The

2 synaptic vesicles then release there neurotransmitter into synaptic through exocytosis. Synaptic transmission is dependent on the amount of vesicle in the vesicle pool. There are several proteins in regulating this process. This experiment focused on synapsin I and munc13. Synapsin. Synapsins are a prevalent synaptic vesicle protein and regulates the vesicle pool, and hence neurotransmitter release (Südhof 2004). Although synapsins are not necessary for life, knock out animals models have shown they are essential for regulating the proper amount of synaptic vesicle production. There are three types of synapsins; synapsin I, synapsin II, and synapsin III. Presynaptic Terminal Figure 1. Left- When the presynaptic terminal is at rest, synapsin is bound to actin and the synaptic vesicles. When the presynaptic terminal is excited the voltage gated, Ca 2+ channels open, which lets Ca 2+ enter the cell, causing the release of neurotransmitter through exocytosis. Ca 2+ also activates secondary messengers like calcium/calmodulin dependent protein kinases. Right- CaM kinase then phosphorylates synapsin, which allows the protein and the synaptic vesicles to dissociate. The synaptic vesicles then migrate to the end of the presynaptic terminal and release their neurotransmitters through exocytosis. Unlike syanpsin II, synapsin I binds to ATP, and unlike synapsin III, synapsin I needs Ca 2+ dependent (Südhof 2004). In general, synapsins bind to actin and synaptic vesicles when the presynaptic terminal is at rest. When an action potential reaches the presynaptic terminal the excited voltage gate Ca 2+ channels, which lets Ca 2+ into the presynaptic terminal. The Ca 2+ activates CaM kinase, which phosphorylates synapsin. When synapsin is phosphorylated the protein dislocates from the synaptic vesicles and actin. The synaptic vesicles are now free to migrate to the synaptic junction, and release there neurotransmitter through exocytosis. Therefore, synapsin plays an important role in the release of neurotransmitter by regulating the pools of vesicles. Another protein that is associated with synaptic transmission is Mun13.

3 Munc13. Failure to produce Munc-13-1 strongly indicates the protein is essential for life (Augusitin et al. 1999). Munc13 plays an essential role in regulation of the glutamatergic vesicles. Munc13-1 mediates vesicle maturation and facilities release through vesicle priming (Dulbova et al. 2005). Figure 2. Before neurotransmitter released the synaptic vesicles nee to dock to the presynaptic membrane by several SNARE proteins that are essential for vesicle fusion. The fusion readiness of synaptic vesicles are regulated by munc13 through the priming of the SNARE complex, moving the synaptic vesicle closer to the presynaptic terminal membrane. This occurs through the following mechanism: After synapsin unbinds from actin and synaptic vesicles, they can migrate to the synaptic terminal and release neurotransmitter through exocytosis. However, before synaptic vesicles can fuse to the membrane of the presynaptic terminal, the vesicle must bind to the SNARE complex (Augusitin et al. 1999). This is known as docking. Then, munc13 will prime the SNARE complex by twisting the SNARE proteins around each other bringing the synaptic vesicles closer to the presynaptic terminal membrane, increasing its fusion readiness. Therefore, both munc13 and synapsin I are involved in regulating synaptic transmission. Rational and hypothesis. Munc13 regulates exocytosis through vesicle priming, while Synapsin I regulate vesicles cycling through its dissociation from actin and synaptic vesicles and may alter the timecourse of vesicle release (Hilfiker et al. 1998, Humeau et al. 2001, Coleman and Bykhovskaia 2009). Synapsin I may mediate these effects on neurotransmitter release by interacting with Munc13. This hypothesis can be explored by comparing the colocalization of Synapsin I and Munc13 at the synapses in a worm tissue when stimulated and unstimulated. Material and Methods Preparing Modified Drews Pac solution and K + Modified Drews Pac solutions. A Drews Pac solution was prepared as outlined in Volkov et al. (2007). This solution was labeled modified Drews Pac solution throughout the experiment. Another solution was prepared and labeled as K + Modified Drews Pac solution. This solution was identical to the Modified Drews Pac solution, except 4 mm of KCL was increase to 90 mm of KCL. The K + Modified Drews Pac solution was used to stimulate the worm tissue.

4 Unexcited Tissue Colocalization Experiment. Live tissue was obtained from an earthworm (Lumbricus terrestris). The worm was anesthetized with 95% ethanol solution and then longitudinally opened on the dorsal side. Next, the digestive and reproductive organs were removed, leaving the longitudinal muscle fibers and ventral nerve cord intact. After the live tissue was thoroughly cleaned using a modified Drewes-Pax solution sections of tissue were pinned to a sylgard filled dish. The tissue was then fixed using 2% paraformaldehyde solution. The tissue was permeabilized using a 5% milk, 0.5% Tris, PBS buffer. Fluorescence immunohistochemical techniques were then used to stain Munc13 and synapsin I. Slides were prepared from the stained tissue and pictures of the results of these florescence immunohistochemistry methods were taken with a Photometrics CoolSNAP ES2 monochromatic camera and an inverted fluorescent microscope. Four separate slides were prepared and photographed from the earthworm used. This experiment was performed over a time span of four weeks, and when the worm tissue was not in use, it was stored in a PBS buffer and refrigerated. Unexcited and Excited Colocalization Comparison Experiment. Live tissue was obtained form an earthworm as outlined in the unexcited colocalization comparison experiment. Then two of tissue samples obtained from the earth worm was stimulated with a K + Modified Drews Pac solution. The other two tissue samples obtained from the earthworm was not stimulated and used as a control. Both treatments were fixed using a 2% paraformaldehyde solution. The 2% paraformaldehyde used for the stimulated tissue treatment was prepared with K + Modified Drews Pac solution, while the control treatment 2% paraformaldehyde was made with Modified Drews Pac solution. Both the control and stimulated tissue treatments were died using fluorescence immunohistochemical techniques specific to Munc13 and synapsin I. Pictures were taken by the same methods for both as they were in the unexcited tissue colocalization experiment. However, due to experiment error, one control sample and one stimulated sample had to be excluded from these data. This experiment was performed over a timespan of two week, and the tissue was stored in the same manner as the unexcited tissue colocalization experiment. Commented [MPT1]: should I include the dyes used? Commented [MPT2]: Is this correct? Measuring Colocalization and Statistical Analysis. The JACoP colocalization plug-in (Bolte and Cordelieres 2006) in ImageJ (Rasband ) was used to measure the colocalization of Munc13 and synapsin I by obtaining the Pearson coefficient of individual synapses. The average Pearson coefficient and standard error was calculated with Microsoft Excel for the unexcited tissue colocalization experiment, and for the stimulated tissue and control tissue in the unexcited and excited colocalization comparison experiment. Microsoft Excel was also used to perform a Two-Tailed, Unequal Variance Student T Test comparing the average Pearson coefficient of the control tissue and stimulate tissue.

5 Results Figure 3. The images of these synapses from worm tissue were obtain from using a Photometrics CoolSNAP ES2 monochromatic camera and an inverted epifluorescence microscope. The tissue was stained using fluorescence immunohistochemistry. The red fluorescent dye targeted Munc13 (I), and the green fluorescent dye targeted Synapsin I (II). The yellow images (III) are overlays of the corresponding synapsin I and Munc13 staining. Both sets of images demonstrate strong colocalization in the neuromuscular junction at the unexcited state (average R = 0.883±0.005, Fig3A, R=0.915, Fig3B, R= 0.956). Unexcited Tissue Experiment Results. The images taken of the neuromuscular junction synapses demonstrated high level of colocalization with low levels of variance in the unexcited state (average R = 0.883±0.005, Fig 3).This high colocalization was visually observable when stained using fluorescence immunohistochemistry (Fig. 3). Then, a comparison of the colocalization of Munc13 and Synapsin I at the synapse of the neuromuscular junction was made for unexcited and excited worm tissue.

6 Figure 4. The images of these synapses from worm tissue were obtain from using a Photometrics CoolSNAP ES2 monochromatic camera and an inverted epifluorescence microscope. The worm tissue was stimulated with a K + modified Drews-Pac solution (A) or was untreated (B). The images in row-c are of the same synapse as row-b but with a higher contrast and brightness to make staining more observable. The tissue was stained using fluorescence immunohistochemistry. The red fluorescent dye targeted Munc13 (I), and the green fluorescent dye targeted Synapsin I (II). The yellow images (III) are overlays of the corresponding synapsin I and Munc13 staining. The images from the control treatment demonstrate strong colocalization in the neuromuscular junction at the unexcited state (Fig4A, R=0.811, Average R=0.848±0.016), but the unexcited state showed decreased levels of colocalization (Fig4B, R=0.595l, Average R=0.529±0.053). Unexcited and Excited Colocalization Comparison Experiment. Images from the control treatment demonstrated a high level of colocalization with a low level of variance (Average R=0.848±0.016, Fig 4A), which was visually observable when stained using fluorescence immunohistochemistry (Fig. 4A). The images of stimulated, worm neuromuscular junction indicated a moderate level of colocalization with a higher level of variance (Average R=0.529±0.053, Fig 4B), which was also observable when stained using fluorescence immunohistochemistry (Fig. 4B, Fig. 4C).

7 Pearson Coeffiecent Control Treatment Stimulated Tissue Figure 5. The control treatment had statically significant higher level of colocalization than the stimulated tissue treatment (percent difference = 46.3%, T=0.999, df =8, P= , Fig 5A). The standard error represented by the error bar is smaller in the control treatment than the stimulated tissue. The observed decrease in colocalization support that Munc13 and Synapsin interaction regulate neurotransmitter release. The decrease in colocalization of the simulated worm tissue was statistically significant (percent difference = 46.3%, T=0.999, df =8, P= , Fig 5A). Discussion The high level of colocalization observed when the tissue was in the unexcited state indicates that Munc13 and Synapsin I are interacting at rest (average R = 0.883±0.005, Fig 3, Average R=0.848±0.016, Fig 4). Moreover, the moderate level of colocalization observed when the tissue was in the excited state indicates that Munc13 and Synapsin are interacting less at rest than the unexcited tissue (Average R=0.529±0.053). This observed reduction in colocalization supports that the interaction between Munc13 and Synapsin I are regulating neurotransmitter release. However, there is the possibility that sampling error caused these results Commented [MPT3]: I think this section is lacking content. Still Thinking about what it need. The small sample size of the stimulated tissue is a concern. There was a total of five slides of unstimulated tissue were prepared and analyzed, but only one slide of stimulated tissue was prepared and analyzed. Therefore, a poor preparation of the stimulated slide could completely account for these results. If more slide were prepared, experimental error would be less likely. Therefore, the Unexcited and Excited Colocalization Comparison Experiment should be repeated in triplicate to confirm the observed role. Finally, these results can be used and hints at the mechanism. For example, from these results, it can be speculated that Munc13 and Synapsin I dissociate from each other when an action potential reaches the presynaptic terminal. Since, the Munc13 priming activity is essential for normal neurotransmitter release (Augusitin et al. 1999), this dissociation may be necessary in mediating normal Munc13 priming activity. However, completely different mechanism could account for the observed reduction in Munc13 and Synapsin I colocalization in the stimulated tissue treatment. Additionally, these results only support that Munc13 and Synapsin I are

8 interacting to regulate neurotransmitter release. A similar experiment could be performed to study Synapsin II or Synapsin III interaction with Munc13. Thus, more experimentation will be needed to determine if, and how synapsins and Munc13 interact with each other. Literature Cited 1. Augustin, I., Rosenmund, C., Sudhof, T. C.., and Brose, N. (1999). Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles. Nature. 400: Bolte S and Cordelieres P (2006). A guided tour into subcellular colocalization analysis in light microscopy. Journal of Microscopy 224, Dulubova, I., Lou, X., Lu, J., Huryena, I., Alam, A. Schneffenburger, R., Sudhof, T. C., and Rizo, J. (20005) A Munc13/RIM/Rab3 triparite complex: From priming to Plasticty? EMBO. 24: Rasband WS, ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, Rasband WS, ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, Sudhof, T. C. (2004). The Synaptic Vesicle Cycle. Neuroscience. 27: