C. elegans Sinaptik İletim Mutants farmakolojik karakterizasyonu için Paradigmalar

Hi, I'm Cody Locke from the Caldwell Laboratory in the Department of Biological Sciences at the University of Alabama. I'm Kyle Lee and I'll be showing you how to prepare worms for outta carb and PTZ exposure assays. I'm Bernard Tu and I will demonstrate to you how to perform out carb assays.

I'm Kaylin Berry and I'm responsible for the sea Elegance videos. In this presentation, I'll be narrating this presentation and demonstrating the PTZ exposure assay. Today my colleagues and I will show how to characterize synaptic transmission mutants with a sea elegance model of epilepsy after exposure to PTZ and Alde carb with a series of representative mutant strains.

So let's get started. Kyle Lee will start us out today by showing the preparation of ALD carb assays on the first day of the procedure. Ensure that at least 30 young adult stage worms of each genotype and of each replicate will be available for ALD carb assays on the second day.

It is best if an experimenter selects 50 or more L four stage worms onto fresh fungicide free NGM plates seated with e coli OP 50 as a food source. Grow the worms for 12 to 24 hours at a consistent and permissive temperature. 20 degrees Celsius to 22 degrees Celsius is best, although 25 degrees Celsius is acceptable.

On the second day, make a 100 millimolar stock solution of Al Decarb and 70%ethanol spread the appropriate amount of Al Decarb onto NGM plates. We consistently use 0.5 millimolar al Decarb by plating 37.5 microliters of 100 millimolar alde carb onto 60 millimeter non vented Petri plates with 7.5 milliliters in gm. Allow the alde carb plates to dry for roughly 30 to 60 minutes at room temperature.

It is not necessary to crack the lids after the plates are dry. Add consistent volumes of e coli onto the center of each alde carb plate and dry for another 30 to 60 minutes. At room temperature, we typically add 25 microliters of e coli OP 50.

This volume of OP 50 creates a sufficiently sized food lawn to keep the worms in a small spot of the center of the plate without overcrowding. Once the food lawn is dry, proceed with alba carb assays. Due to the subjective nature of ALD carb assays, it is highly recommended that experiments be performed blindly.

To do this. A colleague of the primary experimenter relabel the original plates containing the worms to be assayed, or the colleague could transfer worms from the original plates to cipher alde carb plates immediately before starting a timer. Now that the plates have been sied boron ABO will proceed with the analysis to begin the analysis count the number of paralyzed worms.

There are two major approaches to defining acute al decarb induced paralysis. The first approach defines paralysis by cessation of pharyngeal pumping. In this video note, a dark structure which is situated within the lumen of the worm pharynx at the right of the screen.

This structure is called the grinder and contracts rhythmically through a cholinergic mechanism. The locomotion of the worm in this video has been greatly slowed from Al Decarb exposure. Yet the pharyngeal grinder of this worm is still clearly moving.

Therefore, this worm is not paralyzed by a cessation of pumping definition. One should see in this video that the pharyngeal grinder located at the right of the screen is not contracting with cessation of pumping as the working definition for al Decarb induced paralysis. This worm should be scored as paralyzed.

However, like the worm in the previous video, this worm is exhibiting slight side to side foraging movements. Despite having its locomotion impaired by Al Decarb, these movements would prevent an experimenter from deeming this worm paralyzed by the second approach to defining al Decarb induced paralysis, full body immobility and unresponsiveness to touch. The second approach to defining al decarb induced paralysis involves consistently prodding each worm with a platinum wire and checking for a response.

If body movements cannot be detected by the naked eye, our preferred approach is to prod our worms twice on the head and twice on the tail every 30 minutes for a total of three hours. As you should see, the worm in this video is not obviously moving its body. Nonetheless, because this worm responds to prodding by clearly moving its tail, we do not call this worm paralyzed.

By the second definition of al decarb induced paralysis. Like the worm in the previous video, this worm is not obviously moving its body and thus requires a prodding test. Yet unlike the worm in the previous video, this worm does not clearly respond to prodding.

Therefore, this worm is paralyzed by the second definition of al decarb induced paralysis. Although the difference between paralyzed and not paralyzed by alde carb can be subtle, experimenters should be able to identify substantial alterations and synaptic transmission by simply observing large numbers of representative worms exposed to Al Decarb immediately after being picked onto Al Decarb plates. Wild type worms are still mobile.

In this video. One will see the same wild type worms from the previous video after one hour of Al Decarb exposure. Note that the worms are slowing to some degree but are still clearly moving.

Unlike wild type worms, the Tomasin one mutants in this video are either fully immobilized or barely moving after one hour of exposure. This result is consistent with tomasin one mutants having excess acetylcholine secretion from an inability to negatively regulate neurotransmitter release at neuromuscular junctions, the excess acetylcholine and the synaptic clefts at neuromuscular junctions combined with inhibition of acetylcholine degradation, Bial Decarb makes tomasin one mutants hypersensitive, although not shown here, similar results have been postulated to arise from alde carb assays with loss of function alleles of on 43, which encodes the C elegance ortho log of calcium cal Moulin dependent protein kinase two. This video shows on 25 mutants, which are deficient in inhibitory GABAergic transmission.

After one hour of exposure to alde carb, similar to tomasin one and M 43 mutants, these mutants demonstrate significant alde carb induced locomotor e impairments and appear to be more affected than wild type worms with exposure to al Decarb for an identical length of time. This result is consistent with M 25 mutants having overstimulated body wall muscles from a combination of al Decarb exposure and reduced GABAergic transmission leading to a greater excitatory to inhibitory transmission ratio at CL ELs neuromuscular junctions and subsequent paralysis. Yet because tomasin one on 43 and on 25 mutants exhibit similar sensitivities to Al Decarb at this time point.

Despite having different causes for overstimulation of body wall muscles, an experimenter could not know the underlying mechanism for hypersensitivity without first knowing the identities of causative mutations. For this video, we returned to the same wild type worms from an earlier video after three hours of al Decarb exposure at this terminal point of our ALD carb assay, one should clearly see that these worms, despite being wild type, are either fully immobilized or barely moving. These worms are now phenotypically similar to the TOM one and M 25 mutants shown at one hour after alde carb exposure, presumably because excitatory cholinergic transmission has reached comparable levels in contrast to wild type worms, Synap Bren one mutants remain considerably mobile at three hours of exposure to alde carb.

This result is consistent with Synaptive Bren one mutants, which lack normal levels of neurotransmitter secretion, having less cholinergic stimulation at their body wall muscles than wood wild type worms following identical levels of alde carb exposure at this time. Point on four mutants, which are specifically deficient in cholinergic transmission, should have similar sensitivities to Al Decarb as Synaptive Bren one mutants. Conversely, Tomasin one and O 25 hypersensitive mutants remain paralyzed, not unlike wild type worms.

It is worth noting that some worms, especially those that are resistant to Al Decarb, may attempt to crawl off the plate. In this case, the experimenter may spread a consistent amount of palmitic acid, a physical barrier to worm locomotion. Around the Al Decarb plates, we spread 25 microliters of 10 milligrams palmitic acid per milliliter of ethanol.

That completes the Al Decarb exposure paradigm. Now, I will demonstrate the Penta Lean Terazol exposure paradigm. Kaylin Berry will be helping us with PTZ assays.

Preparation of worms for PTZ assays is essentially the same as the preparation of worms for alde carb assays. The only significant differences between the two assays are the following. We use DDH two O as the PTZ solvent instead of 70%ethanol.

Also, because we frequently plate larger volumes of PTZ than ALDE carb, we allow the plates to dry for at least one hour before adding OP 50. Palmitic acid may also be used with PTZ. Also, it is important to note that PTZ is much less stable than alde carb and must be stored at minus 20 degrees Celsius.

To begin the analysis count the number of epileptic light convulsing worms every 30 minutes for a total of one hour. It is best to score numbers of worms with different types of convulsions in separate categories such as full body convulsions, anterior convulsions, which we call head Bobs combination of anterior convulsions with body wall muscle paralysis, which we call tonic-clonic or full body paralysis, which we call tonic tonic. Seizure-like activity as seen with the worm on the right often follows tonic-clonic convulsions as seen with the worm on the left tonic.

Seizure-like activity could simply result from muscle fatigue following repetitive tonic-clonic convulsions. However, our unpublished data indicates that some worms can become tonic on PTZ in a manner similar to al decarb induced paralysis without having been previously tonic-clonic. Here we show a representative synaptic transmission mutant worm crawling in the absence of PTZA GABA receptor antagonist and common seizure inducer in various animal models of seizures and epilepsy.

Note that the tip or nose of the worm is swinging from side to side while foraging. In this video, we show another worm from the same synaptic transmission mutant strain used in the previous video. Notice that this worm, which is in the presence of 2.5 milligrams PTZ per milliliter of water, mostly continues to move its head from side to side while foraging.

Yet this worm occasionally halts foraging from side to side and instead repetitively thrusts its anterior forward. Finally, one should see a worm which carries the same mutation as the worms in the previous two videos in the presence of 10 milligrams PTZ per milliliter of water. Notice that this worm has decreased side to side foraging and is thrusting its anterior forward with a greater frequency and intensity than with the lower concentration of PTZ.

These results demonstrate dose dependency of PTZ, which we observe with all PTZ sensitive synaptic transmission mutants. Importantly, exposure to PTZ is not sufficient to induce epileptic like convulsions in wild type worms. Likewise, we have attempted but not observed PTZ induced epileptic like convulsions in worms with specific deficits in cholinergic transmission such as on four or worms with excessive cholinergic transmission such as tomasin one.

Therefore, it should be feasible to further characterize synaptic transmission mutants with alde carb resistance from either deficits in general synaptic function or specific deficits in cholinergic transmission based on their relative sensitivities to PTZ. Perhaps surprisingly, a strong reduction of function ONC 25 mutant, which lacks detectable GABA expression in C elegance. Inhibitory motor neurons does not exhibit spontaneous epileptic light convulsions as seen in this video.

An ONC 25 mutant on 10 milligrams PTZ per milliliter of water. However, exhibits robust tonic-clonic convulsions, although not shown anterior only convulsions with some movement preceded ONC 25 associated tonic-clonic convulsions and mimicked the convulsions of synap Bren one mutants. Therefore, an experimenter should not assume that all inhibitory GABA mutants will exhibit tonic-clonic convulsions or closely associated tonic seizure-like activity in the presence of PTZ, especially considering the likelihood of obtaining hypomorphic mutant alleles.

Now that we have demonstrated hypersensitivity to alde carb and to PTZ by inhibitory GABA mutants, but only hypersensitivity to alde carb and normal sensitivity to PTZ by mutants lacking negative regulation of cholinergic transmission. One might wonder what UNC 43 loss of function mutants look like on PTZ since they are also hypersensitive to Alda carb like our other synaptic transmission mutants on 43 mutants do not exhibit obvious epileptic like convulsions in the absence of PTZ, although these mutants do have rare non-repetitive, but still spontaneous full body contractions yet following exposure to 10 milligrams PTZ per milliliter of water on 43, loss of function mutants present, robust, repetitive, and dose dependent full body convulsions. At this time, these results are ambiguous but seem to indicate that on 43 must not have a specific role in inhibitory GABAergic transmission or a role in only negative regulation of neurotransmitter release.

In the C elegance nervous system, we have just shown you how to characterize CL elgan synaptic transmission mutants with complimentary neural stimulants, alde carb, a cholinesterase inhibitor, and PTZA GABA receptor antagonist. When following either a complementary pharmacological paradigm, remember that the degree of sensitivity to one neural stimulant cannot predict the degree of sensitivity to the other neural stimulant Utilization of alde carb assays should help distinguish inhibitory GABA mutants, which head Bob on PTZ, but are hypersensitive to Alde carb from general synaptic function deficient mutants, which also head Bob on PTZ, but are resistant to alde carb. Furthermore, one should realize that inhibitory GABA mutants head Bob on PTZ while worms that lack negative regulation of cholinergic transmission appear wild type on PTZ.

Therefore, the PTZ assay could also be employed to distinguish between these classes of synaptic transmission mutants, which are both hypersensitive to Alde carb. In summary, a complimentary approach with PTZ and Alde carb assays should allow an experimenter to characterize C elegant synaptic transmission mutants in a manner which is impossible with either drug alone. Thanks for watching and good luck with your experiments.