c elegans 2 ppt

Cell Signaling in C. elegans – Part 2

C. elegans Pattern of Locomotion

• Involves the following:
    - Alternative flexing and relaxation of four longitudinal bands of muscle

• Control Mechanism:
    - Process controlled by both excitatory and inhibitory motor neurons

Acetylcholine

• Definition: Acetylcholine (ACh) is recognized as the primary excitatory neurotransmitter in C. elegans.

• Production: ACh is secreted by presynaptic cholinergic neurons.

Acetylcholine Receptor

• Function: ACh activates nicotinic ACh receptors (nAChRs) on postsynaptic cells.

• Types of nAChRs:
    - They are heteromeric receptors, consisting of five distinct subunits.

• Mechanism:
    - Act as ligand-gated ion channels, permitting $ext{Na}^+$ ions to enter the cell.

Excitatory and Inhibitory Motor Neurons

• Role of Excitatory Motor Neurons:
    - Release acetylcholine (ACh) to stimulate muscle contraction.

• Counterbalance Mechanism:
    - ACh release is counterbalanced by inhibitory neurotransmitters.

• Inhibitory Neurotransmitters:
    - GABA (gamma-aminobutyric acid) is released from inhibitory motor neurons.

Antagonists and Agonists

• Antagonist:
    - A chemical substance that inhibits a biological response by binding to and blocking receptors on cells.

• Agonist:
    - A substance that binds to receptors and activates them, leading to a biological response.

Acetylcholine Receptor Agonist

• Levamisole:
    - An antiparasitic drug that specifically targets the acetylcholine receptor.

• Effects of Levamisole:
    - Opens nAChR ion channels in nematode nerve and muscle cells.
    - Results in hypercontraction and paralysis of the organism.

Postsynaptic Target Cell Synaptic Signaling

• Mechanism of Signaling:
    - ACh neurotransmitter diffuses across the synapse from the presynaptic cholinergic neuron to the postsynaptic cell.

• Question of Interest:
    - Are unc-11 and/or unc-63 components of the levamisole-sensitive nAChR?

Research Objective

• Primary Aim: Investigate if unc-11 and/or unc-63 are components of the levamisole-sensitive nicotinic ACh receptor (nAChR).

• Contextual Information:
    - Levamisole is used to paralyze worms due to its interaction with the levamisole-sensitive nAChR.

• Experimental Design:
    - Treat wild type (WT) and mutant worms (knockout for unc-11 and unc-63) with and without levamisole in a liquid medium to determine the number of paralyzed worms in each condition.

• Hypothesis:
    - If unc-11 and/or unc-63 gene products are essential components of the levamisole-sensitive nAChR, then the removal of those gene products will lead to a reduced effect of levamisole compared to wild type worms.

Laboratory Procedures - Part 2

• Examination Focus:
    - Are UNC-11 and/or UNC-63 components of the levamisole-sensitive nAChR?

• Materials and Setup:
    - Each group will work with three sets of worms: WT, UNC-11 knockout (KO), and UNC-63 KO.
    - Two experimental conditions: presence and absence of levamisole.

• Experimental Steps:
    - Transfer ten worms from each condition into wells of a multi-well plate.
    - Ensure movement in each group.
    - Add 100 µl of levamisole to the experimental well, or nothing to the control well (mix gently).
    - Start the timer and note paralysis every 30 seconds for 5 minutes.

• Important Note:
    - Avoid prolonged exposure of the worms to the microscope light to prevent damage.

Post-Laboratory Analysis

• Data Visualization:
    - Plot the percentage of paralysis versus time for each of the experimental groups (treated with and without levamisole).
    - Display findings in a line graph with distinct lines for each experimental condition.

• Sample Data Points:
    - Time intervals: 0 s, 30 s, 60 s, 90 s, 120 s, 150 s, 180 s, 210 s, 240 s, 270 s, 300 s.
    - Percentage paralysis: from 0% to 90%, with the graph depicting various experimental groups such as WT, WT with levamisole, UNC-11 KO with and without levamisole, UNC-63 KO with and without levamisole.