Bio Project Lab Final Presentation

Slide 4 - Caffeine is a CNS stimulant

• Most used psychoactive stimulant worldwide

• Milder stimulant, in candy + energy drinks 

• Commonly sourced in coffee beans 

• Generally safe but users can develop tolerance + dependence w/ chronic use

Slide 5 - Caffeine acts as an antagonist of adenosine receptors

• Binds to where adenosine would have bound on adenosine receptors 

• Blocks effects of adenosine (relaxation + sleepiness) 

• Caffeine → increased energy + alertness 

• Caffeine also stimulates dopamine release, contributing to potential for dependence + increased dopamine activity

Slide 6 - Humans can develop a caffeine dependence

• Inability to control caffeine use 

• Withdrawal symptoms (when consumption stops)= headaches + fatigue

• Caffeine tolerance → higher dosages needed for similar effects 

• Healthcare professionals + WHO characterize caffeine dependence as clinical disorder

Slide 7 - C. elegans and humans share many genetic similarities

• 80% of genes are homologs w/ humans 

• Conserved metabolic signaling pathways + neuronal function 

• Due to these genetic similarities, we want to see if C. elegans has the same physical dependence to caffeine as humans

Slide 8 - previous studies

• Past experiments have been performed w/ caffeine + C. elegans → shows that they are a good model organsm for our experiment 

• This is due to:

  • Transparent bodies → analysis of toxins w/o dissection 

  • Proven harmful and preferred caffeine dosages for C. elegans

Slide 11- Experimental Timeline

Prep 

• Creation + maintenance of OP50 liquid culture 

• Chunking + bleaching 

• Chemical prep → diaceytl, NaN3, specific caffeine + nicotine concentrations 

C. elegans used 

• WT 

• UT1 associative learning deficient mutants 

NGM 

LB Agar used to grow OP50 

NaN3 → paralysis chemical 

Bleaching solution 

S Basal Buffer → NaCl, dipotassium phosphate, potassium dihydrogen phosphate, H2O

Slide 13 - Conditioning + Chemotaxis assay Timeline

Conditioning

• C. elegans picked or pipetted w/ S Basal Buffer onto plate w/ OP50 

• 1.5 ul of diacetyl placed on lid (but not for controls) 

• Conditioned for 3-4 hours 

Withdrawal 

• Nematodes moved to platww/ just OP50 using same method as before 

• 1 hour 

Chemotaxis 

• Plates set up w/ A,B,E, * section

• A→ diacetyl + NaN3

• B→ NaN3

• E→ empty 

• * → initial placement 

• Ran for 40 min and then SI was calculated

Slide 15 - Nicotine experiment plates

• Control → nematodes are alive 

• Nicotine + diacetyl plate → shows that diacetyl works as conditioned stimulus

Slide 17- results for nicotine CCP WT C. elegans

• Chemotaxis ran w/ nicotine 

• SI was calculated

• WT avg. SI of 0.44

•  p > 0.05 (T-test), not a significant difference

Slide 20 - Caffeine experiment plates for WT C. elegans

• Control diacetyl → plain NGM plate w/ diacetyl on lid 

• Control caffeine → caffeine plate w/o diacetyl

• Experimental → caffeine w/ diacetyl on lid 

• Chemotaxis was ran

Slide 21 - Caffeine CCP WT. C. elegans

• SI for controls + each caffeine concentration were calculated 

• 5mM caffeine = highest SI, avg = 0.5 

• 10 mM avg = 0.32

• Diacetyl controls 0.24, 0.18

• N.s. difference between SI of experimental + control plates (1 way ANOVA)

Slide 25 - Caffeine CCP UT1 results

• Plain + caffeine control → SI =0 

• Diacetyl control → SI = 0.24, higher than 10 mM caffeine 

• No sig. Difference between experimental + control plates 

Slide 27 - conditioned + unconditioned nematodes sought diacetyl in chemotaxis assay

• In all experiment groups, conditioned + unconditioned → C. elegans had high SI for diacetyl in chemotaxis assay 

• Negative controls failed → therefore data could not support or refute hypothesis 

• Negative controls may have failed due to: 

  • Short conditioning times 

  • More likely explanation: diacetyl used for conditioning instead of hexane

Slide 28 - C. elegans preference for diacetyl may explain our unexpected results

• Negative control may have failed due to using diacetyl for conditioning stimulus rather than hexane as used in Salim et al. paper 

• We did not have enough time to run experiment to establish whether or not diacetyl is a neutral stimulus like Salim et al. did w/ hexane 

• C. elegans have a natural attraction to diacetyl due to their ODR-10 gene 

• It is likely that due to this natural attraction, both conditioned + unconditioned control nematodes will move towards diacetyl due to their natural attraction towards it