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Fall 2019 Grad Seminar Presentation
- 1. Graduate Seminar October 3rd 2019 Madison Honer
- 2. What are Circadian Rhythms? ● circa (around) dies (day) ● Exhibiting a 24-hour rhythm ● Sleep-wake, hormone release, eating habits ● Not limited to just humans https://www.circadian.org/,
- 3. Where are they controlled? Controls Circadian Rhythms by releasing melatonin Regulates release of hormones, body temperature, physiological cycles Where left and right optic nerve cross, crucial for vision https://biologydictionary.net/suprachiasmatic-nucleus/
- 4. Suprachiasmatic Nucleus → Hippocampus? ● No direct synaptic link ○ must be indirect input or diffusible factors ● Limbic system relays the information ○ SCN → Paraventricular nucleus → Hippocampus ● SCN influences the circadian system therefore hippocampus is as well
- 5. How does the rhythm work? BMAL1 CLOCK EBOX PER CRY PER CRY CLOCK BMAL1 Suprachiasmatic Nucleus Cell PER CRY
- 6. https://www.frontiersin.org/articles/10.3389/fnmol.2012.00046/full Light stimulus Ca2+ influx Activates cAMP, MAPK, ERK1/2 CRE transcription Memory Consolidation How does this work related to memory?
- 7. How do we test for this? Circadian Rhythms BMAL1 SCN Hippocampal Memory “Other molecular clock knockout mouse strains retain some rhythmicity due to compensatory functional isoforms and present with subtle or no memory deficits”
- 9. Hypothesis: Let’s Determine ● Is Bmal1 required for hippocampus dependent ... ○ Memory ■ Contextual fear memory ■ Learning and Spatial Memory ○ Signaling ■ Long Term Potentiation at the Schaffer Collateral Synapse ■ cAMP and MAPK Activity
- 10. Experimental Approach Hippocampus-Dependent Memory ● Fear Memory ○ Fear Conditioning Experiments ● Learning and Spatial memory ○ Morris’ Water Training and Novel Object Test Hippocampus Signaling Activity ● Long Term Potentiation ○ Electrophysiology ● cAMP and MAPK Activity ○ Western Blot and cAMP ELISA
- 11. Nocturnal Animals Still Have Rhythm!
- 12. Bmal1−/− mice locomotor activity (light:dark)
- 13. Bmal1−/− mice periodograms do not reveal a 24 h period
- 14. Bmal1−/− mice locomotor activity (dark:dark)
- 15. Bmal1−/− mice periodograms do not reveal a 24 h period
- 16. Is Bmal1 required for…? Hippocampus-Dependent Memory ● Fear Memory ● Learning and Spatial memory Hippocampus Signaling Activity ● Long Term Potentiation ● cAMP and MAPK Activity
- 18. cAMP/MAPK/ CREB Transcription Hippocampus Inhibits Fear Memory Consolidation Contextual Fear Memory
- 19. Test: ZT4 light phase and ZT16 red light night phase at 30 mins and 24 hours Variable: % Freezing (all four paws on the ground, only movement was breathing) https://www.researchgate.net/figure/Contextual-fear-conditioning-A-Experimental-design-B-Fear-training-scheme-C_fig1_329525783 Contextual Fear Conditioning Experiments Explore 2 minutes 0.7-mA foot shock 1 minute recovery
- 20. Bmal1−/− mice demonstrate impaired learning and memory in hippocampus-dependent tasks Contextual Fear Conditioning
- 21. Is Bmal1 required for…? Hippocampus-Dependent Memory ● Fear Memory ○ Yes, Bmal1-/- mice display impaired contextual fear memory ● Learning and Spatial memory Hippocampus Signaling Activity ● Long Term Potentiation ● cAMP and MAPK Activity
- 22. Spatial and Learning Memory https://www.ncbi.nlm.nih.gov/pubmed/19160506 ● Time of day ● Lighting conditions LOWEST Signaling Activity HIGHEST Signaling Activity
- 23. Morris Water Training Maze Test: ZT4 light phase Variable: Escape Latency
- 24. Bmal1−/− mice take longer to find the platform with no quadrant preference Morris Water Maze
- 25. Test: ZT4 light phase Novel Object Test 24 https://journals.plos.org/plosone/article/figures?id=10.1371/journal.pone.0185135
- 26. Bmal1−/− mice spend more time interacting with novel object “C” Novel Object Test
- 27. Is Bmal1 required for…? Hippocampus-Dependent Memory ● Fear Memory ○ Yes, Bmal1-/- mice display impaired contextual fear memory ● Learning and Spatial memory ○ Yes, Bmal1-/- mice display impaired learning and spatial memory Hippocampus Signaling Activity ● Long Term Potentiation ● cAMP and MAPK Activity
- 28. Long Term Potentiation ● Strengthening synapses connection by repetitive signaling ● LTP depends on time of day, as we sleep synapse remodeling strengthens or weakens the synaptic connection ● Synaptic Plasticity → Memory formation + Learning
- 29. Electrophysiology https://www.researchgate.net/publication/51048966_Preparation_of_Acute_Hippocampal_Slices_from_Rats_a nd_Transgenic_Mice_for_the_Study_of_Synaptic_Alterations_during_Aging_and_Amyloid_Pathology Test: Stimulation of the Schaffer collateral efferent fibers at ZT4 to ZT10 Variable: fEPSP: Field Excitatory Postsynaptic Potential
- 30. There is no difference between Wild Type and Bmal1-/- Input/Output Values
- 31. No difference between the normalized slopes of the second fEPSP at each impulse interval
- 32. Bmal1−/− hippocampus demonstrated significantly reduced LTP response
- 33. LTP at the Schaffer-collateral synapse is impaired in the Bmal1−/− hippocampus Black= 5m post stimulation Red= 60m post stimulation
- 34. Is Bmal1 required for…? Hippocampus-Dependent Memory ● Fear Memory ○ Yes, Bmal1-/- mice display impaired contextual fear memory ● Learning and Spatial memory ○ Yes, Bmal1-/- mice display impaired learning and spatial memory Hippocampus Signaling Activity ● Long Term Potentiation ○ Yes, Bmal1-/- mice display impaired LTP at Schaffer Collateral Synapse ● cAMP and MAPK Activity
- 35. Hippocampal ERK and cAMP Activity HIGHEST MAPK activity HIGHEST cAMP activity LOWEST MAPK activity LOWEST cAMP activity
- 36. ERK Western Blot and cAMP ELISA https://www.mybiosource.com/learn/westernblotting https://ruo.mbl.co.jp/bio/e/support/method/elisa.html
- 37. There is no diurnal oscillation of ERK activity or cAMP in Bmal1−/− hippocampi
- 38. Bmal1−/− hippocampi showed no change in pMAPK activity between ZT4 and ZT16
- 39. Explore 2 minutes 0.7-mA foot shock 1 minute recovery Paired and Unpaired Contextual Fear Conditioning Experiments 0.7-mA foot shock Unpaired 30 minutes later Explore 2 minutes 0.7-mA foot shock Paired
- 40. Phosphorylated ERK and ERK intensities from isolated hippocampi of individual mice
- 41. There is no increase in phosphorylated ERK activity in Bmal1−/− hippocampi 30 min after contextual fear conditioning
- 42. Is Bmal1 required for…? Hippocampus-Dependent Memory ● Fear Memory ○ Yes, Bmal1-/- mice display impaired contextual fear memory ● Learning and Spatial memory ○ Yes, Bmal1-/- mice display impaired learning and spatial memory Hippocampus Signaling Activity ● Long Term Potentiation ○ Yes, Bmal1-/- mice display impaired LTP at Schaffer Collateral Synapse ● cAMP and MAPK Activity ○ Yes, Bmal1-/- mice display no daily change in cAMP and + MAPK activity
- 43. Is Bmal1 required for…? Hippocampus-Dependent Memory ● Fear Memory ○ Yes, Bmal1-/- mice display impaired contextual fear memory ● Learning and Spatial memory ○ Yes, Bmal1-/- mice display impaired learning and spatial memory Hippocampus Signaling Activity ● Long Term Potentiation ○ Yes, Bmal1-/- mice display impaired LTP at Schaffer Collateral Synapse ● cAMP and MAPK Activity ○ Yes, Bmal1-/- mice display no daily change in cAMP and + MAPK activity
- 44. What does this mean for the future? ● SCN is the master regulator ● The connection between the SCN and signaling oscillations in the hippocampus still unclear ● Hippocampus is required for spatial memory, but recognition memory needs to be further investigated ● Sleep deprivation studies ● CLOCK, PER and CRY knockout studies ● Genetic Predisposition for Circadian Rhythm disorders https://sleepopolis.com/education/circadian-rhythm-health/
Editor's Notes
- The Circadian Rhythm is the body’s innate timing device. Circadian Rhythms are controlled internally, but are highly impacted by environmental cues.
- Positive and negative transcription/ translational loops Period and cryptochrome clock/bmal proteins produced by the SCN cells bind together to to ebox to promote the transcription of genes PER and CRY Their protein products bind together and block bmal1 and clock from binding to ebox and suppresses the production of per and cry Per and cry proteins break down over time Clock and bmal can act again and promote the expression and starts the cycle all over again This process takes 24 hours to complete before it begins again
- -/- artyhmic in constant conditions and cannot follow light dark cycle
- Horizontal lines are each day Vertical lines are actogram= measure of activity of the mouse running on a wheel
- Horizontal lines are each day Vertical lines are actogram= measure of activity of the mouse running on a wheel
- Horizontal lines are each day Vertical lines are actogram= measure of activity of the mouse running on a wheel
- Horizontal lines are each day Vertical lines are activity actogram= measure of activity of the mouse running on a wheel All tests to be preformed on a day night cycle diurnal Chi square periodogram determines if there is a circadian period or just randomness This program will compute the circadian period of a time series and will determine the statistical significance of the computed period.
- Recurring rounds of transcription and protein synthesis may contribute to the persistence of memories
- When blocked, or altered light: dark entrained conditions, results vary memory Recurring rounds of transcription and protein synthesis may contribute to the persistence of memories
- High MAPK Activity + Fear Memory = ZT4 (4 hrs after light onset) Low MAPK Activity + Fear Memory= red light + ZT 16 (16 hrs after light onset)
- Zeitgeber time is a unit of time based on the period of a zeitgeber, such as the 12:12 light:dark cycle. Thus, while Bmal1−/− mice are able to learn and remember contextual fear conditioning, the strength of their memory is comparable to Bmal1+/+ freezing levels during the trough of fear memory.
- Zeitgeber time is a unit of time based on the period of a zeitgeber, such as the 12:12 light:dark cycle.
- 90s
- Zeitgeber time is a unit of time based on the period of a zeitgeber, such as the 12:12 light:dark cycle. Thus, while Bmal1−/− mice are able to learn and remember contextual fear conditioning, the strength of their memory is comparable to Bmal1+/+ freezing levels during the trough of fear memory.
- Zeitgeber time is a unit of time based on the period of a zeitgeber, such as the 12:12 light:dark cycle. Thus, while Bmal1−/− mice are able to learn and remember contextual fear conditioning, the strength of their memory is comparable to Bmal1+/+ freezing levels during the trough of fear memory.
- Input and output curve and paired pulse factilitation, no significant difference Field Excitatory Postsynaptic Potential Different stimuli As the stimulus increases, so does the postsynaptic potential, indicating that bmal does not effect schafer fibers
- No significant difference between normalized slopes of the second excitatory potential for paired pulse facilitation control
- For the first 30 mins after ltp induction the response of -/- mice were reduced Based off of stimulus To induce LTP three stimuli separated by 5 minutes were delivered
- Ltp traces from individual slices The slope of the negative-going phase of the field excitatory postsynaptic potential (EPSP) corresponds directly to the activation of depolarizing synaptic currents in CA1 pyramidal neurons in response to glutamate release from SC terminals.