SLEEP L2 - general

Question 1

measuring neuronal activity with genetic calcium indicators (GcaMP)

Answer 1

• monitoring neuron activity is crucial for understanding their function, particularly whether they fire action potentials
• while its challenging to directly observe APs in vivo, calcium imaging provides a reliable indicator of neuronal activity, as increased calcium levels typically accompany neuronal activation
• to visualise calcium dynamics, researchers use genetically encoded calcium indicators (GECIs) such as GCaMP, a fusion protein of enhanced GFP and calmodulin linked to a peptide M13
• when calcium binds calmodulin, it alters the fluorescent properties of GCaMP, emitting a stronger fluorescent signal at 515nm compared to baseline fluorescence at 485nm
• in experiments, GCaMP is genetically expressed in specific neurons using viral vectors like AAV, allowing researchers to track calcium fluctuations in response to neuronal activity
• photometry involves stimulating the brain area of interest w/ light at 485nm and recording the resulting fluorescence signal using an optical setup and amplifier
• by measuring changes in fluorescence intensity, researchers can infer neuronal activity

Question 2

optogenetics

Answer 2

• involves the use of light to control cells in living tissues, typically neurons, that have been genetically modified to express light-sensitive ion channels
• inspired by the study of algae behaviours in response to light –> researchers discovered that certain algae possess light-activated ion channels called channelrhodopsins, which allow them to swim towards or away from light sources
• channelrhodospins are 7TM domain receptors; when exposed to specific wavelengths of blue light, the channels open allowing sodium ions to enter the cell and trigger cellular excitation
• initially investigated by researchers at the max planck institute in germany, this phenomenon primarily focused on understanding algae’s phototactic response to sunlight –> towards the end of their discovery, they speculated that this could have implications for neuroscience, suggesting its potential application in studying neurons

Question 3

channelrhodopsin

Answer 3

• Ed boyden during his PhD under guidance of Karl Deisseroth in California, came across the PNAS paper detailing the discovery of channelrhodopsin by german research group
• Boyden proposed the idea of introducing channelrhodopsin into mouse neurons to induce APs upon exposure to light
• obtained plasmid encoding channelrhodopsin and transfected it into cultured neurons –> when blue light was applied, the neurons indeed fired APs, confirming the feasibility of remote neuronal activation using light
• serves as a reminder of the value of interdisciplinary collaboration and the importance of exploring ideas beyond one’s own field
• upon light exposure, channelrhodopsin opens, allowing cations such as sodium to enter the cell while potassium ions exit, resulting in neuronal excitation
• since its initial discovery in early 2000s, Deiseroth and others have systemically engineered various variants of channelrhodopsin by altering its aa sequence; these modifications enable sensitivity to different wavelengths of light, incl red light which penetrates deeper into brain tissue
• this has led to an industry focused on developing channelrhodopsin variants
• while channelrhodopsin excites neurons, researchers have also epxlored methods to inhibit neuronal activity:
• eg. light-activated pumps, derived from simple eukaryotic organisms in the sea –> when exposed to yellow light, these pumps transport chloride ions into the cell, hyperpolarizing it and effectively inhibitng neuronal firing (technique aka as optogenetic inhibition)

Question 4

chemogenetics

Answer 4

• involves use of DREADDs, where DREADD receptors are expressed in specific brain regions using viral vectors such as AAVs –> receptors are then activated by synthetic ligans (eg. CNO), allowing researchers to modulate neuronal activity over extended periods and observe behavioural changes in animals
• while chemogenetics offers the advantage of long-term experiments, it lacks the precision of optogenetics
• optogenetics enables precise control over neuronal activity by introducing light-sensitive proteins such as channelrhodopsin into neurons using AAV
• by coupling these proteins w/ cre recombinase, researchers can selectively target specific neuron types; laser light pulses at precise wavelengths and frequencies are then used to stimulate these neurons, allowing researchers to investigate their role in behaviour w. high temporal resoltuion
• one unexpected benefit of using channelrhodopsin is its fusion w/ enhanced yellow fluorescent protein (EYFP), which facilitates axonal transport; when expressed in neurons, this fusion allows researchers to trace neural circuitry through a process known was anteretrograde mapping
• e.g when channelrhodopsin EYPF is expressed in glutamate neurons associated w/ wakefulness, researchers can observe the projections of their axons to specific brain regions such as the nucleus accumbens, providing insights into the neural circuits regulating arousal