Exam 2 Steidl Flashcards
What is the clinical and scientific significance of Dr. Steidl’s research?
Clinical: addiction is caused by the hijacking of reward pathways by the substances. If you understand the pathways involved in reward you can develop therapies.
Scientific: Understand if cholinergic and glutamergic inputs to a reward contribute equally to reward seeking behaviour
Characterize the mesolimbic and mesocortical dopamine pathways in the rat brain
- Parkinson’s disease affects dopaminergic neurons close to the substantia niagra.
- The VTA sends dopaminergic axons to the prefrontal cortex and other limbic targets.
What does cocaine do?
Inhibits dopamine reuptake
What is morphine and what does it do?
It is a mu-dopamine receptor antagonist. It disinhibits VTA neurons by increasing the production of GABA and suppressing GABA release.
Describe the study that led the researchers to conclude that dopamine neurons respond to environmental cues that predict reward.
- The researchers gave juice (which is the reward) to the monkey and at first, it responded to the reward.
- Then the neurons responded to the sound of the machine instead of the juice. So they did an experiment where they added a CS (sound) to the reward, and found that the monkey responded to the CS instead of the reward.
- Then they did an experiment where they had the CS(sound) but no reward. The neurons responded to the CS but gradually tapered off.
- Thus, they found that dopamine neurons respond to the predictor of the reward (CS/sound).
Role of reward predictors in eliciting craving and relapse. What happens at the cellular level?
Reward predictors elicit craving and precipitate a relapse if a person is with the conditioned stimulus associated with the reward. This occurs due to LTP and the strengthening of synapses.
What section of the midbrain did Dr. Steidl’s group focus on?
Dopamine neurons in the LDTg and PPTg
What three types of inputs go to the VTA?
Cholinergic, glutamergic and GABAergic
What types of neurons send dopamine when LDTg and PPTg are stumulated?
Inputs from the glutamergic and cholinergic neurons send dopamine to the limbic structures.
What is the limitation of using electrical stimulation to stimulate LDTg and PPTg?
You won’t know which neurons you are stimulating, you could be stimulating fibers of passage, and back stimulating others.
What are fibers of passage?
Fibers that don’t synapse onto the area that you are focusing on so they are not relevant for your study.
How did Dr. Steidl use exctitatory optogenetic actuators to understand which neurons are involved in the reward pathway?
He used Ch2R. Activating it with blue light opens a cation channel, which depolarizes the neuron. Seeing which neurons depolarize would tell you which ones are involved in the reward pathway.
How do you identify the VTA neurons?
Depolarization but injecting current
How do the researchers know that the blue light to activate the Ch2R works?
Electrical stimulation gets the same result as using a blue light.
What is the efficacy of light sensitive sensors and actuators compared to an electrical stimulus/electrode?
Light sensitive sensors and actuators are not as efficient as electrical stimuli or electrodes.
What previous study showed that VTA neurons are driven by LDTg neurons, influencing reward and behaviour? What could they not conclude?
They used a rabis virus and injected it into the VTA, which encoded ChR2. The virus expressed the ChR2 and a fluorescent protein. This selectively activated LDTg. They could not conclude which neurotransmitters are involved.
Why did the researchers use a rabis virus when seeing if LDTg neurons drive VTA neurons?
It gets taken up into the terminals and into the cell body and they wanted the virus to be selectively taken up by the terminals of the LDTg neurons.
Describe the viral construct and experimental setup that Dr. Steidl used when seeing if their optogenetic technique excites LDTg inputs into the VTA?
They used an AAV viral vector that has CH2R and eYFP gene under the control of the promoters and enhancers of CamK2. They injected the AAV virus into teh LDTg neurons. The LDTg neurons expressed Ch2R.
When validating their optogenetic technique, why is it a problem if they applied the blue light in the vicinity of the VTA?
Not all of the axons will get the stimulation from the blue light.
How did Dr. Steidl confirm if his optogenetic technique in injecting ChR2 into the LDTg worked? and also what were the experimental results?
He confirmed using intracranial self stimulation to see if the rodent presses the lever that gives the blue light. The blue light was located close to the VTA.
Results:
- ChR2 construct: increased lever presses if only you have the reinforced lever.
- only using eYFP transfected cells doesn’t increase lever presses because there is no ChR2 so you are not exciting anything.
How did Dr. Steidl confirm if pressing the lever depends on the activity of the VTA dopamine neurons?
Add Flupinthixol, which is a dopanine receptor antagonis. With the antagonist, lever pressing decreases.
One day later, the lever presses were better than normal. This means that exciting LDTg inputs to the VTA depend on dopamine.
What is the purpose of microdialysis and how does it work?
You can measure the amount of a specific molecule that is secreted in a specific area. There is fluid that goes down. At the bottom, the dialysis membrane has pores that are just big enough to let the molecule into the tube. The fluid carries the molecule back up to the top. Then you analyze how much molecule the fluid brought out and conclude the levels of the molecule.
How did the Dr. Steidl use microdialysis and What were the results, what did he conclude with this approach?
He used it to measure if stimulating ChR2 with blue light in the LDTg neurons increased dopamine in the nucleus accumbens. Construct without ChR2 and only eYFP shows that levels of dopamine is very little. Construct with ChR2 + eYFP significantly increases dopamine levels. Therefore, ChR2 activation increases dopamine in the nucleus accumbens.
Describe how Dr. Steidl selectively activated cholinergic and glutamergic inputs to the VTA.
He used DIO because he wanted expression in the neurons that he wants to target/study. He expressed cre which was driven by the enhancers and promoters of genes specific to cholinergic neurons (ChAT) or specific to glutamergic neurons (VGLUT2).
How do you know if Dr. Steidl targeted LDTg neurons in cholinergically driven CRE mice?
- eYFP is expressed in LDTg and so is ChAT therefore there is colocalization of the two as seen on the merge since you see the green (eYFP) and red (ChAT) at the same time) so you know that the illumination is specific to the cholinergic neurons.
- The graph shows that there is an equal number of neurons that expressed eYFP and eYFP + ChAT which means that eYFP was only in cholinergic neurons.
When targeting LDTg neurons with ChAT::Cre mice, why did they visualize the VTA terminals?
Look at the VTA terminals to make sure that the eYFP and ChR2 go there otherwise putting blue light in VTA is pointless.
When targeting LDTg neurons with ChAT::Cre mice, why did they look at VAChT and TH and what does it show?
It marks nerve terminals for cholinergic neurons. Everything that is green is supposed to be red, which means the eYFP and the nerve terminals colocalize with the TH labeling.
Was labeling for cholinergic neurons restricted to the LDTg?
Yes. Slide 20
What was the viral construct for targeting LDTg neurons in glutamergic mice?
Used enhancers and promoters under the control of VGluT2 in glutamergic neurons so CRE is only expressed in glutamergic neurons.
How do you know if Dr. Steidl targeted LDTg neurons in glutamergically driven (VGluT2::Cre) CRE mice ?
They saw co-localization with VGluT2 and eYFP which means that ChR2 was targeted in glutamergic cells only. Slide 21-23.
Why is it a problem to inject ChR2 in LDTg with ChAT?
There is no glutamergic marker because all cells express glutamate so there is non localization here. And you don’t want to see that.
What was the procedure and what were the results when they used mCherry and higher resolution of colocalization?
They used two antibodies: 1. against VGlut2 (gold) and 2. against mCherry (grey). Staining with VGlut2 (gold) was higher than staining of mCherry (gold). But mCherry and VGluT2 are in the presynaptic terminals together.
Describe the set up of the place preference test.
there are two different chambers in the box that the mouse can go into and it should be interested in both equally.
Did VTA optogenetic excitation of LDTg-cholinergic axons induce place preference?
- mouse stays in the blue chamber longer for three sessions. Even without the light stimulus, it remembers the chamber and stays there for longer.
- Session three showed significant results for number of times the mouse entered the blue chamber. But its preference didn’t change.
Did VTA optogenetic excitation of LDTg-glutamergic axons induce place preference?
- Mouse spent less time in the blue chamber.
2. Activation of glutamate axons increases number of times the mouse enters the chamber.
Did VTA optogenetic excitation of LDTg-cholinergic axons reinforce chamber entries? What do the results indicate about the two different inputs?
- They gave one light pulse.
- No difference in cholinergic neurons.
- Chamber entries increased in glutamergic axons.
- Results show that the two different systems lead to different behaviours. Glutamergic- increases curiosity. Cholinergic-increases liking for the place.
