PSYC 318-Lecture#4

PSYC 318-Lecture#4

Optogenetics
1. excitatory opsins
  1. drive action potential
  2. ChR2, C1V1
2. inhibitory opsins
  1. inhibit action potential
  2. Halo, NpHR, Arch
Dopamine Neuron Stimulation caused robust cFOS expression in two places:
1. NAc & PFC (not basolateral amygdala)
  1. basolateral amygdala receives dopaminergic input but doesn’t change their cFOS expression very much
2. dopamine cre mouse line + place optogenetic proteins in dopaminergic mouse lines
3. used cFOS to show which brain region showed inc. activity
  1. a protein that is made any time a neurone is more active than they usually are)
    1. type of immediate early gene
4. conclusion
  1. this experiment didn’t show very much
  2. ?
Why is cre used so much?
1. we don't use viral DNA that encodes GCaMP under a dopamine specific promotor
  1. b/c this would not be helpful for researches studying serotonin & other molecules since this is specific to dopamine
    1. you don't want to make one tool that is useful only for your experiment
2. instead we take
  1. 1. viral DNA that encodes GCaMP when cre is present
    1. a.k.a. cre-dependent virus
    2. same cre-dependent virus can be used in hundreds of different mouse lines
  2. 2. a dopamine cre mouse
    1. the same dopamine-cre mice can be used to drive expression of any foreign protein: GFP, ChR2, GCaMP, OptoD1, etc….
3. 1. & 2. enable immense combinatorial control
  1. hundreds of diff. cre mice have been developed that allow us to target specific populations of neurones across the brain.
  2. Cre dependent viruses can be reused in any of these mouse lines, which enable us to manipulate neural activity in any type of neurone.
4. practical aspect
  1. Gene promotor fragments are often too long to fit inside most viruses
  2. ex: TH
    1. string of DNA cells use to determine if they should make dopamine-related enzymes (like TH) are too long to put in AAV
      1. long gene promoters
    2. solution: people need to make transgenic mice and add artificial chromosomes (BACs) to cells during embryonic development
stimulating or inhibiting midbrain dopamine neurones
1. Stimulating or inhibiting midbrain dopamine neurones promotes social interaction
2. Stimulating or inhibiting midbrain dopamine neurones but these manipulations don’t affect novel object exploration
Which dopamine projections promote social interactions: NAc or PFC?
1. inject cre-dependent, ChR2-encoding virus into midbrain of dopamine-cre mice
  1. Optogenetic proteins i.e. CHR2 located = axon terminals
    1. can diffuse everywhere within a cell, therefore, located far from the soma of infected cells
    2. purpose
      1. Activating opsins located in axon terminals is an effective way to get pathway-specific activation or inhibition
2. put optical fibre and shine light into a projection target of the dopamine neurones (NAc or PFC)
3. conclusion
  1. Activating dopamine neurones in the NAc inc. social behaviour, but activating dopamine neurones in the PFC, doesn’t change social behaviour
Summary of Social interaction manipulations
1. VTA ChR2 = dopamine cell body stimulation (inc. social interaction)
2. VTA-NAc CHR2
  1. dopamine axons located in NAc
    1. (inc. social interaction)
3. NAc WGA/ ChR2
  1. axon terminals that are synapsing in NAc that came from the VTA
    1. not specific to dopamine axons)
4. VTA NpHR
  1. dopamine cell body inhibition (dec. social interaction)
Optical stimulation of dopamine axons in the PFC causes a conditioned place aversion
1. test
  1. every time they locked him on the left hand side, they stimulated dopamine axons in the PFC
  2. control
    1. control animals: PFC neurones weren't light sensitive
      1. no difference in time spent in between the two chambers
  3. test animals
    1. animals avoided the conditioned chamber (were PFC was stimulated)
Elevated Plus Maze
1. test animals: stimulation of dopaminergic axons in PFC
  1. causes them to explore the open arms less
    1. stimulation of anxiety like behaviour
2. control animals:
3. conclusion: study shows the importance of studying different pathways
4. Stimulation of dopamine neurons that project to the PFC creates a place aversion and inc. anxiety like behaviours
Summary of Social interaction manipulations
1. Stimulation of dopamine neurons that specifically project to the NAc promotes social interactions.
2. During social interactions, dopamine neurons that project to the NAc are active.
Optical stimulation of dopamine axons in PFC promotes an “anxiety-like” effect: dec. exploration of open, exposed areas.
If dopamine release in the NAc promotes social behaviour, what receptors there are important?
1. 90% of neurons in NAc are projection neurons
  1. Half express the dopamine D1 receptor
    1. Blocking dopamine D1 receptors in the NAc blocks inc. in social interaction that is caused by optical dopamine neuron stimulation.
  2. half express the dopamine D2 receptor
  3. Def NAc projection neurones:
    1. axons leaves the local area
Dopamine neurons can release glutamate and GABA in addition to dopamine, so how can you be sure that dopamine D1 receptor is sufficient?
1. Rhodopsin
  1. light-sensitive g-protein coupled receptor
    1. signaling cascade that is very different from dopamine’s.
  2. intracellular portion
    1. mediated by Gt, with dec. cGAMP
2. Opto-D1
  1. has the extracellular (light sensitive) side from rhodopsin
  2. intracellular (g-protein signaling side) from the dopamine D1 receptor
  3. D1 receptor that is normally activated by the dopamine now responds to light
  4. complicated merger: have to many many different cuts because it crosses the membrane so many times. (not manipulating protein, manipulating a string of DNA)
3. D1 receptor
  1. transmembrane protein, one protein crosses the membrane many times
  2. extracellular portion binds dopamine
  3. intracellular portion
    1. mediated by Gs which inc. cAMP
Dopamine D1 receptor activation in the NAc is sufficient to inc. social interactions.
1. Opto-D1-eYFP
  1. cre-dependent virus that encodes
2. Drd 1:: Cre
  1. a transgenic mouse that expresses ‘cre’ under control of the dopamine D1 receptor gene promotor
3. cells make both normal D1 receptor as well as Opto-D1 receptor shine light in the cage, causes animals to spend more time with one another.
4. place Opto-D1 receptor in any neurones in the NAc that usually make D1 receptors.
Activating dopamine D1 receptors can have many consequences for cells. Does the firing rate of these neurons change following this opto-activation?
Is an increase in firing of dopamine D1 receptor-containing NAc projection neurons sufficient to promote social interactions?
1. transgenic mouse that expresses cre-recombinase under control of the dopamine D1 receptor gene promotor.
  1. shine light, all neurones that make dopamine D1 receptors spike more, but doesn't initiate the dopamine signalling cascade
Summary: Activating dopamine D1 receptors in the NAc promotes social interactions, as does direct activation of dopamine D1 receptor-containing NAc projection neurons

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	Creado por Pascale Bockelmann hace casi 8 años