Zusammenfassung der Ressource
PSYC 318-Lecture#4
- Optogenetics
- excitatory opsins
- drive action potential
- ChR2, C1V1
- inhibitory opsins
- inhibit action potential
- Halo, NpHR, Arch
- Dopamine Neuron Stimulation caused robust cFOS expression in two places:
- NAc & PFC (not basolateral amygdala)
- basolateral amygdala receives dopaminergic input but doesn’t change their cFOS expression
very much
- dopamine cre mouse line + place optogenetic proteins in dopaminergic mouse lines
- used cFOS to show which brain region showed inc. activity
- a protein that is made any time a neurone is more active than they usually are)
- type of immediate early gene
- conclusion
- this experiment didn’t show very much
- ?
- Why is cre used so much?
- we don't use viral DNA that encodes GCaMP under a dopamine specific promotor
- b/c this would not be helpful for researches studying serotonin & other molecules since this is specific
to dopamine
- you don't want to make one tool that is useful only for your experiment
- instead we take
- 1. viral DNA that encodes GCaMP when cre is present
- a.k.a. cre-dependent virus
- same cre-dependent virus can be used in hundreds of different mouse lines
- 2. a dopamine cre mouse
- the same dopamine-cre mice can be used to drive expression of any foreign protein: GFP, ChR2,
GCaMP, OptoD1, etc….
- 1. & 2. enable immense combinatorial control
- hundreds of diff. cre mice have been developed that allow us to target specific populations of
neurones across the brain.
- Cre dependent viruses can be reused in any of these mouse lines, which enable us to
manipulate neural activity in any type of neurone.
- practical aspect
- Gene promotor fragments are often too long to fit inside most viruses
- ex: TH
- string of DNA cells use to determine if they should make dopamine-related enzymes (like
TH) are too long to put in AAV
- long gene promoters
- solution: people need to make transgenic mice and add artificial chromosomes (BACs) to cells during embryonic development
- stimulating or inhibiting midbrain dopamine neurones
- Stimulating or inhibiting midbrain dopamine neurones promotes social interaction
- Stimulating or inhibiting midbrain dopamine neurones but these manipulations don’t affect novel
object exploration
- Which dopamine projections promote social interactions: NAc or PFC?
- inject cre-dependent, ChR2-encoding virus into midbrain of dopamine-cre mice
- Optogenetic proteins i.e. CHR2 located = axon terminals
- can diffuse everywhere within a cell, therefore, located far from the soma of infected cells
- purpose
- Activating opsins located in axon terminals is an effective way to get pathway-specific activation or
inhibition
- put optical fibre and shine light into a projection target of the dopamine neurones (NAc or PFC)
- conclusion
- 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
- VTA ChR2 = dopamine cell body stimulation (inc. social interaction)
- VTA-NAc CHR2
- dopamine axons located in NAc
- (inc. social interaction)
- NAc WGA/ ChR2
- axon terminals that are synapsing in NAc that came from the VTA
- not specific to dopamine axons)
- VTA NpHR
- dopamine cell body inhibition (dec. social interaction)
- Optical stimulation of dopamine axons in the PFC causes a conditioned place aversion
- test
- every time they locked him on the left hand side, they stimulated dopamine axons in the PFC
- control
- control animals: PFC neurones weren't light sensitive
- no difference in time spent in between the two chambers
- test animals
- animals avoided the conditioned chamber (were PFC was stimulated)
- Elevated Plus Maze
- test animals: stimulation of dopaminergic axons in PFC
- causes them to explore the open arms less
- stimulation of anxiety like behaviour
- control animals:
- conclusion: study shows the importance of studying different pathways
- Stimulation of dopamine neurons that project to the PFC creates a place aversion and inc. anxiety
like behaviours
- Summary of Social interaction manipulations
- Stimulation of dopamine neurons that specifically project to the NAc promotes social interactions.
- 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?
- 90% of neurons in NAc are projection neurons
- Half express the dopamine D1 receptor
- Blocking dopamine D1 receptors in the NAc blocks inc. in social interaction that is caused by optical
dopamine neuron stimulation.
- half express the dopamine D2 receptor
- Def NAc projection neurones:
- 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?
- Rhodopsin
- light-sensitive g-protein coupled receptor
- signaling cascade that is very different from dopamine’s.
- intracellular portion
- mediated by Gt, with dec. cGAMP
- Opto-D1
- has the extracellular (light sensitive) side from rhodopsin
- intracellular (g-protein signaling side) from the dopamine D1 receptor
- D1 receptor that is normally activated by the dopamine now responds to light
- complicated merger: have to many many different cuts because it crosses the membrane so many
times. (not manipulating protein, manipulating a string of DNA)
- D1 receptor
- transmembrane protein, one protein crosses the membrane many times
- extracellular portion binds dopamine
- intracellular portion
- mediated by Gs which inc. cAMP
- Dopamine D1 receptor activation in the NAc is sufficient to inc. social interactions.
- Opto-D1-eYFP
- cre-dependent virus that encodes
- Drd 1:: Cre
- a transgenic mouse that expresses ‘cre’ under control of the dopamine D1 receptor gene
promotor
- 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.
- 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?
- transgenic mouse that expresses cre-recombinase under control of the dopamine D1 receptor
gene promotor.
- 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