Karteikarten
PSYC 318- Lecture#3
Karten in diesem Stapel (34)
- 12nd PaperQ: Are dopamine neurones active during social interactions?
- 2Methods:a) metal wires
b) problem- difficult to know what kinds of neurones you are recording from (b/c dopamine neurones are mixed w. other kinds cell types in midbrain - 3Q: How can we find out when dopamine neurones fire Q: How can we make neurones glow when they fire action potentials?a) during an action potential Ca2+ ions enter & inc. intracellular Ca2+ levels for milliseconds
b) GCaMP (engineered = fluorescent 'genetically encoded' calcium indicators) glow when bindings to Ca2+
c) get dopamine neurones to express synthetic DNA - 4GCaMP= 'genetically encoded' calcium indicatora) 'genetically encoded' = protein that can be made from DNA in cells
b) fused from 3 diff. proteins = GFP, calmodulin, M13
c) GCaMP #6: sensitive enough to detect single changes in action potentials (monitoring fluorescence) - 5Method GCaMPa) inject AAV into midbrain a.k.a. where dopamine is located ( cell body infecting virus)
b) virus contains DNA for GCaMP
c) DNA only expressed under the condition that infected cell also expresses 'cre' ( a.k.a. 'cre' is present)
d) neurones express 'cre', 'stop signal' will be excises & GCaMP proteins will be made - 6Q: How do we determine precisely when genetically-identified dopamine neurones fire?a) inject virus into transgenic mouse where dopamine neurones express cre-recombinase
b) 'tyrosine hydroxylase-cre mouse'
c) tyrosine hydroxylase = enzyme makes dopamine (TH gene is only read in neurones producing dopamine & norepinephrine)
d) are-dependant virus affects diff. kinds of cells in the midbrain, but only dopamine neurones express 'cre' so only they will be affected & produce GCaMP
e) GCaMP will floresce for 100ms after each action potential - 7Overview of Techniquea) DIO indicated 'cre' needed to read DNA
- 8Dopamine Neurone activity is detected during juice reward consumptiona) red lines = mouse licked juice containing substance
b) mice expressing eYFP (& not GCaMP) are control animals (fluorescence is not sensitive to Ca2+ levels)
c) - 9Dopamine neurones are active during social interactionsa) red lines = social interactions
- 10Dopamine neurones are also active in interactions w. novel objectsmeasured w/ GCaMP
- 11Dopamine neurone is diff. between social & novel object interactions ( but dopamine neurones in midbrain are active in both )a) dopamine activity during a social interaction determines how quickly an animal will come return to the interaction once again
b) measure VTA object activity - 12Does stimulation of dopamine neurones promote social interactions?test: insert metal wires in midbrain delivery excitatory impulses ( but everything passing through would be stimulated )
test: give dopamine receptor agonist (see if dopamine receptor activation drives social interactions); problem= synaptic & extra synaptic dopamine receptors would be activated (all over body)
big problem: activating receptors with drugs produces a diff. 'activation profile' compared w/ NT i.e. many NT receptor desensitize/inactivate - 13Optogeneticsdef: use of light to control neurones which have been made sensitive to presence of light through foreign DNA insertion
i) foreign DNA encodes light sensitive-proteins (opsins) - 14How Optogenetics Works #1a) take light sensitive gene from algea
b) take gene for this protein
c) insert DNA into specific neurones in the brain
d) you can cause neurones to fire by flashing light
* w/ right combination of neurones you can activate whole brain circuit - 15How Optogenetics Works #2a) excitatory opsins ChR2, C1V1
i) you can either pulse light
ii) leave light on
b) inhibitory opsins (Halo, NpHR, Arch)
i) long light pulse - 16Does optical exciting dopamine neurones promote social interactions?a) cre-dependent viral DNA the encodes excitatory opsin ChR2 (channelrhodopsin-II), delivered w/ AAV
- 17Optical stimulation of dopamine neurones promotes social interactions & optical inhibition of dopamine neurones dec. social interactionsbut these optical stimulations/ inhibitions do not affect novel object interaction
- 18Dopamine neurones project to many diff. place. Which pathway is driving this effect?mesotelencephalic dopaminergic system
a) tried to identify regions that showed elevated activity in response to same ontogenetic stimulation of dopamine neurones that influence behaviour - 19Immediate Early Genes ( IEGs ) a.k.a. Activity-Dependent Genes)a) def: definitions that are transcribed in response to elevations activity (from norm)
b) activated to variety of cellular stimuli, standing response mechanism
c) IEG (index of neural activity)
i) inc. in neural activity (action potentials) = IEG expression
ii) IEG presence means cells was recently (mins-hrs.) firing above baseline
iii) most commonly measured: cFOS & Arc are two of the most commonly measured IEGs in neurobiology - 20VTA stimulation -> downstream cFos quantificationa) nuclear stain (makes all cells)
b) ChR2 in dopamine in NAc axon terminals
c) antibody for cFOS (marks recently active cells)
d) antibody for dopamine related enzyme (confirms presence of enzyme) - 21Dopamine neurone stimulation causes cFOS expression in 2 placesNAc & PFC but not basolateral amygdala
- 22Which dopamine neurone projection promotes social interactions NAc or PFC?a) ChR2 (optogenetic proteins) can diffuse far; located in axon terminals of cell
b) activating opsins located in axon terminals is an effective way to get pathway-specific activation/inhibition - 23Shining light into NAc will activate all axons there that have ChR2 #1a) possible that axons that travel through NAc but don't terminate there are important
- 24Shining light into NAc will activate all axons there that have ChR2 #2a) virus AAv infects cell bodies & encodes mCherry & WGA-Cre
b) WGA-Cre is a protein jumps across synapses
c) WGA-Cre is used to transport Cre backwards to all NAc projecting neurones
* rabies virus would have worked here too
* IRES sites indicates to cell this is the start of a distinct protein - 25Summary of social interaction manipulationsa) dopamine cell body stimulation
b) dopamine axon terminal stimulation in NAc
c) dopamine axon terminal stimulation in NAc (w/o fibers of passage problem
d) dopamine cell body inhibition - 26Optical stimulation of dopamine axons in PFC causes a conditioned place preference aversionconditioned/unconditioned chamber
- 27Optical stimulation of dopamine neurones in PFC promotes an "anxiety-like" effectdec. exploration of open areas
- 28stimulation of dopamine neurones projecting to PFCa) doesn't promote social interactions
b) creates place aversion & inc. anxiety like behaviours - 29stimulation of dopamine neurones projecting to NAca) promotes social interactions
b) & are active during social interactions - 30Do NAc projecting neurones selectively fire during social interactions?result = image calcium transients in dopamine terminals in NAc
- 31If dopamine release in NAc promotes social behaviour, what receptors there are importanta) 90% of neurones in NAc are projection neurones?
b) 50% express D1 receptor/ 50% express D2 receptor
c) block dopamine D1 receptors in NAc (w. D1 antagonist) blocks inc. in social interaction caused by dopamine neurone stimulation - 32Dopamine neurones release dopamine, glutamate, & GABA- how do you know D1 receptor is sufficient?D1 receptor sensitive (responds) to light
a) extra cellular (light- sensitive side) rhodopsin
b) intracellular (g-protein signalling side) dopamine - 33Dopamine D1 receptor activation in NAc is sufficient to inc. social interactionsActivating dopamine D1 receptors can have many consequences for cells
a) Does firing rate of these neurones change following opto-activation?
b) shining light in NAc to activate onto D1 receptors inc. neural activity in NAc neurones located next to metal wire - 34Is an increase in firing of dopamine D1 receptor-containing NAc projection neurones sufficient to promote social interactions?yes, it promote social interactions
Informationen
- Beschreibung
- Psychology Karteikarten am PSYC 318- Lecture#3, erstellt von Pascale Bockelmann am 11/01/2017.
- Erstellt von
- Pascale Bockelmann
- Aufrufe
- 18
- Bewertung