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Description
Flashcards by Pascale Bockelmann, updated more than 1 year ago
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Created by Pascale Bockelmann
almost 8 years ago
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| Question | Answer |
| 2nd Paper | Q: Are dopamine neurones active during social interactions? |
| Methods: | 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 |
| Q: 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 |
| GCaMP= 'genetically encoded' calcium indicator | a) '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) |
| Method GCaMP | a) 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 |
| Q: 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 |
| Overview of Technique | a) DIO indicated 'cre' needed to read DNA |
| Dopamine Neurone activity is detected during juice reward consumption | a) red lines = mouse licked juice containing substance b) mice expressing eYFP (& not GCaMP) are control animals (fluorescence is not sensitive to Ca2+ levels) c) |
| Dopamine neurones are active during social interactions | a) red lines = social interactions |
| Dopamine neurones are also active in interactions w. novel objects | measured w/ GCaMP |
| Dopamine 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 |
| Does 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 |
| Optogenetics | def: 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) |
| How Optogenetics Works #1 | a) 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 |
| How Optogenetics Works #2 | a) excitatory opsins ChR2, C1V1 i) you can either pulse light ii) leave light on b) inhibitory opsins (Halo, NpHR, Arch) i) long light pulse |
| Does optical exciting dopamine neurones promote social interactions? | a) cre-dependent viral DNA the encodes excitatory opsin ChR2 (channelrhodopsin-II), delivered w/ AAV |
| Optical stimulation of dopamine neurones promotes social interactions & optical inhibition of dopamine neurones dec. social interactions | but these optical stimulations/ inhibitions do not affect novel object interaction |
| Dopamine 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 |
| Immediate 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 |
| VTA stimulation -> downstream cFos quantification | a) 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) |
| Dopamine neurone stimulation causes cFOS expression in 2 places | NAc & PFC but not basolateral amygdala |
| Which 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 |
| Shining light into NAc will activate all axons there that have ChR2 #1 | a) possible that axons that travel through NAc but don't terminate there are important |
| Shining light into NAc will activate all axons there that have ChR2 #2 | a) 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 |
| Summary of social interaction manipulations | a) 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 |
| Optical stimulation of dopamine axons in PFC causes a conditioned place preference aversion | conditioned/unconditioned chamber |
| Optical stimulation of dopamine neurones in PFC promotes an "anxiety-like" effect | dec. exploration of open areas |
| stimulation of dopamine neurones projecting to PFC | a) doesn't promote social interactions b) creates place aversion & inc. anxiety like behaviours |
| stimulation of dopamine neurones projecting to NAc | a) promotes social interactions b) & are active during social interactions |
| Do NAc projecting neurones selectively fire during social interactions? | result = image calcium transients in dopamine terminals in NAc |
| If dopamine release in NAc promotes social behaviour, what receptors there are important | a) 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 |
| Dopamine 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 |
| Dopamine D1 receptor activation in NAc is sufficient to inc. social interactions | Activating 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 |
| Is an increase in firing of dopamine D1 receptor-containing NAc projection neurones sufficient to promote social interactions? | yes, it promote social interactions |
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