PSYCH 318
Behavioral Neuroscience II
Salah El Mestikawy
Week 5, Class 1: VGLUT3 and basal ganglia
VGLUT3 in TANS and addiciton
Glutamatergic neurons
• The « old view » of glutamatergic pathways was pretty straightforward
• Functions of glutamate in the brain
o Major excitatory neurotransmitter in the CNS (20% of glutamate)
o Involved in all brain functions (cognitive, memory, sensori‐motor, limbic, etc…)
o Gliotransmitter
o Metabolism (80% of glutamate)
• Key actors in the brain!
Vesicular glutamate transporters – VGLUTs
• Vesicular transporters mechanistic
o Neurotransmitter transporters are secondary transporters
Accumulation of protons inside the vesicle by primary transporters leads to:
• A charge difference
• A pH difference
The Proton gradient has two components!
• ΔμH = ΔΨ + ΔpH
Recall 3 large transport families
• First – Sugar Porter:
o Derived from bacterial proteins that transport sugar
o VMAT
Uses mostly pH
o VACHT
• Second – Amino Acids Auxine Permeases:
o VIAAT or VGAT (vesicular Gaba transporter)
VGAT uses both charge and pH
o Carry both GABA and Glycine
• Third – Vesicular Glutamate Transporterd
o Glutamate
Uses mostly charge
Vesicular glutamate transport in brain vesicles
• mM affinity for glutamate
• No transport for aspartate, glutamine or GABA
• Dependent on the Δψ component of pH gradient (not the ΔpH)
o Specific transporters: Use mostly charge difference.
o Any cation will do the job
• Inhibition by dyes: Rose Bengal, Trypan Blue, Evans Blue (10‐100nM)
The VGLUTs family shows reasonable homology: VGLUT1,2&3 are pretty conserved.
Cells can be used for research:
• Purify cells, assay for glutamate accumulation.
• Low initial material means difficult assay.
• Identical Vesicular Transport of glutamate by VGLUT1‐3.
o Clones had same properties as the cells in brain.
o Why 3 subtypes for the exact same job?
VGLUT1&2 in the rodent brain
• Analyzing anatomy: In‐situ hybridization
o Detect (using radioactive probes) biosynthesis of vesicular glutamate transporters
(parasagittal descents)
VGLUT1:
• Expressed in Cortical areas:
o Cerebral cortex, hippocampus and little in thalamus/brain stem
nuclei
VGLUT2:
• Almost complimentary to VGLUT1 at the mRNA level, mostly sub‐
cortical expression
Recall thalamus projects to thalamo‐striatal terminus
Few regions of overlap
• Antiserums:
o Images are different!
mRNA and protein distributions are DIFFERENT!
o Cortex projects massively to striatum.
Proteins travel
Mismatch is seen in mRNA vs Protein levels
o No vglut in striatum or accumbens
o Medium spiny neurons are GABAergic
Striatum and accumbens are mainly GABAergic
The rest is cholinergic.
o Glutamatergic cells are principal cells
Leave structure where the soma are. Ultrastructural localisation of VGLUT1 & VGLUT3
• Post‐synaptic density can be visualized
• VGLUT1&VGLUT2 are contained in asymmetrical synapses
o These are characteristic of classical glutamatergic neurons.
o VGLUT1 & VGLUT2 are « glutamatergic »
If VGLUT1 and VGLUT2 account for all « classical » glutamatergic neurons…
• Why the need for VGLUT3 ?
VGLUT3 is expressed by (± all) 5‐HT neurons
• VGLUT3 mRNA is expressed in the Dorsal and medial raphe.
o Area for all serotonergic neurons.
• Another experiment: Label VGLUT3 mRNA in blue and SERT in red (serotonin transporter)
o Many (maybe all?) serotonergic neurons express both SERTand VGLUT3.
o Serotonergic neurons have the potential to transmit info via serotonin or glutamate!
VGLUT3 is expressed by some GABAergic interneurons (Basket cells CCK‐positive) of the
hippocampus and the cortex
• Hypocampus: Mainly glutamatergic pyramidal cells, and <10% GABAergic interneurons.
• Scientists saw a pattern of interneurons.
• Top pic: Some interneurons express VGLUT3 mRNA
• Bottom pic: Colocalization of VGLUT3 & VIAAAT is seen
o Why would you release opposite neurotransmitters at the same time?
VGLUT3 is expressed by all cholinergic interneurones from the striatum
• All the cholinergic interneurons from the striatum express VGLUT3!
o Can communicate with both glutamate and acetylcholine
• Cholinergic interneurons are also named TANs (tonically active neurons)
o This is because they fire regularly (or in bursts)
• What is the impact of these “bilingual” systems on addiction?
Not one, but three different Glutmatatergic systems
• System 1, VGLUT1: Cortical
• System 2, VGLUT2: Subcortical
• System 3, VGLUT3: Heterologous
o Presence of VGLUT3 in cholinergic interneurons, GABAergic interneurons and
serotonergic cells.
Question :
• What is the role of VGLUT3 in « heterologous » neurons ?
• VGLUT3 is found in :
o Cholinergic interneurons (striatum)
o GABAergic basket cells (CCK‐positive) interneurons (hippocampe, cortex)
o Serotoninergic neurons (raphe nuclei)
VGLUT3 in Striatal cholinergic interneurons: A new function of glutamate
The Striatum and Brain Disorders:
• Function of the striatum:
o Locomotor:
Planning and modulation of locomotor activity
o Limbic Regulation:
Egocentric navigation
Goal directed behavior
Habit formation
Reward
• Neuropathologies involving the striatum
o Neurological Diseases
Parkinson disease
Huntington disease
Progressive supranuclear palsy
o Psychiatric Disorders
Addiction
Schizophrenia
Obsessive Compulsive Disorder
The old view on Striatal cholinergic interneurons (TANS):
• Convergence of DA and ACh onto SMS to regulate the output (D1 and D2‐positive MSNs) of
striatal activity
• Striatum receiving somatotropic projections from the cortex
o D1: Pro‐Reward
o D2: Anti‐Reward
• Cholinergic Interneurons: Fire regularly (TAND) express VGLUT3 and also project on MSN
• Thought Acetylcholine & Dopamine had opposite effects on MSN.
This is wrong!!
Imaging: see a massive colocalization
• VGLUT3 and SERT and
• VGLUT3 with VaChT
o VGLUT3 and VAChT are present in the same terminals
o VGLUT3 is found in TANS
Are VGLUT3 and VAChT present on similar or different vesicles?
• Use antibody that binds to VGLUT3, pass through immune beads…
• They are found in the same vesicles!!!
o What functional consequences does this have?
Vesicular Synergy:
• VGLUT3 impacts on accumulation of acetylcholine.
o Glutamate stimulates Acetylcholine vesicular accumulation
• Vesicular uptake of acetylcholine:
o In presence of glutamate, the amount of accumulated acetylcholine can double or
triple.
o Experiment:
Take normal mice, do vesicular accumulation of acetylcholine, you get 100%
Same thing with presence of glutamate, 200%
Absence of VGLUT3 channel, no more increased stimulation by glutamate.
Vesicular synergy is clearly due to VGLUT3
• Recall acetylcholine uses delta pH to load vesicles.
o 1 acetylcholine through VAChT exudes 1 proton.
o Using a negatively charged molecule like glutamate, you can increase the proton
gradient by lowering the charge gradient/
Used as a counter‐ion to increase the size of the proton gradient
Convergence of transporters increases the size of cholinergic transmission
Optogenetics:
• Record in Striatum
• Found that stimulation of TANS (have VGLUT3) creates a small glutamatergic current
VGLUT3 molecular roles in TANS:
1) VGLUT3 amplify ACh transmission
2) Glutamatergic signaling
• CONCLUSION : Because of VGLUT3 presence, TANS are bilingual!
o They communicate with Ach and glutamate!
Vesicular synergy extension in the brain
• Growing discovery, other examples have been found.
o Dopaminergic neurons from VTA and substancia Nigira express VGLUT2 at a certain
point in life.
• VGLUTs colocalization with other vesicular transporters has 2 major consequences:
o Co‐transmission
o Vesicular synergy
• Any counter ion that will be uploaded in synaptic vesicles as Ach, DA or 5‐HT will have the
same effect
VGLUT3 roles in TANs
1. VGLUT3 amplify cholinergic tones (+200% !)
2. Glutamatergic signaling
• Vesicular synergy – a new role of glutamate in neurotransmission
o (presynaptic regulation of neuromodulation)
• Because of the presence of VGLUT3 & VAChT , TANS communicate with Ach and glutamate
o Co‐transmission
o TANS are bilingual!
What are the functional consequences of VGLUT3 presence in TANS?
• VGLUT3 knockout mice lost mRNA and protein expression
o A total knockout, can be informative.
• Is there compensation/imbalance of surrounding system
o Important to check since neurotransmitters work together.
o Will changing one component affect others?
Study: Checked 35 markers, none were modified.
Cholinergic cells looked fine.
Can interpret results as a consequence of VGLUT3 loss, not interactions
No major anatomical changes in the striatum of VGLUT3‐KO (VGLUT3 Knock‐out)
VGLUT3 and dorsal striatum
• TANS in the dorsal striatum (sensori‐motor) contribute to the regulation of locomotion and
goal directed behaviors
• Question: does VGLUT3 impact on locomotor activity ?
Basal locomotor activity:
• Study:
o Locomotor activity recording:
Put mouse in chamber with two rays of infrared beams.
Recording “horizontal” & vertical beam cuts to observe activity.,
o Knockouts are slightly hyperactive (a significant difference)
Which system is responsible for this small hyperactivity?
o Use another mouse:
Less achetylcholine, hypocholinergic.
Block ach degradation.
You should restore WT phenotype provided effects observed are due to
acetylcholine.
o Result suggests hyperactivity of VGLUT3 knockout mice caused by acetylcholine
depletion.
o VGLUT3‐KO are slightly hyper active
o Hyperactivity is abolished by donepzil (AChE inhibitor)
• Haloperidol‐induced catalepsy
o Haloperidol is a DA reverse agonist
o VGLUT3‐KO are less sensitive to haloperidol induced catalepsy
VGLUT3 and addiction
• TANS in the nucleus accumbens contribute to the regulation of motivated behavior and
reward
• Question: does VGLUT3 impact on reward behavior ?
Addiction:
• As defined by the DSM‐IV:
• Indicated by the presence of three or more of the criteria listed below in the last 12
months.
Tolerance: Does the patient tend to need more of the drug over time to get
the same effect?
Withdrawal symptoms: Does the patient experience withdrawal symptoms
when he or she does not use the drug?
Continued use of drug despite harm: Is the patient experiencing physical or
psychological harm from the drug?
Loss of control: Does the patient take the drug in larger amounts, or for
longer than planned?
Attempts to cut down: Has the patient made a conscious, but unsuccessful,
effort to reduce his or her drug use?
Salience: Does the patient spend significant time obtaining or thinking about
the drug, or recovering from its effects?
Reduced involvement: Has the patient given up or reduced his or her
involvement in social, occupational or recreational activities due to the drug?
• A compulsive pattern of drug‐seeking/drug‐taking behavior that takes place at the expense
of most other activities. It leads to a loss of control despite negative consequences and
reoccurring episodes of abstinence and relapse.
• Addiction is fundamentally a pathology of the reward system
• One can be addicted to:
To substances: psychostimulants (cocaine, amphetamine), opiods, alcohol,
nicotine, sugar, food
• Only a few compounds in nature have addictive properties.
To “behaviors”: gambling, computer games, sex, work (falling in love?)
• Love is a good thing! Addiction can be a strength too!
• Controversy: Can we model addiction in mice?
• Often, addiction conjures up thoughts of pleasure.
• However, addiction is not related to pleasure.
Overtime “liking” of reinforce decreases! However, “wanting” increases.
Hedonia (pleasure or liking) vs Incentive salience (wanting)
Addiction is the pathology of the wanting. Rationale for suspecting VGLUT3 implication in addiction
• Dorsal striatum involved in Locomotor activity
• Ventral striatum involved in reward
o Addiction is often viewed as a dysregulation of reward circuits
o VGLUT3 is expressed by TANS.
These cholinergic interneurons play a central role in the regulation of reward
circuits
Are TANS implication in addiction?
• Cell ablation by immunotoxin‐mediated cell targeting technology
o Specifically kill cholinergic interneurons in the accumbens.
o Monitor CPP: Conditioned place preference to cocaine
• Ablation of TANS in the nucleus accumbens increase sensitivity to cocaine
Ach silencing in the striatum minimally impact on reward
• VAChT‐KO (in the striatum) minimally alters cocaine sensitivity
o Knockout in striatum of cholinergic interneurons produces almost no effect.
How do TANS regulate reward and addiction?
VGLUT3 in TANS:
• TANS use 2 transmitters
o Glutamate
o Acetylcholine
Behavior:
• Addiction as defined by DSMIV or experienced in human cannot be modeled in rodents.
• But part of it (endophenotypes) can be modeled:
‐ Locomotor activation
• Cocaine induced hyper‐locomotor activity can be measured.
‐ Sensitization
• Drug Memory
‐ Positive association with a given environment
‐ Salience (craving or wanting)
‐ Extinction
‐ Relapse
Study:
• Inject cocaine
o Cocaine: Increase 7X~
o VGLUT3 knockout + Cocaine: locomotor activity is increased ~18X
• Conclusion:
o VGLUT3‐KO are more sensitive to cocaine‐induced locomotor activity
Conditioned Place Preference
• Learn to associate particular environment with drug treatment
• Pairing drug with environment:
o Use a Y‐maze.
o Pair one compartment with cocaine, other with Saline
Alternate pairings
Test: Is there liking for a particular compound?
• VGLUT3‐KO mice have an increased preference for the cocaine‐associated compartment!
o (WT mice can reach KO preference with increased concentrations.)
Cocaine self‐administration
• Implant catheter in vascular system of mouse.
• Connect it to syringe with cocaine.
• Light = on, system active.
• Nose‐poke = cocaine injection.
o Mice decides when it wants cocaine
• VGLUT3‐KO mice do much more nose‐pokes before plateau compared to WT.
o Change rules: 3 pokes for a single injection.
Does moue know to change behavior?
How much are they willing to work for cocaine?
o Day 10: to get cocaine reward:
1 time, 1 nose poke
2X, 2 nose pokes, etc.
• When does the mouse stop playing the game?
• How much does it want to work/want the drug?
WT pokes 40X for reward
VGLUT3: go all the way to 80!
• Very strong will to get drug!
o Control: Does the mouse really like the drug, or is pushing the pedal what it wants?
Light turn off, system inactive
Extinction curve! Mice learny quickly that poking is useless.
• Conclusion:
o VGLUT3 has important role in reward and cocaine‐reinforcing properties.
o Behavior!
VGLUT3 and addiction
• VGLUT3‐KO induces major facilitation of cocaine‐self administration
o Higher level of acquisition of self‐administration
o Improved performance of FR1 and FR3
o Increased motivation to obtain the drug
o Comparable levels of extinction
o Augmented susceptibility to relapse
VGLUT3 and underlying molecular mechanisms
• TANS use 2 transmitters
o DA release, DA neurons firing
o DA signaling cascade
o MSN D1 or D2 morphology
o Cortico‐striatal plastiticity
All addictive compounds or behaviors have in common that they induce a DA release in the NAc
• Dopamine release in the accumbens is a very important addiction marker
o (but not in dorsal striatum)
What properties of DA neurons can be observed?
• DA neurons have two modes of firing in the VTA:
o Single Spike – Regular, Isolated spiking
Or
o Bursting
Addictive compounds or behaviors have in common that:
• They increase burst firing of DA neurons in the VTA
o They induce a DA bursting in the VTA
o DA neurons fire regularly:
Turn on light saying cocaine will follow, neurons will burst
o DA neurons increase firing when reward is predicted
o **May also increase for wanting of a drug, not confirmed**
• Meanwhile TANS pause in the NAc
o Cholinergic neurons will pause after the cue!
In vivo (anesthesized mouse)
• Track DA release related to stimulation of the accumbens.
o Use voltametry
• DA release is doubled with VGLUT3 Knockout
• Likely occurs in accumbens as DA release is massive.
o VGLUT3‐KO increases DA release (without modification of firing in the VTA)
o VAChT‐KO decreases DA release
D1 & D2 neurons
• Some biochemical markers allow you to
measure D1 & D2.
o Ex: Receptor itself.
Increased number of D1, no
change in D2 when looking at
N. Acc.
Suggests D1 (pro‐reward
pathway) activated.
• D1 neurons are coupled to CAMP, cascade.
o P‐ERK is a clear marker of D1 pathway
exclusively in the striatum
o VGLUT3‐KO have an increased P‐ERK
response in the accumbens
Study:
• Measure # of dendritic spines
o (Active pathway will increase this #)
• WT, VGLUT3‐KO, Saline, Cocaine.
o KO have ~50% more spines than WT!
A LOT!
Especially since initially they’re already covered with spines!
• Spines related to increased protein synthesis
o Spines are where cortico‐accumbens pathways connected to each other.
o Where glutamatergic terminals will reach the MSNs and enter the system.
• Increased spines:
o When you record activity of cortico accumbens pathway, you find an increased
activity:
Increased frequency of glutamatergic signaling
Increased AMPA activity of NMDA.
• Whole pathway (D1 MSN pro‐reward pathway) is constitutively activated in VGLUT3‐KO mice
VGLUT3‐KO Constitutive modification of the brain:
• Increase of dendritic spines
• Activation of glutamatergic cortico accumbens pathway
Dopamine release in the NAc
• Constitutive modification of the brain of VGLUT3‐KO, even before receiving cocaine:
o Increased DA release
o Increased density of D1 receptors
o Increased reactivity of the D1‐pathway (pro reward)
o Increase of dendritic spines
o Activation of glutamatergic cortico accumbens pathway
• NB: DA encodes for the prediction of reward and for the wanting; cortico‐accumbens
glutamatergic pathway encodes for the context
How we think it’s working:
• Working hypothesis:
o TANS release Ach that stimulates DA release (through nicotinic receptors:
established) and glutamate that inhibits DA release through mGLUR present on DA
terminals (to be established).
o Alternatively mGLUR can be present on Ach terminals
Shown: spine of D1 positive MSN
• VTA dopaminergic terminal controls the activity of neck of the spine
• On top of spine, you have the cortico‐excitatory pathway which arrives from the cortex.
• To the right, TANS control what’s going on:
o Release both Ach (red) and glutamate (green)
Acetylcholine has a stimulatory effect on the release of dopamine.
Glutamate has an inhibitory effect (because of voltammetry experiment)
• Get rid of VGLUT3 – Only release Ach.
o Increase in DA release/nicotinic receptor because only excitation component is on.
Recall:
• Ach will stimulate DA release, activate pathway, increase # of spines, increase the
glutamatergic transmission.
o One pathway codes for wanting of drug
o Other codes for context
• When this goes wrong:
o Saline VGLUT3 KO mouse already has the brain of an addict!
o VGLUT3 mutation = at risk for addiction.
DA and Ach release
• Diminished Ach release in the striatum of VGLUT3‐KO
• No change of DA release
VGLUT3 in TANS and Ach/glutamate cotransmission:
• In TANS VGLUT3: accelerate ACh vesicular accumulation and transmission + glutamate fast
transmission (AMPA) + glutamate slow transmission (mGLUR)
• In the NAC TANS use preferentially glutamate to regulate reward
• In the dorsal (sensori‐motor, DM) TANS use preferentially ACh to regulate locomotor activity
• In the dorsal (limbic, DL) ?
VGLUT3 and addiction
• Absence of VGLUT3 increases:
o Addiction endophenotypes
o DA release (through glutamate: mGLUR ?)
o D1DR and D1 activated cascade (pro‐reward)
o Dendritic spines
o Glutamatergic transmission
• DA and glutamate activation in the NAc are biochemical hallmarks of addiction
• DA
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