Cocaine on the BrainAlterations in D1/D2 Medium Spiny Neurons of the Nucleus Accumbens Shell Due to Cocaine Exposure

HMB420: Kathleen, Urshita, Majid

I can’t get enough of this stuff!

Goals- Understand what this neurobiological system is

- Basal Ganglia mNAc Anatomy and Connectivity

- Understand the research methods- Optogenetics; Pharmacogenetics; DREADS; Two-Photon

Microscopy, Patch-Clamps, Paired Pulse Facilitation, etc.

- Understand what was found- NAc D1/D2 changes due to cocaine- Inputs from V. Hippocampus and BL Amygdala

- Understand how this could shape behaviour - Theories for addiction

?

NAc

Action Initiation or Inhibition- Which of several possible

behaviors will be performed

Emotional & Motivational - reinforcement of wanted

behavior - suppression of unwanted

behavior

What are the Basal ganglia?- Striatum

- Putamen- Caudate

- Nucleus Accumbens (NAc)

- Medial Shell

Anatomy of the NAc● NAc Shell (medial, lateral & ventral)

○ limbic system - regulation of Reward behaviours

● Medium Spiny Neurons (MSN) ~90%○ GABA-ergic

■ D1 or D2 receptors○ large ∆ spine density, morphology excitability

■ ∆ behaviours (i.e. sensitization)

● Inputs from ○ Ventral Tegmental Area○ Prefrontal Cortex○ Basolateral Amygdala○ Ventral Hippocampus

Not as well understood

Direct and Indirect PathwaysDirect - GO!- NAc D1 MSNs= EPSP= facilitate action

Indirect - STOP!- NAc D2 MSNs=IPSP= facilitate action

Reason for Experimentation

Can such addiction related behaviour be seen as changes in the mNAc and associated circuits?

Is cocaine’s behavioural sensitization reflected in NAc D1/D2

MSN synaptic connectivity?

Question #1

Pilot Data; Confirmation- Rewarding/ addictive drugs increase locomotion in mice

- behavioral sensitization => addiction

- Ensured;- Behavioral sensitization and; - Synaptic changes of MSN post cocaine

- Patch clamp D1/D2 MSN mEPSCs- Recorded at −80 mV- TTX - block AP - Gabazine - block

GABAA receptors

Function/ Structure Change; Methods

Example of method; Whole cell patch using diffusion contrast + fluorescence in Purkinje neuron

- Two-photon microscopy:- D1/D2- MSNs with

fluorescent dye- 3D model to compare

density and volume

Example of 3D synapse modeling

Function/ Structure Change; Results - Cocaine increased D1-

mEPSCHz - No mEPSCAP change

- Cocaine increased D1-MSNs density

May reflect greater numbers of excitatory connections

Sensitization is strongly associated with functional and structural

changes in NAc D1-MSNs synaptic connectivity.

Answer #1

Question #2

Are cocaine induced changes restricted to certain cell types or

cortical inputs?

Input connectivity; Method - Optogenetics:

- AAV-ChR2 in the BLA, VH or PFC. - Only BLA, VH and PFC afferents to mNAc.

Input connectivity; Results- Enhanced BLA D1/2-EPSCs - Dampened VH D1/2-EPSCs - No change with PFC

“Initially stronger VH inputs got

weaker, and BLA inputs became

much stronger?! What does this

mean?!”

Hopefully you are thinking,

Answer #2

Cocaine differentially alters ratio of D1/D2-MSNs NAc afferents, with cell type and input specificity from BLA/

VH inputs.

Question #3

Is observed synaptic plasticity reflected as altered qEPSC

changes?

Postsynaptic Changes; Method - Use light evoked qEPSC visualized

with Extracellular Strontium uptake- Direct estimate qEPSC efficacy - Indirect estimate qEPSC connection

LTP

Spine#

- Paired-pulse experiment check

Postsynaptic Changes; Results - Increased BLA qEPSCHz at D1-MSNs

- unaltered BLA qEPSCAP

- VH qEPSCAP decreased at D1-MSNs- unaltered qEPSCHz for VH inputs

- Consistent with patch mEPSC recordings

- Not due to vesicle release differences - Specific postsynaptic effects at D1-MSNs

Suggest cocaine enhances BLA NAc afferents by more inputs, and

decreased VH inputs by dampened inputs onto D1-MSNs.

Answer #3

Question #4

Is observed BLA and VH input changes reflected as altered

synaptic structure?

Subcellular Changes; Method - Two-photon mapping:

- detect spine volume and synapse volume via Alexa Fluor 594

- detect synaptic calcium levels via calcium indicator (Fluo-4FF)

Subcellular Changes; Results- Cocaine selectively enhanced synapse density at D1-MSNs- Repeated exposure to cocaine selectively enhances the

number of BLA connections onto D1-MSNs.

- Spine volume was initially much larger at D1-MSNs for VH inputs in saline-treated mice

- Difference was abolished after repeated cocaine exposure (reduction in spine volume at D1-MSNs)

- Drug exposure suppresses the strength of VH connections onto D1-MSNs

- Synapse density similar at D1-MSNs and D2-MSNs

Answer #4

Repeated cocaine exposure enhances BLA inputs and

suppresses VH inputs onto D1-MSNs

Question #5

Is NMDAR activation required for behavioral sensitization and observed BLA/VH synaptic

plasticity?

Synaptic Induction; Method - In vivo Pharmacology:

- NMDAR antagonist (MK-801) to test for cocaine induced sensitization

- inject MK-801 to test cocaine induced EPSC changes at BLA and VH

- Test if NMDAR is required for the changes in synaptic connectivity

Synaptic Induction; Results - Long term MK-801

- Abolished sensitization - blocked changes to BLA & VH EPSCs

- Acute pretreatment MK-801 - Did not prevent sensitization

NMDAr needed for sensitization

Answer #5

NMDAR activation required for cocaine-induced behavioral

sensitization and changes to BLA/ VH connections onto MSNs.

Is BLA/VH activation responsible for observed sensitization, mEPSC

frequency, and synaptic remodeling of the mNAc?

Question #6

Structure/Function reversal; Method - In vivo pharmacology:

- DREADD (hM4D) with N-oxide (CNO)

- pre-treat CNO/ saline before cocaine - Measure light-EPSC from D1/D2-MSNs- Measure mEPSCHZ and mEPSCAP

- Two photon image for density and volume

Structure/Function reversal; Results Inhibiting BLA activity:

- Abolished behavioural sensitization & only altered BLA EPSCs

Inhibiting VH activity- Remain sensitized but altered VH EPSCs

BLA activity is necessary for cocaine sensitization- Inhibiting BLA activity:

- Eliminated cocaine-induced increase in mEPSCHz at D1-MSNs

- Abolished cocaine-induced increase in spine density at D1-MSNs

- BLA connectivity accounts increased D1 mEPSCHz and D1 spine density.

Supports BLA/ VH EPSCs being dissociable, with only BLA inputs

causing sensitization, through structural and functional BLA D1-

MSNs innervation.

Answer #6

Overall Findings1. Cocaine alters synaptic connectivity in the NAc

○ Sensitization = increased mEPSCHz & spine density at D1-MSNs

2. Connectivity changes are cell type and input specific○ Increased BLA D1 EPSC, dampened VH D1 EPSC

3. Number and strength of connections are altered○ Increased BLA D1 qESPCHz, dampened VH D1 qESPCAP

4. Subcellular structures are altered○ Increased BLA D1 spine density, reduced VH D1 spine volume

5. Connectivity depends on NMDA receptors○ NMDAR antagonist inhibits sensitization, BLA & VH EPSCs changes

6. Depend on amygdala activity○ Only BLA activity needed for sensitization, & altered BLA D1 EPSCs

7. Amygdala activity causes enhanced connectivity○ Only BLA activity needed to increase mEPSCHz, and spine density

NAc MSNs synaptic rearrangement

during sensitization is cell type and input-specific!

So… What does this mean?!● Goal-directed/ motivated behavior regulated by differential

activation of D1/ D2-MSNs in the NAc

● Drug exposure rebalances connectivity to BLA inputs○ VH inputs usually dominant onto D1 neurons○ Thus; motivated behavior may shift from VH contextual inputs to BLA

emotional inputs!

● Types of neurons and individual spines can have huge behavioral effects

● How neural circuits are reorganized in many neurological / neuropsychiatric diseases?

Huh! So maybe I develop more emotional based drive-learning

Where knowing something is bad does not matter, because doing it feels

right, so I keep doing it!

Criticisms/ Future workComparison to VTA?Relevance in overall circuit?Time dependent plasticity?D2 MSN interactions?

Any questions about me?

Andrew F MacAskill, John M Cassel & Adam G Carter . “Cocaine exposure reorganizes cell type– and input- specific connectivity in the nucleus accumbens. Naure Neuroscience. 2014

Will add Bibliography dropbox link

References