Marisela Morales

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Interactions Between Reward and Stress Systems

• Marisela Morales
• NIDA Intramural Research Program
• Cellular Neurobiology Branch
• Cellular Neurophysiology Section

National Advisory Council on Drug Abuse
The Science of Drug Abuse Addiction
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Identification of neuronal pathways , neurons
and molecules that may be affected or
participate in the biology of drugs of abuse
Diversity. Brain is made of neurons with
different phenotypes
Connectivity. Different phenotypes of neurons
establish functional interactions (synapses) that
determine specific neuronal pathways (specific
behaviors)
Information. Exchange of information among
different neurons in a neuronal pathway is
mediated by molecules
Drugs of abuse affect the structure and function
of the brain
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Interactions between the stress and reward
systems
Different models of stress have shown that it
increases vulnerability to addictive drugs
Stressors increase drug self-administration
Prenatal stress increases amphetamine
self-administration in the adult rat
Single or repeated exposure to stressful stimuli
can augment the motor stimulant action of
amphetamine, cocaine, or morphine
Stressors reinstate drug seeking (model of
relapse). Recent findings
Foot shock reinstates cocaine seeking, however,
transient inhibition of the VTA blocks drug
seeking (McFarland, 2004)
Foot shock reinstates cocaine seeking and induces
release of CRF, glutamate and DA in VTA of
cocaine-experienced rats (Wang, et al., 2005)
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Investigate neuronal pathways , type of neurons
and molecules that might mediate functional
interactions between stress and reward systems
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Stress responses are mediated by
corticotrophin-releasing factor (CRF) originated
from different cell types located in several
brain areas
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Mesocorticolimbic DA system
Hippocampus
Prefrontal cortex
Nucleus accumbens
Ventral Tegmental Area (VTA)
Olfactory tubercle
Amygdala
Dopamine neurons
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Interactions between stress and reward systems.
Brain area?
VTA
(1) Application of CRF into VTA increases
locomotor activity(Kalivas et al., 1987
CRF cell
Do CRF target VTA cells?
GABAergic or DAergic neurons?
(2) Footshock induces CRF release in VTA (Wang et
al., 2005)
(3) In vivo administration of drugs of abuse or
acute stress increase strength at excitatory
synapses on DA neurons (Saal et al., 2003)

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Do CRF cells establish functional interactions
(synapses) with cells located in VTA?
(1) Rat brain sections were incubated with
specific antibodies to label neurons containing
CRF
(2) VTA ultra thin sections (70 nm in thickness)
were obtained from labeled brain tissue
(3) Material was analyzed under the electron
microscope
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Do CRF cells establish functional interactions
(synapses) with cells located in VTA?
Yes
Postsynaptic dopamine?
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Do CRF cells establish synapses with dopaminergic
neurons in VTA?
(1) Rat brain sections were incubated with
antibodies against CRF and tyrosine hydroxylase
(TH, marker of dopamine neurons in VTA)
(2) VTA ultra thin sections (70 nm in thickness)
were obtained from double labeled brain tissue
(3) Material was analyzed under the electron
microscope
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Do CRF cells establish synapses with dopaminergic
neurons in VTA?
Yes
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Asymmetrical synapses
EXCITATORY
INHIBITORY
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Aim
To investigate neuronal pathways , type of
neurons and molecules that might mediate
functional interactions between stress and reward
At the molecular level, CRF mediates its
biological effects by interacting with three
different proteins

• CRF receptor 1 (CRF-R1)

• CRF receptor 2 (CRF-R2)

• CRF binding protein (CRF-BP)

Which of these molecules mediate the functional
interactions between CRF and VTA dopaminergic
neurons?

• Are these proteins present in DAergic neurons in
  VTA?

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Method
DNA
mRNA
Protein
(Double in situ hybridization)

• Brain sections were hybridized with a
  non-radioactive anti-sense TH riboprobe to label
  DAergic neurons

• Same sections were hybridized with a radioactive
  anti-sense CRF-R1, CRF-R2 and CRF-BP riboprobes
  to determine expression of any of these molecules
  within DAergic neurons

Results
CRF-R2 mRNA was not detected in VTA neurons
CRF-R1 and CRF-BP mRNA were detected in VTA
neurons
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Expression of CRF Receptor 1 (CRF-R1) mRNA in the
Ventral Tegmental Area
Regional Distribution
TH mRNA
CRF-R1 mRNA
Hybridization with radioactive antisense RNA
probes to detect CRF-R1 mRNA
Hybridization with non radioactive antisense RNA
probes to detect TH mRNA
VTA Ventral Tegmental Area SNC Substantia
Nigra Compacta
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Expression of CRF receptor 1 (CRF-R1) mRNA in
dopaminergic neurons in the VTA
Hybridization with non radioactive antisense RNA
probes to detect TH mRNA
Hybridization with radioactive antisense RNA
probes to detect CRF-R1 mRNA
TH mRNA
CRF-R1 mRNA
Arrows indicate cellular co-expression of TH
(dark color) and CRF-R1 (green grains) in VTA
71.46 of all CRF-R1 expressing neurons are
dopaminergic in VTA
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At the molecular level, CRF mediates its
biological effects by interacting with three
different proteins

• CRF receptor 1 (CRF-R1)

• CRF receptor 2 (CRF-R2)

• CRF binding protein (CRF-BP)

CRF binding protein

• Peripheral CRF-BP plays a role in lowering free
  circulating CRF levels

• CRF-BP is expressed in different type of cells
  in many brain regions
• (What is the role of CRF-BP in the brain?)

• Studies with mouse midbrain slices indicates
  that CRF-BP is required for CRF to potentiate
  synaptic transmission by N-MDA (N-methyl-D- aspart
  ate) receptors in VTA dopaminergic neurons

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Expression of CRF Binding protein (CRF-BP) mRNA
in the Ventral Tegmental Area
Regional Distribution
TH mRNA
CRF-BP mRNA
Hybridization with radioactive antisense RNA
probes to detect CRF-BP mRNA
Hybridization with non radioactive antisense RNA
probes to detect TH mRNA
VTA Ventral Tegmental Area SNC Substantia
Nigra Compacta
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Expression of CRF Binding Protein (CRF-BP) mRNA
in VTA Dopaminergic Neurons
CRF-BP mRNA
TH mRNA
Hybridization with non radioactive antisense RNA
probes to detect TH mRNA
Hybridization with radioactive antisense RNA
probes to detect CRF-BP mRNA
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Expression of CRF Binding Protein (CRF-BP) mRNA
in VTA Dopaminergic Neurons
TH mRNA
CRF-BP mRNA
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Summary

• Within the VTA, CRF axonal terminals establish
  mainly asymmetrical (presumably excitatory)
  synapses with dopaminergic dendrites

Implications Following stress, synaptical
release of CRF in VTA may directly activate
dopaminergic neurons, inducing release of
dopamine within the mesocorticolimbic system

• Within the VTA, CRF-R1 and CRF-BP are
  preferentially expressed in dopaminergic neurons

Implications At the cellular level, CRF may
affect dopaminergic neurotransmition by
interacting with CRF-R1 and CRF-BP located with
VTA dopaminergic cell bodies

• We suggest CRF excitatory synapses on
  dopaminergic dendrites as a locus for the known
  interaction of stress mechanisms and the
  mesocorticolimbic dopamine system (a system
  implicated in addiction, a number of
  stress-related psychiatric syndromes) and
  co-morbidity between the two

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Why is this important?
We provide evidences indicating that stress
system may directly activate the reward system
through CRF-R1 and CRF-BP
New targets for medication development
CRF-BP is a molecule that interacts with CRF and
is selectively present in DAergic neurons
involved in the rewarding effects of drugs of
abuse
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(No Transcript)
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Current and future studies
(1) Brain circuitry involved in the direct
interaction between stress and reward systems
Identification of CRF neurons that synapse on
VTA dopaminergic neurons brain distribution,
cellular phenotype (receptors, neurotransmitters,
etc.), afferents, etc.
Determination the neurotransmitters (glutamate,
GABA) present in CRF axonal terminals, and
establish at the ultratructural level the
distribution of CRF-R1 and CRF-BP

• (2) Evaluation of effects of drugs of abuse on
  the CRF, CRF-R1 and CR-BP system

(3) Evaluation of the participation of CRF,
CRF-R1 and CRF-BP system in cocaine and
methamphetamine induced behaviors (collaboration
with Dr. Roy Wise)
(4) Set up in vitro studies to determine
functional molecular interactions among CRF,
CRF-R1 and CRF-BP
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Acknowledgement

• Patricia Tagliaferro Ph.D.
• (Ultrastructural studies)
• Emma Roach
• (In situ hybridization studies)

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