1. 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

2. 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

3. 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)

4. Investigate neuronal pathways , type of neurons and molecules that might mediate functional interactions between stress and reward systems

5. Stress responses are mediated by corticotrophin-releasing factor (CRF) originated from different cell types located in several brain areas

6. Mesocorticolimbic DA system
Reward responses are mediated by dopamine (DA) produced by neurons located in the ventral tegmental area (VTA)
Mesocorticolimbic DA system
Hippocampus
Prefrontal cortex
Nucleus accumbens
Ventral Tegmental
Area (VTA)
Olfactory tubercle
Amygdala
Dopamine neurons

7. 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?
The mesolimbic dopamine system consist of a group of dopamine neurons that resides in the ventral tegmental area. These dopaminergic neurons send dopaminergic projections to limbic structures, such as the nucleus accumbens, prefrontal cortex and amygdala. In return, the VTA receives neuronal innervations from several brain areas.
(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)

8. 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

9. Do CRF cells establish functional interactions (synapses) with cells located in VTA?
Yes
CRF (-) axonal terminal
CRF (+) axonal terminals
Presynaptic
CRF
Postsynaptic
dopamine?
CRF (-) dendrite
Synapse

10. 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

11. Do CRF cells establish synapses with dopaminergic neurons in VTA?
Yes
Symmetrical
synapse
CRF (+) axonal terminals
83 %
17 %
TH (+) dendrites
Asymmetrical
synapses
EXCITATORY
INHIBITORY

12. Aim: CRF receptor 1 (CRF-R1) CRF receptor 2 (CRF-R2)
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?

13. (Double in situ hybridization)
DNA
mRNA
Protein
Method
(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

14. Regional Distribution
Expression of CRF Receptor 1 (CRF-R1) mRNA in the Ventral Tegmental Area
Regional Distribution
SNC
VTA
SNC
VTA
TH mRNA
CRF-R1 mRNA
Hybridization with non radioactive antisense RNA probes to detect TH mRNA .
Hybridization with radioactive antisense RNA probes to detect CRF-R1 mRNA
VTA = Ventral Tegmental Area
SNC = Substantia Nigra Compacta

15. 71.46% of all CRF-R1 expressing neurons are dopaminergic in VTA
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

16. CRF binding protein (CRF-BP)
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- aspartate) receptors in VTA dopaminergic neurons

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

18. Expression of CRF Binding Protein (CRF-BP) mRNA in VTA Dopaminergic Neurons
TH mRNA .
Hybridization with non radioactive antisense RNA probes to detect TH mRNA
Hybridization with radioactive antisense RNA probes to detect CRF-BP mRNA

19. Expression of CRF Binding Protein (CRF-BP) mRNA in VTA Dopaminergic Neurons
TH mRNA
CRF-BP mRNA

20. 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

21. Why is this important? New targets for medication development
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

23. 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

24. Acknowledgement (Ultrastructural studies) Emma Roach
Patricia Tagliaferro Ph.D.
(Ultrastructural studies)
Emma Roach
(In situ hybridization studies)
CHECK names
Support: NIDA IRP