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

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

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

12. Aim: To investigateneuronal 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. Method DNA mRNA Protein (Double in situ hybridization) • Brain sections were hybridized with a non-radioactive anti-sense TH riboprobeto label DAergic neurons • Same sections were hybridized with a radioactive anti-sense CRF-R1,CRF-R2andCRF-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. VTA VTA SNC SNC Expression of CRF Receptor 1 (CRF-R1) mRNA in the Ventral Tegmental Area Regional Distribution 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. 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-R1expressing neurons aredopaminergic in VTA

16. 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. VTA VTA SNC SNC Expression of CRF Binding protein (CRF-BP) mRNA in the Ventral Tegmental Area Regional Distribution 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 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

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 themesocorticolimbic 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? 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 • Patricia Tagliaferro Ph.D. • (Ultrastructural studies) • Emma Roach • (In situ hybridization studies) Support: NIDA IRP