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Marisela Morales NIDA Intramural Research Program Cellular Neurobiology Branch Cellular Neurophysiology Section Interactions Between Reward and Stress.

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Presentation on theme: "Marisela Morales NIDA Intramural Research Program Cellular Neurobiology Branch Cellular Neurophysiology Section Interactions Between Reward and Stress."— Presentation transcript:

1 Marisela Morales NIDA Intramural Research Program Cellular Neurobiology Branch Cellular Neurophysiology Section Interactions Between Reward and Stress Systems The Science of Drug Abuse & Addiction National Advisory Council on Drug Abuse

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 Stressors increase drug self-administration Different models of stress have shown that it increases vulnerability to addictive drugs Single or repeated exposure to stressful stimuli can augment the motor stimulant action of amphetamine, cocaine, or morphine Prenatal stress increases amphetamine self-administration in the adult rat Stressors reinstate drug seeking (model of relapse). Recent findings Foot shock reinstates cocaine seeking and induces release of CRF, glutamate and DA in VTA of cocaine-experienced rats (Wang, et al., 2005) Foot shock reinstates cocaine seeking, however, transient inhibition of the VTA blocks drug seeking (McFarland, 2004)

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) Ventral Tegmental Area (VTA) Amygdala Olfactory tubercle Nucleus accumbens Prefrontal cortex Hippocampus Mesocorticolimbic DA system Dopamine neurons

7 Interactions between stress and reward systems. Brain area? CRF cell Do CRF target VTA cells? (1) Application of CRF into VTA increases locomotor activity(Kalivas et al., 1987 (2) Footshock induces CRF release in VTA (Wang et al., 2005) VTA (3) In vivo administration of drugs of abuse or acute stress increase strength at excitatory synapses on DA neurons (Saal et al., 2003) GABAergic or DAergic neurons?

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 terminals CRF (-) axonal terminal CRF (-) dendrite Presynaptic CRF Postsynaptic dopamine? 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 CRF (+) axonal terminals TH (+) dendrites Asymmetrical synapsesSymmetricalsynapse 17 % EXCITATORYINHIBITORY 83 %

12 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)  Are these proteins present in DAergic neurons in VTA? Which of these molecules mediate the functional interactions between CRF and VTA dopaminergic neurons? Aim: To investigate neuronal pathways, type of neurons and molecules that might mediate functional interactions between stress and reward

13 Method (Double in situ hybridization)  Brain sections were hybridized with a non-radioactive anti-sense TH riboprobe to label DAergic neurons CRF-R2 mRNA was not detected in VTA neurons CRF-R1 and CRF-BP mRNA were detected in VTA neurons Results  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 DNA mRNAProtein

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

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 mRNACRF-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)  Peripheral CRF-BP plays a role in lowering free circulating CRF levels CRF binding protein  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 VTA = Ventral Tegmental Area SNC = Substantia Nigra Compacta Regional Distribution Hybridization with radioactive antisense RNA probes to detect CRF-BP mRNA Hybridization with non radioactive antisense RNA probes to detect TH mRNA SNCSNC VTA VTA TH mRNACRF-BP mRNA

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

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

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

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

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23 Current and future studies (2) Evaluation of effects of drugs of abuse on the CRF, CRF-R1 and CR-BP system (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.] (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 Determination the neurotransmitters (glutamate, GABA) present in CRF axonal terminals, and establish at the ultratructural level the distribution of CRF-R1 and CRF-BP

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


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