# Presentation: 079963

## Marisela Morales

NIDA Intramural Research Program
Cellular Neurobiology Branch
Cellular Neurophysiology Section

**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**

## 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**

** **

**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)**

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

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

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

## Mesocorticolimbic  DA system

**Ventral Tegmental**
**Area (VTA****)**

**Amygdala**

**Olfactory tubercle**

**Nucleus accumbens**

**Prefrontal cortex**

**Hippocampus**

**Mesocorticolimbic  DA system**

**Dopamine neurons**

## Interactions between stress and reward systems. Brain area? 

**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?**

**Notes:**
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.

## 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**

## Do CRF cells establish functional interactions (synapses) with cells located in VTA?

**Yes**

**Postsynaptic**
**dopamine?**

## 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**

## Do CRF cells establish synapses with dopaminergic neurons in VTA?

**Yes**

**Asymmetrical**
**synapses**

**17 %**

**EXCITATORY**

**INHIBITORY**

## Aim:

** ****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**_

## 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**

**mRNA**

**Protein**

## Expression of CRF Receptor 1 (CRF-R1) mRNA in the Ventral Tegmental Area

**Expression of CRF Receptor 1 (CRF-R1) mRNA in the Ventral Tegmental Area**

**TH mRNA**

**CRF-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**

## 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

## At the molecular level, CRF mediates its biological effects by interacting with three different proteins

**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**

## 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**

**TH mRNA**

**CRF-BP mRNA**

## 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**

## Expression of CRF Binding Protein (CRF-BP) mRNA in VTA Dopaminergic Neurons

**Expression of CRF Binding Protein (CRF-BP) mRNA in VTA Dopaminergic Neurons**

**TH mRNA**

**CRF-BP mRNA**

## 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**

## 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**

## Slide 22

## 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**

## Acknowledgement

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

- Support: NIDA IRP

**Notes:**
CHECK names