Introduction 
Norepinephrine (NE) is one of the most abundant neurotransmitters in the central and peripheral nervous systems, and has been implicated in many aspects of physiology and behavior. I have taken a genetic approach to studying NE by using dopamine beta-hydroxylase knockout (Dbh -/-) mice. Dbh functions in the NE biosynthetic pathway and is required to converts dopamine to NE; thus, Dbh -/- mice completely lack NE and provide a good model to study NE function. I have been primarily using the Dbh -/- mice in behavioral, pharmacological, and molecular paradigms to study central NE function.

Why study NE? 
NE was the originally characterized in the peripheral nervous system and was one of the first neurotransmitter discovered. It has profound effects on almost all aspects of the sympathetic nervous system, including regulation of cardiovascular function and energy metabolism. NE is also abundant in the central nervous system. Most noradrenergic neurons originate in the brainstem in a region called the locus coeruleus (LC). These neurons project to almost every region of the brain; in fact, it is nearly impossible to find a brain structure that completely lacks noradrenergic input. Because it was the first neurotransmitter discovered and is so widespread, NE has been extensively studied for over 30 years by various means. There are three major types of NE receptor, all of which are seven-pass transmembrane, G protein-coupled receptors. Stimulation of alpha-1 and beta-noradrenergic receptors increase intracellular Ca++ and cAMP respectively, and are localized on target cells of noradrenergic neurons. Activation of alpha-2 receptors decreases cAMP. These receptors function as inhibitory autoreceptors and are also found on target neurons. There are multiple subtypes within each of these receptor classes, and the total number of identified NE receptors now stands at 9. Fairly specific agonists and antagonists exist for most of these receptors and have been extensively characterized. There are also neurotoxins that are specific for noradrenergic neurons that have been used for many years to study the consequences of destroying NE-containing neurons. Knockout technology has resulted in the generation of mice lacking two different NE biosynthetic enzymes (Dbh and tyrosine hydroxylase) as well as almost every receptor subtype. What makes this system so enticing is that because of the rich history of NE experimentation, there is an abundance of pharmacological tools available for its study.

Why use genetics?
Neurotransmitter function has classically been studied in one of three ways: neuronal lesioning, which eliminate the cells and their transmitters, neuronal stimulation, which activates neurons and causes neurotransmitter release, and pharmacological intervention with agonists and antagonists that activate or block neurotransmitter receptors. Using these techniques, a lot has been learned about the role of NE in many processes. However, there are caveats associated with these techniques that make the interpretation of some experiments difficult. Cell lesions deplete not only the neurotransmitter of interest, but also any other transmitters co-expressed in the neurons, and similarly neuronal stimulation causes release of all transmitters. It is nearly impossible to eliminate all of the neurons of a specific transmitter type by lesioning, and the ones that remain can activate mechanisms to compensate for cell loss. Pharmacological agents can suffer from lack of both receptor and regional specificity.

Over the last 15 years, transgenic and knockout technology has established a viable alternative for studying neurotransmitter function. Genes coding for biosynthetic enzymes required for neurotransmitter synthesis, neuropeptides, and neurotransmitter receptors have been successfully knocked out in mice. Dbh -/- mice have been useful in determining many critical functions of NE in vivo. These include roles predicted by lesioning and pharmacology such as cardiovascular function, smooth muscle contraction, and brown fat thermogenesis, and novel roles such as embryonic development, maternal behavior, and immune function.

The major concern with using neurotransmitter knockouts is that because the animals develop in the absence of the transmitter, compensation by other transmitters may mask some phenotypes and create others de novo. In an effort to circumvent these problems, conditional knockouts have been developed that can restrict the gene deletion to particular times during the life cycle and regions of the brain. The Dbh -/- mouse is, in essence, a reversible knockout. A compound called L-threo-3,4-dihydroxyphenylserine (DOPS) can be converted to NE by the enzyme L-aromatic acid decarboxylase (AADC), thus bypassing the requirement for DBH. Acute administration of DOPS to adult Dbh -/- mice that have lacked NE since birth is able to reverse nearly all of the phenotypes associated with the mutation. This demonstrates that the defects caused by knocking out the Dbh gene are solely due to a specific lack of NE and are not caused by developmental abnormalities or compensatory changes in other neurotransmitters. Therefore, the principal caveat to using genetics to study neurotransmitter function is addressed in Dbh -/- mice. Combining the pharmacological tools made available by years of NE study with newly developed molecular genetic tools such as Dbh -/- mice has created a powerful system with which to investigate NE function.

Drug Addiction
The mesolimbic dopamine (DA) system has been primarily implicated in the reinforcing effects of drugs of abuse.  While this pathway and DA signaling are the focus of most research in this area, it is also clear that norepinephrine (NE), via interactions with the dopaminergic system, plays an important role in modulating the neurochemical and behavioral responses to drugs of abuse in animal models.  This has become of particular interest for two reasons.  First, a common polymorphism in the human Dbh gene is a critical determinant of DBH enzymatic activity and appears to influence behavioral and cognitive responses to cocaine.  Second, the DBH inhibitor disulfiram (Antabuse) has shown striking promise as a treatment for cocaine dependence. We have found that Dbh -/- mice have alterations in DA signaling and are hypersensitive to psychostimulants.  The goal of this research is to further understand how NE influences behavioral and cellular responses to psychostimulants and to explore the possibility that DBH inhibition might be an effective pharmacotherapy for cocaine addiction.  We are also interested in assessing the contribution of NE to the effects of other stimulant-like such as amphetamine and the wake-promoting agent and anti-narcoleptic drug modafinil.

Epilepsy and Depression
Although depression is the most common co-morbid condition associated with epilepsy from an epidemiological standpoint, the relationship between these diseases has never been demonstrated experimentally, and possible underlying mechanisms are unclear. The creation of an animal model of epilepsy and depression co-morbidity is essential to understanding the mechanisms of this interaction.  Because NE is both antidepressant and anticonvulsant, we hypothesize that a loss of NE could contribute to both epilepsy and depression.  We are currently attempting to create animal models of epilepsy by (1) testing seizure susceptibility in rats selectively bred for depression-like phenotypes, and (2) testing for depression-like phenotypes in epileptic rodents.  We are also using Dbh -/- mice to assess the contribution of NE to the co-morbidity of these diseases.  Finally, since it is unclear how best to treat individuals suffering from both epilepsy and depression, we are evaluating the effects of various antidepressant drugs on seizure susceptibility.

Neurodegenerative Disease
Alzheimer’s disease (AD) and Parkinson’s disease (PD) are neurodegenerative disorders that are characterized by the loss of cholinergic and dopaminergic neurons, respectively, in the brain.  A well established but less recognized feature of both of these diseases is the loss of noradrenergic neurons in the the brainstem locus coeruleus (LC), the major source of norepinephrine (ND) in the brain.  Cell culture models have revealed that NE can protect neurons in cell culture from death, and animal models have revealed that LC lesions exacerbate both AD and PD-like neuropathology and behavioral deficits.  Thus, it is of interest to determine the role of NE in neurodegenerative disease.

We are currently assessing Dbh -/- mice and norepinephrine transporter knockout mice that also carry a transgene expressing mutant beta-amyloid to assess the contribution of NE to AD-like neuropathology and behavioral deficits.  In addition, we are using the MPTP model of PD in Dbh -/- mice to understand how NE modulates dopamine neuron death and behavioral deficits in PD.  We hypothesize that Dbh -/- mice will be more sensitive than controls to AD and PD like phenotypes, while NET KO mice will be less sensitive.

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Ahern T, Javors MA, Eagles DA, Martillotti J, Mitchell HA, Liles LC, Weinshenker DThe effects of chronic norepinephrine transporter inactivation on seizure susceptibility in mice. Neuropsychopharmacology (in press).

Schank JR, Ventura R, Puglisi-Allegra S, Alcaro A, Cole CD, Liles LC, Seeman P, Weinshenker DDopamine b-hydroxylase knockout mice have alterations in dopamine signaling and are hypersensitive to cocaine. Neuropsychopharmacology (in press).

Martillotti J, Weinshenker D, Liles LC, Eagles DA (2005) A ketogenic diet and knockout of the norepinephrine transporter both reduce seizure severity in mice. Epilepsy Res (in press).