Elsevier

Biological Psychiatry

Volume 46, Issue 9, 1 November 1999, Pages 1124-1130
Biological Psychiatry

Norepinephrine: New Vistas for an Old Neurotransmitter
Genetic approaches to studying norepinephrine function: knockout of the mouse norepinephrine transporter gene

https://doi.org/10.1016/S0006-3223(99)00245-0Get rights and content

Abstract

Norepinephrine is an important chemical messenger in the nervous system. It regulates affective states, learning and memory, endocrine and autonomic functions. It has been implicated in depression, aggression, and addiction, as well as cardiac and thermal dysregulation. The norepinephrine transporter functions by uptaking norepinephrine back into the cell for cyclic use, and is a direct target of a number of antidepressants and psychostimulants. Functional deletion (knockout) of monamine transporters results in increases in extracellular levels of neurotransmitters, thereby prolonging their actions. For the norepinephrine transporter knockout mice, this altered state of the norepinephrine system should simulate the therapeutic effects of norepinephrine selective antidepressants and some of the effects of psychostimulants. Careful use of such an animal model can hopefully provide valuable insight into the multiple roles norepinephrine plays in normal and pathological physiology.

Introduction

The evolutionarily conserved chemical messenger norepinephrine (NE) exerts a wide variety of effects on animal physiology. As a central nervous system (CNS) neurotransmitter, NE is synthesized primarily in the brainstem nuclei locus ceruleus and subceruleus, which project not only rostrally to virtually every region of the midbrain and forebrain and dorsoventrally to the cerebellum, but also caudally to lumbar segments of the spinal cord (Carpenter and Sutin 1983). Additionally, the A1, C1, A2, C2, C3, A4, A5, and A7 noradrenergic cell groups (according to Dahlstrom and Fuxe 1964’s standardized classification of monoaminergic cell groups in the brain) provide more projections in the brain. This extensive projection pattern throughout the neuroaxis defines the global function central NE plays as a general regulator of neurotransmission. As established by extensive investigation Charney 1998, Edwards 1997, Haller et al 1998, NE is directly involved in mood stabilization, sleep regulation, expression of aggression, and the general degree of alertness and arousal, as well as in exerting central control over the endocrine and autonomic nervous systems.

In the peripheral nervous system, NE serves as the major neurotransmitter released by postganglionic neurons of the sympathetic branch. Its main function is to orchestrate the body’s responses to stimulation, culminating in increased heart rate and blood pressure, and enhanced energy mobilization and neural reflexes (Kuhar et al 1999). This set of stress responses is inseparable from the “fight or flight” response resulting from the secretion of corticosteroids by the adrenal cortex and the release of epinephrine (adrenaline) and NE by the adrenal medulla, which is innervated only by the sympathetic branch and affords NE a dual function as a stress hormone (McEwen 1987). Together with epinephrine and corticosteroids, the blood-borne NE helps coordinate different parts of the body for adaptation to a stressful situation, such as glycolysis by the liver and skeletal muscles.

Considering the widespread projections of the NE system and the global role NE plays for neural and endocrine activation, malfunction of the NE system can predictably result in general incapacitation. Such may be the case of clinical depression, which is characterized and/or accompanied by persisting melancholy, anhedonia, psychomotor retardation, sleep disturbances, eating disorders, and not infrequently, suicide (Charney 1998). It is commonly believed that clinical depression is accompanied by hypofunctioning of the NE system as well as of the serotonin system (Leonard 1997). It stands to reason that a heightened noradrenergic tone may also be associated with altered affects. Insofar as there is scanty clinical evidence, animal research suggests a definitive link between aggression and locus ceruleus activation (Siegel et al 1999). There is also evidence suggesting that NE system may be involved in the modulation of reward-related behaviors such as addiction, aside from the well-established involvement of the dopamine system (Markou et al 1998). Local NE dysfunction can also give rise to various pathological cardiovascular conditions and thermoregulatory imbalances (Cannon et al 1998; Colucci 1998; Grassi 1998).

Norepinephrine functions are mediated by NE binding proteins such as adrenergic receptors and NE transporters. Of particular interest to us is the neuronal plasma membrane NE transporter (NET), which is a major target of psychoactive drugs such as psychostimulants and antidepressants (Table 1). Based on pharmacological evidence, the existence of one and possibly more NETs was postulated (Iversen et al 1965). The first NET was cloned in 1991 by Amara and colleagues, breaking the ground for future cloning of numerous members of the large Na+/Cl-dependent transporter family (Pacholczyk et al 1991). Members of this protein family are typically presumed to possess 12 transmembrane (TM) domains based on the hydrophobicity profiles of their amino acid sequences (Amara and Kuhar 1993). Ligand-binding domains in these proteins have largely been delineated. The first five TM domains of the amino terminal have been shown to be involved primarily in general uptake mechanisms and are well conserved in the family. The next three TM domains play a key role in determining tricyclic antidepressant and cocaine binding, while the carboxy terminal region appears to mediate substrate interaction Buck and Amara 1995, Giros et al 1994.

Search for a second NET has been elusive until the recent cloning of a human extraneuronal transporter by Schömig and colleagues. This transporter serves as an effective epinephrine and NE reuptake mechanism and binds the rodent stress hormone corticosterone with high affinity (Gründemann et al 1998). Additionally, several other cloned organic cation transporters (rOCT1, hOCT1, rOCT1A, and hOCT2) have been shown to bind NE with appreciable affinity Busch et al 1996, Gorboulev et al 1997, Zhang et al 1997a, Zhang et al 1997b. In spite of the fact that these transporters have been well characterized in vitro, the in vivo roles they play are still not fully understood. One approach is to target these genes in mice by employing the technique of homologous recombination (Muller 1999), commonly known as knockout (KO).

Section snippets

Experimental approaches

There are a number of techniques available for creating KO mice. Depending on the intended purpose, various designs of the targeting construct can be achieved. To start with, conventional KO (Bronson and Smithies 1994) allows one to examine the consequences of inactivation of either one (heterozygote) or both (homozygote) copies of the gene since inception, which may resemble the clinical situation of inherited genetic diseases. Furthermore, gene expression levels can be increased by designing

Requisite characterizations

Once the KO mice are generated, certain requisite characterizations need to be performed. First of all, the presence of the disrupted NET gene can be confirmed by either Southern blotting (Figure 2) or polymerase chain reaction (PCR). It should be pointed out that normal PCR usually does not confirm homologous targeting because the presence of a new fragment can also result from random integration of the construct. Secondly, the absence of transcription or the presence of modified transcripts

Anticipated phenotypes

Although there was reasonable concern over whether the NET KO mice would survive to serve as useful animal models, we have observed a pattern of survival based on available KO models. It seems to be an emerging principle that excess or overloading of chemical messengers does not usually lead to demise of the mice Bengel et al 1998, Giros et al 1996, while deficiency of essential chemicals often results in death Thomas et al 1995, Wang et al 1997, Zhou et al 1995. Indeed, the NET KO mice survive

Advantages and limitations of the genetic model

There are obvious advantages of using genetically altered mice to help delineate the function of NET. To start with, results obtained from these mice will generally be more definitive than pharmacological manipulations as they eliminate well-known drawbacks of drug treatments. The various roles NET and hence NE play in physiology should be better elucidated. Another important use of these KO mice stems from the fact that conventional heterozygous KO mice afford a close simulation of hereditary

NET KO mice as animal models

Once the NET KO line is established, a number of functional tests is in order to establish them as useful animal models. Since the NET binds a number of psychostimulants and antidepressants with high affinity (Table 1), we anticipate concomitant changes in drug response tests in NET KO mice (Crawley and Paylor 1997). A good frame of reference would be long-term drug treatment data in wild-type (WT) mice compared to the effects of disrupting NET. An open field test can easily be carried out to

Acknowledgements

This work was supported in part by grants from the National Institutes of Health NS19576 and unrestricted gifts from Bristol-Myers Squibb and Zeneca Phamaceuticals (MGC). Raul R. Gainetdinov is a visiting scientist from the Institute of Pharmacology, Russian Academy of Medical Sciences, Baltiyskaya, 125315, Moscow, Russia.

This work was presented at the conference, “Norepinephrine: New Vistas for an Old Neurotransmitter,” held in Key West, Florida in March 1999. The conference was sponsored by

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