Introduction

Top Abstract Introduction Tyrosine Hydroxylase Dopamine--Hydroxylase Vesicular Monoamine Transporter Norepinephrine Transporter Uptake-2 Deficiency Catechol-O-Methyltransferase Monoamine Oxidase Conclusions References

Catecholamine molecules are crucial neurotransmitters in both the central and peripheral nervous systems. Norepinephrine (NE) and epinephrine (EPI) in the CNS modulate many physiologic effects, including emotion, anxiety, neuronal excitability, and the regulation of central autonomic outflow. In the periphery, the sympathetic nervous system exerts widespread control throughout human physiology, from cardiovascular regulation to energy balance. The principal neurotransmitter in the sympathetic nervous system is NE. Release of NE from sympathetic nerve terminals constricts arterioles, thickens salivation, increases heart rate and contractility, promotes glycogenolysis and gluconeogenesis, constricts the abdominal vasculature, decreases gastrointestinal motility, causes ejaculation, and stimulates lipolysis in adipose tissue.

Elucidation of the biosynthetic pathway of NE and discovery of its receptor subtypes were triumphs of classical pharmacology. The power of pharmacologic techniques lies in the selectivity and specificity of the pharmacologic agents used. Historically, studies using pharmacologic tools were often circumscribed by a limited armamentarium of highly selective agonists or antagonists. Thus, in vivo pharmacologic differentiation of specific protein targets was often problematic. Furthermore, pharmacologic agents sometimes had effects not specific to the protein under study. Lesioning techniques facilitated the elucidation of organ and system physiology and have been used to create models of human autonomic disorders, such as neuropathic postural tachycardia syndrome (Carson et al., 2001). However, lesioning models may often be limited by transience or incompleteness.

Homologous recombination models have become invaluable tools to the integrative physiologist and pharmacologist. Such models have versatility to yield absent, mutated, or overexpressed proteins throughout the lifespan of the animal. There are also an array of new genetic-engineering capabilities to provide local anatomic targeting or temporally controlled targeting of gene expression. Genetic models hold the promise of providing animals and reagents that can be used to elucidate the specific role of a given gene product in an intact animal or cell culture system. Although one must acknowledge both that the physiology of rodents differs in some respects from the physiology of the human (i.e., prolonged orthostasis) and that the techniques available for analysis of human physiology may not be applicable or available for the study of rodent physiology, integrated physiological studies of genetically modified animals, and the determination of the resultant phenotypes may aid the clinician in recognizing novel genetic disorders due to either gain-of-function or loss-of-function mutations.

This article aims to address knockout mouse models of gene products involved in the biosynthetic and metabolic pathways of norepinephrine. Although a large number of diverse gene products must be involved in the moment-to-moment function of noradrenergic neurons (for example, second messenger systems and ion channels), treatment of such models is beyond the scope of this article. Since the physiologic consequences of polymorphisms and of genetic manipulation of specific adrenergic receptors and receptor subtypes have been surveyed recently (Rohrer and Kobilka, 1998; Kable et al., 2000; Koch et al., 2000; Garland and Biaggioni, 2001), these will be omitted in this article.

	Tyrosine Hydroxylase

Top Abstract Introduction Tyrosine Hydroxylase Dopamine--Hydroxylase Vesicular Monoamine Transporter Norepinephrine Transporter Uptake-2 Deficiency Catechol-O-Methyltransferase Monoamine Oxidase Conclusions References

The biosynthetic pathway leading to the production of NE begins with the amino acid tyrosine. The initial and rate-limiting step in the production of NE is the hydroxylation of tyrosine by tyrosine hydroxylase to L-dihydroxyphenylalanine (DOPA) (Fig. 1A). Tyrosine hydroxylase (TH) deficiency has been reported in humans and is characterized by generalized rigidity, hypokinesia, and low cerebrospinal fluid levels of the NE and dopamine (DA) metabolites homovanillic acid and 3-methoxy-4-hydroxy-phenylethylene glycol (Brautigam et al., 2000; Dionisi-Vici et al., 2000). TH deficiency can be effectively treated with supplementation of the dopamine precursor DOPA. Pharmacologic inhibition of TH may be achieved using metyrosine (-methyl-p-tyrosine), a false substrate for TH. Metyrosine has seen clinical use in attenuating the hypertension caused by pheochromocytoma.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Deficiencies in norepinephrine synthesis and release. A, the initial and rate-limiting step in the production of NE is the hydroxylation of tyrosine by tyrosine hydroxylase to DOPA. DOPA is subsequently decarboxylated by AADC to dopamine. Dopamine is mobilized to vesicles through VMAT, where it is subsequently hydroxylated by DBH to form NE. The majority of released NE is removed from the synaptic cleft through reuptake into the presynaptic neuron by NET. NE that diffuses from the synapse may also be taken into other cells by EMT. COMT is an extraneuronal enzyme that catalyzes the methylation of NE to normetanephrine (NMN), whereas NE that is recycled into the neuron by NET is either repackaged into vesicles or oxidized by MAO to dihydroxyphenyl glycol (DHPG). B, loss of DBH leads to an inability of the neuron to synthesize norepinephrine, leading to a buildup of dopamine. Because other aspects of neuronal function are presumably intact, DA may be packaged and released in place of NE. Without maternal supplementation with DOPS, DBH deficiency is lethal to nearly 90% of mouse pups. C, loss of VMAT function in a noradrenergic neuron would impair the ability of dopamine to be transported into vesicles where it could be converted to norepinephrine by DBH. Complete loss of VMAT function in mice is lethal.

Zhou et al. (1995) described the phenotype of TH-deficient mice. Initial screening of offspring failed to reveal any transgenic animals completely lacking TH. In TH-deficient embryos, mortality occurred on embryonic day 11.5 (E11.5) through 15.5. Kobayashi et al. (1995) observed that when TH-deficient animals were delivered by caesarean section at E18.5, 19% of animals survived, some took a breath, but all were dead within 1 day of birth. Measurement of TH activity in heterozygotes revealed a 50% reduction in TH activity, which suggests a gene-dosing effect; 50% of the gene results in 50% of the protein. TH activity was absent in the homozygous animals. In neonatal heterozygotes, both NE and EPI levels were reduced in the head, but body levels were normal. In postnatal (/) mice, NE, EPI, and DA levels were greatly reduced. The specificity of TH impairment was demonstrated by complete rescue of embryos through administration of L-DOPA to pregnant dams and by rescue with human TH. Both rescue techniques demonstrate that mortality was due to loss of the TH. Rescued animals that did survive were smaller than their wild-type counterparts and did not survive past 5 weeks (Zhou et al., 1995). Examination of deceased embryos revealed congested blood in the liver and major vessels, atrial dilation, and disorganized cardiomyocytes in some of the embryos. Evaluation of heart rate by EKG in newborn TH (/) animals revealed a bradycardia (Kobayashi et al., 1995). Combined, these data indicate that loss of catecholamines and attendant-altered cardiac function result in the observed embryonic mortality. The presence of residual levels of DA suggests an alternative mechanism for the synthesis of DA.

Evaluation of adrenal function in TH (/) animals revealed the expected reduction in adrenal catecholamine levels, a reduction in plasma corticosterone, and elevated message levels for the neuropeptides enkephalin and neuropeptide Y. Despite the reduction in corticosterone levels, adrenocorticotropin levels were not significantly altered in animals lacking catecholamines (Bornstein et al., 2000).

The enzyme tyrosinase may be acting as an alternative source for the production of DA and may be responsible for the residual DA in the TH-deficient animals (Rios et al., 1999). Tyrosinase is primarily localized to melanocytes and is capable of hydrolyzing DOPA, leading to formation of melanin and pigment in mice. Mice that were lacking both TH and tyrosinase were examined. To prevent the embryonic mortality associated with TH deficiency, animals were rescued through administration of DOPA to the maternal drinking water. Surviving embryos were examined at postnatal day 6 to allow clearance of maternally derived catecholamines. In TH-deficient animals, catecholamines were detected histologically, whereas in albino TH-deficient mice, no catecholamines were detected histologically or through high-performance liquid chromatography. This demonstrates that tyrosinase may be an alternate source for catecholamines, although tyrosinase-derived catecholamines are clearly not sufficient for animal survival.

Because TH-deficient animals are deficient in DA, NE, and EPI, Kim et al. (2000) used the DBH promoter to selectively return TH to noradrenergic neurons, thus creating animals with impaired DA signaling. Offspring lacking TH in dopaminergic neurons did survive to term but were hypoactive and hypophagic and died by 3 weeks of age. Chronic administration of DOPA rescued animals and normalized activity. DA-deficient animals exhibited an increased sensitivity to DA agonists and DOPA despite normal DA receptor and transporter densities. These data indicate that DA is not required for the acquisition of postsynaptic DA receptors but does play a role in regulation of receptor responsiveness.

Although NE levels are near normal in TH (+/) animals, Kobayashi et al. (2000) demonstrated that repeated electrical stimulation of NE neurons resulted in decreased NE release in TH (+/) animals but not in WT controls. Furthermore, using a variety of learning paradigms, memory deficits were observed in the TH (+/) animals, all of which could be normalized although administration of the norepinephrine reuptake inhibitor desipramine. The ability to normalize the phenotype pharmacologically suggests a functional reduction in synaptic NE, the neurological consequences of which can be reversed by elevating synaptic NE levels. Lastly, no structural abnormalities were detected in these animals, further supporting a functional effect.

	Dopamine--Hydroxylase

Top Abstract Introduction Tyrosine Hydroxylase Dopamine--Hydroxylase Vesicular Monoamine Transporter Norepinephrine Transporter Uptake-2 Deficiency Catechol-O-Methyltransferase Monoamine Oxidase Conclusions References

DOPA synthesized by TH can be decarboxylated by L-aromatic amino acid decarboxylase (AADC) forming dopamine (Fig. 1A). In noradrenergic neurons, dopamine is subsequently hydroxylated by DBH to form NE. In a subset of neurons, EPI is synthesized through methylation of NE by phenylethanolamine-N-methyl transferase (PNMT). Human DBH deficiency was first described in humans in 1986 (Robertson et al., 1986). In humans, DBH deficiency is characterized by orthostatic hypotension, ptosis of the eyelids, retrograde ejaculation, and dramatically elevated plasma levels of DA. DBH deficiency in humans has been effectively treated L-dihydroxyphenylserine (DOPS) (Biaggioni and Robertson, 1987; van den Meiracker et al., 1996). DOPS is converted directly to norepinephrine through decarboxylation by AADC. Despite restoration of plasma NE levels in humans with L-DOPS, EPI levels are not restored, leading to speculation that PNMT may somehow require DBH for appropriate functioning (van den Meiracker et al., 1996) (Fig. 1B).

DBH-deficient mice experience an 88% embryonic mortality (Thomas et al., 1995). Of the 12% of mice that were born, there was a further 60% mortality by 5 weeks. Fetal morphology seemed to be normal in DBH-deficient animals, although disorganized cardiomyocytes were observed in one animal. Other than exhibiting ptosis and reduced body mass, DBH-deficient adult mice appeared normal. NE levels were reduced approximately 20% in (+/) fetuses and 97% in (/) fetuses, whereas NE was undetectable in surviving offspring of (/) mothers, supporting that maternal transfer of NE may be occurring in the (+/) mothers. Replenishment of NE through administration of L-DOPS in the maternal drinking water dose dependently rescued (/) fetuses. Increased mortality of pups born to DBH-deficient mothers was observed, possibly due to a reduction in the level of parentally transferred NE. Further study revealed that NE-deficient mothers failed to exhibit maternal behavior. Mothers would give birth but failed to gather the pups (Thomas and Palmiter, 1997a). Maternal behavior could be rescued if L-DOPS was administered on the evening before giving birth but not if administered the morning after. Once animals exhibited maternal behavior, L-DOPS was not required for the demonstration of maternal behavior with subsequent litters. It is possible that postpartum hemodynamic compromise caused or contributed to these behavioral findings. DBH-deficient mice also exhibited metabolic abnormalities, including a reduced ability to metabolize brown fat and an elevation in basal metabolic rate. When animals were placed in a cold environment, they lost body temperature more rapidly than WT controls, presumably due to the combined deficiencies in thermogenesis and the failure to piloerect or to constrict the peripheral vasculature (Thomas and Palmiter, 1997b). Alterations in neuronal excitability were reported in DBH-deficient mice. After exposure to seizure-inducing stimuli, both the time to the first onset of seizure symptoms and the time to tonic-clonic seizures were reduced in mice lacking NE (Szot et al., 1999). The latency to seizure symptoms could be normalized with prior administration of L-DOPS.

Examination of the cardiovascular consequences of the lack of NE or Epi revealed increases in both basal and agonist-stimulated cardiac contractility in DBH-deficient mice, although basal heart rate was unchanged (Cho et al., 1999). As in mice lacking DA (Kim et al., 2000), -adrenergic receptor density was unchanged, although more receptors were in the high-affinity state. Levels of -adrenergic receptor kinase, the enzyme that inactivates -receptors through phosphorylation, were reduced. Thus, increased contractile responses to agonist stimulation could be due to agonist stimulation of high-affinity -receptors.

Because DOPS is used clinically to treat human DBH deficiency, the ability of DOPS to supplement NE and to treat the phenotypes resulting from lack of NE in DBH-deficient mice was examined (Thomas et al., 1998). After treatment with DOPS, NE levels were elevated above wild-type levels in a variety of tissues, although no restoration of NE levels was seen in the adrenal gland. These data help resolve the issue of whether there is an associated PNMT deficiency, suggesting that the failure to produce EPI may not be due to an associated deficiency in PNMT but to an inability of DOPS to adequately penetrate to the adrenal medulla. Using a variety of physiological tests that are known to be modulated by NE, including measurement of uncoupling protein 1 levels, hind-limb extension, postdecapitation convulsions, ptosis, and male fertility, it was shown that administration of DOPS normalized all phenotypes examined. Multiple treatments of DOPS partially replenished CNS levels of NE (Thomas et al., 1998).

During development, sympathetic noradrenergic neurons innervating sweat glands convert from a noradrenergic phenotype to a cholinergic phenotype. In the adult, the sympathetic neurons that stimulate the sweat response are cholinergic. DBH (/) mice were used to address whether NE is required for the conversion of noradrenergic neurons to a cholinergic phenotype (Tafari et al., 1997). NE was undetectable in these animals, although as seen previously, DA levels were elevated. No difference in sweating was observed between DBH (+/) and (/) mice, suggesting that NE is not required for the conversion of sympathetic noradrenergic neurons to a cholinergic phenotype. Similar results were obtained in TH-deficient mice, animals used as controls for the elevated DA levels seen in DBH-deficient mice.

The question of whether residual catecholamines formed by tyrosinase might be sufficient for the generation of the sweat response has also been addressed (Tian et al., 2000). A decrease in the number of functional sweat glands was seen in mice lacking both TH and tyrosinase relative to mice lacking TH alone, supporting that residual catecholamines are allowing production of the sweat response. Normal levels of the vesicular acetylcholine transporter, a marker of the cholinergic phenotype, were observed, demonstrating that the conversion from the noradrenergic phenotype to a cholinergic phenotype was still occurring. Sweat glands were still formed but were not functional. Taken together, these data suggest that NE is not required for the conversion of noradrenergic sympathetic neurons to the cholinergic phenotype but is required at some levels for completely normal sweat gland function.

	Vesicular Monoamine Transporter

Top Abstract Introduction Tyrosine Hydroxylase Dopamine--Hydroxylase Vesicular Monoamine Transporter Norepinephrine Transporter Uptake-2 Deficiency Catechol-O-Methyltransferase Monoamine Oxidase Conclusions References

The vesicular monoamine transporter (VMAT-2) mobilizes monoamines from the neuronal cytoplasm into vesicles, where they are then available for release at the synapse. Blockade of VMAT with reserpine leads to a gradual and prolonged depletion of NE from the nerve terminal and has been used therapeutically in the treatment of hypertension. Mice lacking VMAT died by the second week after birth (Takahashi et al., 1997) (Fig. 1C). VMAT levels were reduced approximately 50% in (+/) animals, suggesting a gene dosage effect. As a result of the mortality in VMAT (/) animals, VMAT (+/) animals were used in further studies. In anesthetized animals, there was both an elevation of HR and MAP, although studies in conscious mice revealed a normal resting HR. Both striatal DA and 3,4-dihydroxyphenylacetic acid levels were elevated; dopamine transporter (DAT) levels were decreased, and dopamine receptor levels were unchanged, supporting near normal synaptic levels of DA. Norepinephrine transporter (NET) and - and -receptor densities were unchanged. MPTP is a substrate for the DAT. After uptake into dopaminergic neurons, MPTP is metabolized to a toxin. MPTP is also a substrate for VMAT; thus, its toxic effect can be reduced by sequestration into vesicles. Despite decreased DAT densities and a concomitant decreased ability of MPTP to enter the neuron, MPTP was more toxic to neurons after a partial loss of VMAT. The sensitivity to amphetamine was likewise elevated.

Long term analysis of VMAT (+/) animals revealed an increased mortality relative to WT litter mates (Itokawa et al., 1999). After 10 months, 28% of VMAT-deficient mice had died, whereas no mortality was seen in the controls. Those dying seemed to die of sudden death. Heart rate and body temperature were not altered in VMAT (+/) animals, but the QT and QTc intervals were lengthened, suggesting a developmental compensation to maintain heart rate. The failure to completely compensate for the changes in NE signaling may lead to arrhythmias and, consequently, sudden death.

	Norepinephrine Transporter

Top Abstract Introduction Tyrosine Hydroxylase Dopamine--Hydroxylase Vesicular Monoamine Transporter Norepinephrine Transporter Uptake-2 Deficiency Catechol-O-Methyltransferase Monoamine Oxidase Conclusions References

The NE transporter is a Na/Cl-dependent 12-transmembrane-spanning protein that resides on presynaptic noradrenergic nerve terminals. NE can be removed from the synapse through either reuptake into the presynaptic neuron, enzymatic degradation, or clearance by simple diffusion. The majority of NE is removed from the synaptic cleft through reuptake into the presynaptic neuron by NET (Eisenhofer et al., 1992). NET is a target of many pharmacologic agents, including antidepressants, such as reboxetine and desipramine, and drugs of abuse, such as amphetamine. We previously reported patients with partial NET deficiency characterized by orthostatic tachycardia, decreased NE clearance, and reduced tyramine responsiveness (Shannon et al., 2000). Studies on the NET-deficient patients have not yet been directed at potential CNS effects outside the autonomic nervous system. The importance of the NET in cardiac regulation is seen through the observation that approximately 80 to 90% of released NE is cleared through the NET (Eisenhofer et al., 1992).

Mice lacking NET are smaller than their WT litter mates and exhibit a reduced body temperature (Xu et al., 2000). CNS NE levels were reduced in various brain regions between 55 and 70% despite elevated TH activity. Thus, despite an elevation in the rate-limiting step in NE biosynthesis, there is a reduction in overall levels of NE, suggesting that the NET plays a key role in maintaining the NE content of neurons. Examination of NE kinetics revealed a 60% reduction in release and a 6-fold reduction in NE clearance, resulting in an overall 2-fold elevation in extracellular NE levels. NET (/) animals behaved like animals administered tricyclic antidepressant agents. Furthermore, the behavioral effects could be further enhanced with serotonin-selective reuptake inhibitors but not with norepinephrine-selective reuptake inhibitors. Overall locomotor activity was decreased, yet the sensitivity to amphetamine and DA agonists was increased (Fig. 2A).

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Deficiencies in norepinephrine transport and metabolism. NET is responsible for clearance of a majority of the released NE from the synaptic cleft. A, loss of NET from the neuron results in elevated synaptic levels of NE. Furthermore, the inability to recycle NE leads to reductions in intraneuronal levels of NE and a concomitant reduction in the production of the NE metabolite DHPG. Increased dependence upon COMT for NE metabolism may lead to elevated levels of the NE metabolite NMN. NE that diffuses from the synapse may be cleared from the circulation through transport into nonneuronal cells via EMT. B, loss of NE clearance by EMT may lead to elevations in spillover of released NE and decreased production of NMN. EMT deficiency may be physiologically important in the clearance of NE from the heart. COMT and MAO are two key enzymes responsible for the metabolism of NE. C, loss of COMT would result in decreased extraneuronal metabolism of NE and a reduced production of NMN. MAO is responsible for the intraneuronal metabolism of NE; thus, loss of MAO will lead to elevations in the intraneuronal levels of NE and a concomitant reduction in DHPG production. Therefore, the loss of these enzymes could result in elevations of synaptic and circulating levels of NE.

Because NE plays an important role in modulation of nociception, Caron's group examined the role that loss of NET would play in analgesia (Bohn et al., 2000). NET-deficient mice had an elevated pain threshold relative to WT controls. A potentiation of opioid-induced analgesia was also present in NET-deficient mice. A similar potentiation of opioid analgesia could be observed in WT animals administered the NE reuptake inhibitor desipramine or the ₂-agonist guanfacine. The potentiation could be blocked with the ₂-antagonist yohimbine, supporting that the analgesic effects if NET deficiency were mediated through stimulation of ₂-adrenergic receptors.

	Uptake-2 Deficiency

Top Abstract Introduction Tyrosine Hydroxylase Dopamine--Hydroxylase Vesicular Monoamine Transporter Norepinephrine Transporter Uptake-2 Deficiency Catechol-O-Methyltransferase Monoamine Oxidase Conclusions References

Although NET is the predominant transporter in neuronal tissue, a second process termed uptake-2 is responsible for removal of NE that has not been recycled into the neuron via NET. The protein responsible for uptake-2 is the extraneuronal monoamine transporter (EMT), also known as the organic cation transporter 3 (OCT-3). EMT differs from NET not only the localization to nonneuronal cells but also in the differentiation of substrate specificities, including sensitivity to inhibition by glucocorticoids. To examine whether the Orct3 gene indeed codes for EMT and is responsible for uptake-2, Zwart et al. (2001) generated mice lacking the Orct3 gene. Both Northern and Western analysis confirmed deletion of both the Orct3 gene and gene product, respectively. Homozygous mice lacking the Orct3 gene appeared normal with respect to size, fertility, and general behavior. Heart size was also normal in Orct3 (/) mice.

Uptake-2 activity in mutant mice was examined using [³H]MPP⁺ as a substrate for uptake-2. Accumulation of [³H]MPP⁺ was dramatically reduced in the heart, an organ that expresses Orct-3, of Orct3 (/) mice, whereas accumulation of [³H]MPP⁺ in the liver, an organ that does not express Orct-3, was not altered. Analysis of [³H]MPP⁺ in other organs, including the lung, kidney, spleen, colon, or brain, failed to demonstrate an alterations in [³H]MPP⁺ accumulation between Orct3 (/) and WT mice. Furthermore, examination of the maternal transfer of [³H]MPP⁺ to embryos revealed a 3-fold reduction of [³H]MPP⁺ in Orct3 (/) embryos, suggesting a functional role of Orct3 in the placenta. Analysis of NE and DA levels from Orct3 (/) and WT embryos and placentas failed to differentiate genotypes. Thus, an obvious role for Orct-3 was observed only in the hearts of Orct3-deficient animals (Fig. 2B).

	Catechol-O-Methyltransferase

Top Abstract Introduction Tyrosine Hydroxylase Dopamine--Hydroxylase Vesicular Monoamine Transporter Norepinephrine Transporter Uptake-2 Deficiency Catechol-O-Methyltransferase Monoamine Oxidase Conclusions References

Catechol-O-methyltransferase (COMT) is one of the key enzymes responsible for the metabolism of catecholamines. COMT is an extraneuronal enzyme that inactivates catecholamines through O-methylation, methylating NE to form normetanephrine. Association of the chromosomal region on which COMT resides has been noted with respect to a variety of psychiatric disorders. In mice lacking COMT, increased levels of DA were observed in the frontal cortex of male but not female mice, whereas CNS levels of NE were not altered (Gogos et al., 1998). In a model of anxiety, increased anxiety was seen in female but not male animals, whereas increased levels of aggression were observed in (+/) males but not (/) males, data that parallels the observation of increased aggression in humans homozygous for the low-activity COMT allele (Fig. 2C).

	Monoamine Oxidase

Top Abstract Introduction Tyrosine Hydroxylase Dopamine--Hydroxylase Vesicular Monoamine Transporter Norepinephrine Transporter Uptake-2 Deficiency Catechol-O-Methyltransferase Monoamine Oxidase Conclusions References

The enzyme responsible for the intraneuronal metabolism of catecholamines is monoamine oxidase (MAO). Oxidation of NE by MAO-A leads to the production of dihydroxyphenyl glycol (Fig. 2C). Two subtypes of MAO exist, MAO-A and MAO-B. An association of the loss of the gene encoding MAO-A has been reported with aggressive behavior. The first report of MAO-deficient mice was due to an incidental insertion of an interferon transgene into the catalytic domain of MAO-A (Cases et al., 1995). Increased aggression was noted in the transgenic offspring. Normalization of the increased aggression in the MAO-A-deficient mice could be achieved through administration of an inhibitor of 5-HT synthesis but not with an inhibitor of NE synthesis. Increased levels of 5-HT, DA, and NE were measured in the CNS of the mice. Furthermore, it was observed that the animals possessed cytoarchitectural abnormalities in the somatosensory cortex (Cases et al., 1998). Analysis of the MAO-A-deficient embryos revealed that during development, catecholaminergic neurons were taking up 5-HT. In contrast to MAO-A-deficient animals, no alterations in levels of 5-HT, NE, DA, or their respective oxidative metabolites were seen after knockout of MAO-B, although urinary levels of phenylethylamine (PEA) were dramatically elevated (Grimsby et al., 1997). Despite no detectible alterations in levels of biogenic amines, animals showed a hyperactive response to inescapable stress, suggesting a functional effect potentially related to elevated PEA levels. Lastly, MAO-B-deficient mice were resistant to the neurotoxic effects of MPTP, further implicating the requirement of metabolism of MPTP by MAO-B in the toxicity of MPTP.

In vivo, difficulties have arisen in separating the specific roles of MAO-A versus that of MAO-B, due to overlapping antagonist specificity. To verify which CNS structures use MAO-A versus MAO-B, mice deficient in MAO-A were examined (Ikemoto et al., 1997). No MAO activity was measured in the locus coeruleus or A1 cell group, confirming that MAO-A is the only oxidase in noradrenergic neurons. MAO-B was localized to a variety of brainstem structures, supporting the view that MAO-B may be playing an important modulatory role in limbic output. Studies in the striatum revealed no changes in DA, 3,4-dihydroxyphenylacetic acid, or HVA levels after removal of MAO-B, demonstrating that MAO-A is the primary oxidase in the striatum (Fornai et al., 1999). After generation of high DA levels through administration of DOPA, it was observed that DA was metabolized by both MAO-A and MAO-B.

The Shih group used cerebral blood flow as a marker of neuronal activity and examined the changes in neuronal activity resulting from loss of either MAO-A or -B (Scremin et al., 1999; Holshneider et al., 2000). After loss of either MAO-A or -B, patterns of neuronal blood flow were altered in a variety of CNS nuclei. A reduced HR and a trend for a reduction in MAP were observed in MAO-A-deficient mice. In MAO-B-deficient mice, there was a trend for a reduction in MAP and HR, although after administration of PEA, MAP was elevated. Levels of PEA, a dietary amine, are elevated 8× in MAO-B knockout animals. PEA administration resulted in an overall decrease in cerebral blood flow. An association of MAO-B and migraines has been observed. Thus, in humans deficient in MAO-B, there may be an increased sensitivity to dietary amines and, thus, reductions in cerebral blood flow and possibly a cardiovascular phenotype. Since both MAOs reside on the X chromosome, abnormalities of oxidase function are presumed to have a more dramatic phenotype in men compared with women.

Mice lacking the MAOs have been used to elucidate interactions and targets of pharmacologic agents within the central nervous system. In one such case, Remaury et al. (2000) used both MAO-A- and MAO-B-deficient mice to demonstrate that MAO-B, but not MAO-A, fits the criteria of an I₂-imidazoline-binding protein.

The MAO-B inhibitor selegiline (Deprenyl) has demonstrated neurotrophic and neuroprotective properties in a variety of in vitro and in vivo models, but the potency at which selegiline exerts such effects is below that at which it acts to inhibit MAO-B. Ekblom et al. (1998) used mice lacking MAO-B and radiolabeled selegiline to search for selegiline binding sites in the mouse central nervous system. Very little selegiline binding was detected in CNS tissue from MAO-B-deficient mice using either scintillation counting or autoradiography. These data are consistent with the hypothesis that the neuroprotective effects of selegiline are mediated via its desmethyl-metabolite.

	Conclusions

Top Abstract Introduction Tyrosine Hydroxylase Dopamine--Hydroxylase Vesicular Monoamine Transporter Norepinephrine Transporter Uptake-2 Deficiency Catechol-O-Methyltransferase Monoamine Oxidase Conclusions References

Knockout animals with impaired monoamine signaling are invaluable tools for the study of catecholamine physiology. Although some studies have examined the overall cardiovascular function in the knockout animals presented above, selective characterization of the central regulatory and peripheral sympathetic changes associated with such animals is needed (Table 1). Furthermore, the demonstrations of subtle yet discernible phenotypes in heterozygote animals suggest the importance and necessity of in-depth physiological studies of these animals. Integrated physiologic and pharmacologic studies of genetically modified animals has the potential to greatly aid the clinician in recognition of human phenotypic counterparts of these genetic models. These studies may also lead to the creation and testing of novel therapeutic interventions specifically targeted to the individual gene deficiency.