Johns Hopkins Advanced Studies in Medicine n S417

ABSTRACT

Affective disorders have hitherto beenexplained in terms of neurotransmitter deficits ordysfunction. However, research in patients withbipolar disorder shows that, while neurotransmit-ter dysfunction is clearly important in the presen-tation of bipolar disorder, it is not the soleexplanation for its etiology. Rather than all of theneurotransmitters being independently abnormalin bipolar disorder, perhaps there is somethingcommon that regulates all of them? This articlewill discuss the cellular plasticity and endopheno-type models of bipolar disorders as well as thepreclinical and clinical research that uses 3 of themost commonly used and effective drugs forbipolar disorder (ie, lithium, valproate, and car-bamazepine) to show that bipolar disorder maybe a neurologic dysfunction at the cellular level,via anomalies in intracellular signaling. The roleof intracellular signaling cascades holds greatinterest because it may explain why so many neu-rotransmitter systems are involved in bipolar dis-order, the frequent comorbidities with bipolardisorder, some of the physical brain changes

observed (also described in this article), whystructurally dissimilar drugs can all be effective intreating bipolar disorder, and the lag timebetween pharmacotherapy initiation and benefi-cial effect and well as the lag time between treat-ment cessation and withdrawal effect. Given thecomplex presentation of mood disorders and theadvances in medical research technology, wenow are starting to understand the true mecha-nisms of action of some of our older treatments aswe better understand the underlying neurobiolo-gy of the disorders. We are on the verge of devel-oping new drugs based on these cellular targets,which, if effective, will allow clinicians to addressthe fundamental biology of the disorder, ratherthan the presenting symptoms.(Adv Stud Med. 2006;6(6A):S417-S429)

NEUROBIOLOGY OF RECURRENT MOOD DISORDERS:CURRENT THEORIES

Psychiatry is a unique branch of medicine in thatthe diagnostic and classification system of psychiatricdisorders is based not on pathophysiologic markersbut clinical manifestation. The bipolar disorders are acluster of affective disorders with a broad spectrum ofpresenting signs and symptoms, with regard to severi-ty, duration, and type. Bipolar disorder is also fre-quently comorbid with other psychiatric and medicaldisorders, which can complicate not only diagnosisbut also treatment. Nonetheless, The Diagnostic andStatistical Manual of Mental Disorders, Fourth Edition,Text Revision (DSM-IV-TR) provides a sensitive andspecific set of diagnostic criteria that allows the clini-cian to detect and diagnose these disorders in mostpatients afflicted with these illnesses.

With the emergence of serotonin reuptakeinhibitors and their related counterparts (eg, dual

PROCEEDINGS

NEUROBIOLOGY AND BIPOLAR DISORDER: BEYOND THE SYNAPSE*

—

Husseini K. Manji, MD†

*This article by Dr Manji is based on a roundtable sym-posium held in Baltimore, Maryland, November 12, 2005.

†Visiting Professor of Psychiatry, Columbia University, DukeUniversity, Chief, Laboratory of Molecular Pathophysiology,Director, Mood and Anxiety Disorders Program, NationalInstitute of Mental Health, Bethesda, Maryland.

Note: Dr Manji contributed to this article in his personalcapacity. The views expressed are his own and do not nec-essarily represent the views of the National Institutes of Healthor the United States Government.

Address correspondence to: Husseini K. Manji, MD, Chief,Laboratory of Molecular Pathophysiology, Director, Mood andAnxiety Disorders Program, NIMH, Porter NeuroscienceResearch Center, Building 35, Room 1C-917, 35 ConventDrive, Bethesda, MD 20892. E-mail: manji@nih.gov.

S418 Vol. 6 (6A) n June 2006

PROCEEDINGS

reuptake inhibitors), affective disorders, in general,have been explained in terms of neurotransmitterdeficits or dysfunction. However, research into theneurotransmitters affected in bipolar disorder showsthat while neurotransmitter dysfunction is clearlyimportant in the presentation of bipolar disorder, it isnot the sole explanation for its etiology. Indeed, thecomplex clinical presentation of bipolar disorder is oneof the first reasons researchers began looking beyond asingle neurotransmitter hypothesis.

THE GENETICS OF BIPOLAR DISORDER:WHERE ARE WE?

Although bipolar disorder is widely regarded as apsychiatrist disorder having amongst the greatestgenetic contribution, no genes have been identifieddefinitively; however, there are many promising link-age regions include 4p16, 4q35, 6q22, 8q24, 12q24,13q31-33, 16p12, 18p11-q12, 18q22-23, 21q22,and 22q11-13.1-3 Questions have been raised con-cerning the definition of genetically relevant pheno-types and the nature of the underlying, partiallyoverlapping sets of susceptibility genes. Thediagnosis of bipolar disorder, as defined bycurrent classification schemas includingDSM-IV, is based on clusters of symptomsand characteristics of clinical course that donot necessarily describe homogenous disor-ders, but rather reflect final common path-ways of different pathophysiological processesinvolving genetic and environmental contrib-utors.4,5 In addition, there is growing evidencethat the boundaries between bipolar disorderand schizophrenia, and between bipolar disor-der and recurrent major depression may notbe as distinct as previously assumed.6-8 Indeed,the pattern of findings emerging from geneticstudies shows increasing evidence for an over-lap in genetic susceptibility across the tradi-tional classification categories, includingassociation findings at various genes.6 Thisshould not be altogether surprising becausethere is not a 1-to-1 relationship betweengenes and behaviors, thus different combina-tions of genes (and resultant changes in neu-robiology) contribute to any complexbehavior (normal or abnormal; Figure 1).9 It isalso critically important to remember poly-

morphisms in these genes (and those to be discov-ered) are simply associated with schizophrenia; thesegenes do not invariably determine outcome, but onlylend a higher probability for the subsequent develop-ment of illness.10 In fact, genes will never code forabnormal behaviors per se, but rather code for pro-teins, that make up cells, forming circuits, that incombination determine facets of abnormal and nor-mal behavior. With mood disorders, there is symp-tom overlap, thus inheritance of multiplesusceptibility genes could lead to similar symptomsamong the different mood disorders, with other fac-tors (eg, epigenetic) affecting the ultimate expressionof which mood disorder presents. Alternatively, dif-ferent mutations in the same susceptibility genescould drive the ultimate presentation, similar to therelationship between migraine and epilepsy.11

The elucidation of genotype-phenotype relation-ships is at an early stage, but current findings highlightthe need to consider alternative approaches to classifi-cation and conceptualization for psychiatric research;as we discuss below, the endophenotype model is onesuch very promising model.

Figure 1.The Heterogeneity and Genetics of Bipolar Disorder

WMA = white matter abnormalities.Data from Hasler et al.9

Environmental Factors:Psychological stressSleep deprivationRepoductive hormonesStimulants

Epigenetic Events:Early onset WMAReward impairmentCognitive and attention

deficitsReduced anterior

cingulate volume

SchizoaffectiveBIPOLARI, IISusceptibility Genes

(? Location of mutation)Protective Genes

Modifying GenesImprinting

Variable Haploinsufficiency

RecurrentUnipolar

(severe depression)

Johns Hopkins Advanced Studies in Medicine n S419

PROCEEDINGS

WILL AN ENDOPHENOTYPE STRATEGY FOR

BIPOLAR DISORDER LEAD TO A MAJOR PAYOFF?

With respect to additional conceptualizationsfor psychiatric research, it is becoming increas-ingly more clear that, although the pathwaysbeginning with genes and then expressedthrough simple biological processes do not nec-essarily have a single quantifiable endpoint (ie,behavior), it may be possible to assay the resultof aberrant genes through more biologically“simpler” approaches.9 The term “endopheno-type” was described as an internal, intermediatephenotype (ie, not obvious to the unaided eye)that fills the gap in the causal chain betweengenes and distal diseases, and therefore may helpto resolve questions about etiology.12 Theendophenotype concept assumes that the num-ber of genes involved in the variations ofendophenotypes representing more elementaryphenomena (as opposed to the behavioralmacros found in the DSM) are fewer than thoseinvolved in producing the full disease.13

Endophenotypes provide a means for identify-ing the “upstream” traits underlying clinical phe-notypes, in addition to the “downstream”biological consequences of genes. The methodsavailable to identify endophenotypes include neu-ropsychological, cognitive, neurophysiologic,neuroanatomical, imaging, and biochemical mea-sures.9 The information revised in this volumesuggests that candidate brain function endophe-notypes include attention deficits, deficits in ver-bal learning and memory, cognitive deficitsfollowing tryptophan depletion, circadian rhythminstability, and dysmodulation of motivation andreward. Moreover, reduced anterior cingulate vol-ume and early onset white matter abnormalitiesrepresent candidate brain structure endopheno-types. Finally, symptom provocation endopheno-types may be based on bipolar patients’ sensitivityto sleep deprivation, psychostimulants, andcholinergic drugs.9

NEUROTRANSMITTER AND NEUROPEPTIDE

ABNORMALITIES IN BIPOLAR DISORDER

In bipolar disorder, studies have produced asurfeit of findings implicating several major neu-

Direct targets of lithium Description

Inositol monophosphatase Rate-limiting enzyme in inositol recycling; lithium's(IMPase) inhibition of IMPase led to the inositol depletion

hypothesis of lithium's actionsInositol polyphosphate Enzyme involved in inositol recycling in

1-phosphatase (IPPase) phosphoinositol signaling; acts before IMPaseBisphosphate nucleotidase Removes phosphate from 30-phosphoadenosine

(BPNase) 50-phosphate (PAP) to form adenosine 50-phosphate (AMP); an increase in PAP inhibits sulfotransferases, which transfer sulfur tobiological molecules

Fructose 1,6-bisphosphatase Key enzyme in glyconeogenesis; catalyzes the (FBPase) removal of the 1-phosphate from fructose 1,

6-bisphosphatase to form fructose 6-phosphatePhosphoglucomutase (PGM) Key enzyme in glycogenolysis and glycogenesis;

catalyzes the formation of glucose-1-phosphate from glucose-6-phosphate during glycogenolysis (and the reverse during glycogenesis)

Glycogen synthase kinase-3 Normally active kinase that is inhibited by the (GSK-3) activity of many signaling pathways; inhibiting

GSK-3 has been linked to neurotrophic sup-port, neuroprotection, and possible modulation of circadian rhythms; preclinical studies suggest antidepressant-like effects of inhibiting GSK-3

Direct targets of valproic acidSuccinate semialdehyde Converts succinate semialdehyde to succinate;

dehydrogenase (SSA-DH) succinate inhibits GABA-T; inhibiting GABA-T increases GABA levels

GABA transaminase Converts GABA to succinate semialdehyde; (GABA-T) however, valproic acid inhibition appears to be

above the therapeutic rangeSuccinate semialdehyde Converts succinate semialdehyde to gamma-

reductase (SSA-R) hydroxybutyrate (GHB)Histone deacetylases Enzymes that acetylates histones; inhibition of

(HDACs) this enzyme results in an increase in nonacety-lated histones and a resultant increase in tran-scription of some genes

Sodium channels Inhibits sodium channel function at times of high-frequency firing

Direct targets of carbamazepineSodium channels Inhibits sodium channel function at times of

high-frequency firingAdenosine receptor Antagonist at A1 subtype; increases adenosine

receptor protein levels in rats; adenosine has modulatory functions on neurotransmitter release and numerous behavioral and cognitive functions

Adenylate cyclase (AC) AC forms cyclic AMP from adenosine triphos-phate, and as such is a principal regulator of the adenylate cyclase signaling pathway; carba-mazepine attenuates cyclic AMP signaling and appears to have an inhibitory effect on AC or an AC-associated protein

Table. Direct Targets of Lithium,Valproate, and Carbamazepine:Drugs Used to Treat Bipolar Disorder

Adapted with permission from Gould et al. Mol Psychiatry. 2004;9:734-755.14

S420 Vol. 6 (6A) n June 2006

PROCEEDINGS

rotransmitters and neuropeptides: abnormal levels ofnorepinephrine, serotonin, acetylcholine, dopamine,glutamate, GABA, corticotropin releasing factor, andcortisol in cerebrospinal fluid, plasma, and/or urine inpatients with bipolar disorder. Rather than all of thesesubstances being independently abnormal, perhapsthere is something common that regulates all of them?One way to approach this idea is to understand the pre-cise biochemical targets of antimanic drugs and moodstabilizers. Three of the most effective and commonlyused drugs for bipolar disorder are valproate (an 8-car-bon fatty acid), lithium (a monovalent cation), and car-bamazepine (an anticonvulsant with a similar chemicalstructure to tricyclic antidepressants).14 The direct tar-gets of these drugs are listed in the Table.14 Note that all3 drugs affect enzymes or proteins involved in intracel-lular signaling cascades (ie, adenylyl cyclase, phospholi-pase A and C, cGMP phosphodiesterase, potassiumchannels, and calcium channels). These cascades areinitiated when a membrane-bound receptor receives asignal, which activates an associated G-protein thatultimately affects gene expression, protein function,and perhaps cellular plasticity via these enzymes (Figure2). The potential role of some of these enzymes isdescribed later in this article.

The role of intracellular signaling cascades holdsgreat interest because it may explain not only all of theneurotransmitter systems involved in bipolar disorderbut also all of the comorbidities with bipolar disorder,and some of the physical brain changes observed.Effects on the more general role of intracellular signal-

ing may also explain why structurally dissimilar drugs(ie, lithium, valproate, and carbamazepine) can all beeffective in treating bipolar disorder and why there is alag time between pharmacotherapy initiation and ben-eficial effect, which is usually several weeks. Similarly,when patients stop taking these drugs, an immediatewithdrawal effect is not seen; relapse can be delayedseveral weeks. The role of intracellular signaling cas-cades in other chronic diseases have been appreciatedin recent years (eg, cancer and diabetes), thus there issome precedent for their role in chronic diseases.15-19

PROTEIN KINASE CProtein kinase C (PKC) is present throughout the

body and acts as a major regulator of neuronalexcitability. It is activated by amphetamines, cocaine,and stress—factors that typically destabilize bipolardisorder—and plays a central role in phosphorylatingproteins, such as neurotransmitter and neuropeptidereceptors, signaling molecules, transcriptional factors,and cytoskeletal proteins.20,21 It is thought to play animportant role in bipolar disorder because of its altereddistribution and functioning in postmortem studies offrontal cerebral cortex and blood platelets from bipo-lar disorder patients.22-24 PKC also appears to be thetarget of lithium’s effect on bipolar mania.25-29 Veryrecent studies in patients with bipolar disorder showthat lithium and valproate do indeed alter PKC activ-ity and distribution and that high levels of PKC activ-ity in prefrontal cortex markedly impair behavioraland electrophysiological measures of working memory,possibly contributing to signs of prefrontal corticaldysfunction, such as distractibility, impaired judg-ment, impulsivity, and thought disorder.30,31

Only 3 PKC inhibitors are currently available forhumans: tamoxifen, ruboxistaurin (now approved fordiabetic retinopathy), and bryostatin-I. Tamoxifen isan estrogen receptor blocker best known for its use inbreast cancer treatment; however, it also inhibits PKCat higher concentrations. Tamoxifen crosses the blood-brain barrier and has good tolerability. In animal mod-els of mania, tamoxifen attenuates the effects ofamphetamine (eg, risk-taking behavior and hedo-nism).32 A pilot study in 13 women with acute bipolardisorder in the manic phase shows that tamoxifen isable to reduce manic symptoms over 28 days (Figure3).33 Current studies are also looking at a possible rela-tionship between PKC polymorphisms and responseto lithium or valproate.

Figure 2. Intracellular Signaling Cascades

Receptor G protein Effector

MultiplePhysiologicProcessesAdenylyl cyclase

Phospholipases C & A2

cGMP phosphodiesterasePotassium channelsCalcium channels

αβ

γ

Johns Hopkins Advanced Studies in Medicine n S421

PROCEEDINGS

GLYCOGEN SYNTHASE KINASE-3Glycogen synthase kinase-3 (GSK-3) is activated

by the insulin receptor and it regulates (as its namesuggests) glycogen and glucose synthesis as well asnerve growth and survival, circadian rhythms, andtargets in the pathophysiology of Alzheimer’s disease(ie, tau phosphorylation, presenilins, and amyloiddeposition). It is present in the adult brain and is reg-ulated by amphetamines, estrogen, and glucocorti-coids. GSK-3 exerts its effects via promotingapoptosis and inducing changes in gene expressionthat lead to long-term changes in synaptic connec-tions and modulation of critical neuronal circuits.34

GSK-3 is a clear target of lithium; in animal models,GSK-3 inhibitors produce an unusual antimanic andantidepressive phenotype.14 As reviewed by Gould etal, several GSK-3 inhibitors are under investigationnow in clinical studies.14

AMPA RECEPTORS

Glutamate is the major excitatory neurotransmitterand has 4 major types of receptors: N-methyl-D-aspar-tate (NMDA), α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA), kainite, and metabotropic.AMPA receptors play a major role in regulating variousforms of neural and behavioral plasticity and areinvolved in both mood disorders and drugs of abuse.35,36

AMPA receptors may sit at the crossroads of rewardpathways in the brain, which may explain the very highrates of comorbid substance abuse with bipolar disor-der. AMPA receptor potentiators reduce the rate ofreceptor desensitization and/or deactivation in thepresence of an agonist (eg, glutamate and AMPA).37,38

AMPAkines are a subclass of AMPA receptor potentia-tors that produce positive modulation of AMPA recep-tors; they are actively under investigation for thetreatment of schizophrenia.39 Preclinical studies with anAMPAkine have shown reduced synaptic and neuronaldegeneration from excitotoxic insults, with other anti-depressant effects.40,41 Preclinical studies also show thatlithium and valproate reduce synaptic expression of aglutamate receptor subunit, while imipramine (an anti-depressant that can trigger manic episodes) increasedexpression of this receptor subunit.42 AMPAkines havealso undergone preliminary evaluation in patients withschizophrenia and we are starting a study withAMPAkines in treatment of depression.

NMDA ANTAGONISTS

N-methyl-D-aspartate antagonists are used as anti-convulsants and are now being studied for the treat-ment of depression. There have been 2 studies ofintravenous (IV) ketamine in depression with veryexciting results.43,44 These studies in treatment-resistantunipolar major depressive disorder revealed robust andrapid antidepressant effects resulting from a single IVdose of an NMDA antagonist; interestingly, onset ofantidepressant effects was seen very rapidly (occurringwithin 4 hours after infusion) and remained signifi-cant for 1 week.44

GLUTAMATE RELEASE INHIBITORS

Riluzole, a neuroprotective agent with anticon-vulsant properties, was originally approved for thetreatment of amyotrophic lateral sclerosis. It acts byinhibition of the glutamate release, blocking presy-naptic calcium and sodium ion channels, by inhibi-tion of the voltage-dependent sodium channels in

Tamoxifen

MPA

Placebo

Baseline Day 7 Day 14 Day 21 Day 28

0

5

10

15

20

25

30

35

Day number

Mea

n C

AR

S-M

tota

l sco

re

Figure 3.The Effect of Tamoxifen Treatment onManic Symptoms

This was a 3-arm, double-blind, randomized, placebo-controlled, 28-day, adjunctivestudy in 13 women with mania. The 3 treatment arms were: 40 mg/day oral tamox-ifen, 20 mg/day (MPA), and oral placebo capsules containing glucagon powder.CARS-M = Clinician-Administered Rating Scale for Mania; MPA = medroxyproges-terone acetate.Reprinted with permission from Kulkarni et al. Psychoneuroendocrinology.

S422 Vol. 6 (6A) n June 2006

PROCEEDINGS

mammalian central nervous system (CNS) neurons,and by inhibition of postsynaptic actions by indirectmechanisms. It has been shown to have neuroprotec-tive properties in animal models of Parkinson’s dis-ease, dementia, ischemia, and traumatic CNS injury.It has shown some antidepressant benefit in a pilotstudy of 14 patients with bipolar disorder (Figure4).45 Similarly, lamotrigine, which works presynapti-cally to regulate glutamate release, has shown signifi-cant antidepressive benefit in long-term,randomized, placebo-controlled studies in patientswith refractory bipolar I depression.46-49

BRAIN MORPHOLOGY AND MOOD DISORDERS

With computed tomography and magnetic reso-nance imaging (MRI), we can now see morphologicchanges in patients with mood disorders. Althoughmood disorders are not classic neurodegenerative dis-orders, such as Alzheimer’s disease, there is measurablebrain atrophy and nerve cell loss. In 1997, Drevets etal showed using MRI quantitative loss of prefrontal

cortex gray matter in the brains of unmedicatedpatients with bipolar and unipolar depression by 39%and 48%, respectively (P <.0002).50 Using positronemission tomography, they also showed decreasedmean measures of metabolism by 16.3% anddecreased mean measures of blood flow by 18.5% inthis region of the brain in patients with bipolar disor-der, relative to control subjects.50 Postmortem studiesof patients with bipolar disorder also reveal decreasedvolume, decreases in the number of neurons, anddecreases in glia in specific areas of the brain.51 It is notcurrently known if these alterations constitute devel-opmental abnormalities conferring vulnerability tosevere mood episodes, compensatory changes to otherpathogenic processes, or the sequelae of recurrentaffective episodes.52 However, a recent report showedthat individuals at high risk of developing mood dis-orders exhibited reduced subgenual prefrontal corticalvolumes, raising the possibility that this endopheno-type may constitute a heritable vulnerability factor inthese patients.52 Overall, the data reviewed clearlyshows that bipolar disorder, undoubtedly a neuro-

chemical illness, is also a dis-order associated withimpairments of cellular plas-ticity.

It has also been hypothe-sized that the atrophy resultsfrom fewer dendritic spineor branches, rather thanactual cell death.53 Of note,these morphologic changeshave also been observed inchildren with bipolar disor-der.54 Studies have shown anassociation between depres-sion duration and hip-pocampal volume.55,56

MacQueen et al note thatreductions in hippocampalvolume may not antedate ill-ness onset, but volume maydecrease at the greatest ratein the early years after illnessonset.55

A recent study of med-icated and nonmedicatedpatients with bipolar disor-der (n = 36) showed the

0

5

10

15

20

25

30

35

40

0 1 2 3 4 5 6 7 8

All patients

Trial completers

Week

* * **

**

**

* **

* **

P <.05 compared to baseline

MA

DR

S t

ota

l sc

ore

50%50%

0

10

20

30

40

50

60

70

80

%

Response Remission

Pat

ient

s, %

Figure 4. Antidepressant Efficacy in Patients withBipolar Depression Treated with Riluzole

Fourteen patients (10 men, 4 women; mean ± SD age = 49.5 ± 9.2 years) with bipolar depression (bipolar I: n = 6;bipolar II: n = 8) entered the study; most were inpatients (n = 8). Eight subjects (57%) completed the 8-week trial. Ofthose patients who showed a response, 50% achieved remission, suggesting a possible “glutamatergic subtype” ofpatients with bipolar disorder.*P <.05 vs baseline.MADRS = Montgomery-Asberg Depression Rating Scale.Adapted with permission from Zarate et al. Biol Psychiatry. 2005;57:430-432.45

Johns Hopkins Advanced Studies in Medicine n S423

PROCEEDINGS

extent of neuroplastic changes that may be occurring.Using MRI and voxel-based morphometry, the resultsshowed morphometric abnormalities in brain areasknown to exhibit increased metabolism in depression.These areas project to the network of prefrontal cortexand other areas (ie, amygdala, hippocampal subicu-lum, and ventral striatum) already identified to bealtered based on postmortem studies of patients withbipolar disorder. Thus, the morphologic changes inbipolar disorder occur in an extended network of neu-roanatomical structures that regulate emotional behav-ior.57

Preclinical studies in animal models of stress showmeasurable effects of valproate and lithium in neu-ronal cell death and expression of neuroprotectivepeptides. Clinical studies are measuring the effect ofbipolar disorder treatments on morphometric brainchanges. In a pilot study, 10 patients with bipolar dis-order, who were in a depressed state, received 4 weeksof lithium treatment. MRI scans showed lithium sig-nificantly increased total gray-matter volume in 8 of10 patients by a mean of 3%.58 In a similar study,healthy volunteers and patients with newly diagnosedbipolar disorder were treated with lithium for 8weeks. MRI analysis showed an overall increase ingray matter volume and in specific brain areas in thosepatients with bipolar disorder, but not in the controls(article in preparation). Collectively, these data sug-gest a causal relationship between volume changesand mood disorders. However, the observed changeswith treatment occur over a period of at least 4 weeks.Therefore, perhaps these treatments restore neuronalconnections through which neurotransmitters, suchas serotonin and norepinephrine, work. If this theoryholds true, this suggests the possibility of altering thelong-term trajectory of the illness if patients are treat-ed early enough.

FUTURE DIRECTIONS

Figure 5 shows 11 of the identified cellular targetsin the expression of mood disorders; 7 of them are cur-rently being investigated for therapeutic options.4 Ofparticular interest is the use of transcriptomics (thegenome-wide study of mRNA expression levels).59

Technological advances in recent years, with tech-niques such as differential display and micorarrayanalysis (Sidebar), allow investigators to measure at agenetic level the pathophysiologic processes of mood

DIFFERENTIAL DISPLAY ANDMICROARRAY ANALYSIS

—

DIFFERENTIAL DISPLAY

In differential display, mRNA from the tissue ofinterest is isolated and reverse transcribed to form itscDNA. The cDNA sequences are amplified by poly-merase chain reaction, and the resultant products areseparated on polyacrylamide gels. The bands canthen be visualized, for example, by autoradiography.If a band is present in a lane representing one sampleand not the other, this band is considered to repre-sent an expressed gene and is excised and furtheramplified. The amplified gene product is used toscreen a library to obtain a full-length cDNA, which,using sequence homology and sequencing, can beused to identify the gene of interest.59

MICROARRAY ANALYSIS

This technique builds on other standard tech-nologies that use cDNA to probe immobilizedsequences of DNA. Microarray analysis involvesimmobilization of the entire expressed gene contentof an organism (not just a cell type or tissue) onto alarge number of locations on a solid support (com-monly a derivatized glass microscope slide or siliconwafer), in the form or arrays of dots. Each dot repre-sents a unique sequence. RNA from the tissue ofinterest is extracted, labeled with a fluorescent dye,an hybridized to the sequences on the array. In theo-ry, the signal detected from each spot would be pro-portional to the amount of that specific RNA in theoriginal sample, so that the technique shows not onlywhat gene is expressed but also how much. As shownin the Figure, microarray can also show which genesare expressed in response to treatments for bipolardisorder, such as lithium or valproate.59

(Continued on page 424)

S424 Vol. 6 (6A) n June 2006

PROCEEDINGS

disorders and the effects of current medications onthese disorders. To date, these techniques have sug-gested that some of the genes involved in bipolar dis-order are in fact classes of genes involved inneurotrophy and cell survival, mitochondria function,metabolism, cytoskeleton, and cell signaling. TheSequenced Treatment Alternatives to RelieveDepression study is a multicenter study designed toevaluate the comparative effectiveness of treatmentswhen used either as augmenting treatments or as newtreatments when remission is not achieved with citalo-pram. Clinical outcomes include symptoms, function,side effect burden, quality of life, and participant sat-isfaction.60 We will be obtaining the DNA from about2000 of these study patients to perform differentialdisplay and microarray analysis.61

CONCLUSIONS

Our understanding of the pathophysiology ofmood disorders has moved beyond simple explana-tions of neurotransmitter deficiencies. Given the com-plex presentation of mood disorders and the advancesin medical research technology, we now are starting tounderstand the true mechanisms of action of some ofour older treatments as we better understand theunderlying neurobiology of the disorders. These pur-suits are not strictly an academic interest. An increas-ing amount of data is suggesting a number of newdrugs for cellular targets, which, if effective, will allowclinicians to address the fundamental biology of thedisorder, rather than the presenting symptoms.

DISCUSSION

MOLECULAR MECHANISMS OF DRUGS FOR

BIPOLAR DISORDER

Dr Treisman: Have you studied whether there’s adifference between lithium and valproate in terms oftheir antidepressant effects?

Dr Manji: Yes, but at this point it’s clearly veryspeculative. In some parts of the brain, we see anequivalent effect, but in the frontal cortex, lithiumseems to have more of a neurotrophic effect than val-proate. The longitudinal data in humans thus far areonly with lithium, but it’s interesting that the leftsubgenual prefrontal cortex is one of the places thatseems to correlate with lithium’s antidepressantresponse. Therefore, that may be something that dif-

(Continued from page 423)

AACondition

RNA isolationand RT to cDNA

PCR amplifycDNAs

SeparatecDNAs onsequencing gel

BB

Clonedifferentiallyexpressed cDNA

Sequence

DatabaseSearch

Functional Studies

Validate transcript ∆s

Figure. Differential Display andMicroarray Analysis

PCR = polymerase chain reaction; RT = reverse transcription.

Lithium Valproate

Microarray Studies

Johns Hopkins Advanced Studies in Medicine n S425

PROCEEDINGS

ferentiates the 2 agents and may explain lithium’santidepressant effects.

Dr Kaye: Do you think tamoxifen is workingthrough cortisone, which we have historically thoughtis probably not good for mood, or through this PKCinhibitory effect, or are they the same?

Dr Manji: In our studies, the animal behaviorsresponsive to tamoxifen really do look like they’rePKC-mediated. In animals, we use PKC inhibitors(which are very clean) and show that the inhibitorsmimic tamoxifen’s beneficial antimanic effects. Wehave not looked as much at glucocorticoids; we’velooked more into estrogen with tamoxifen.

In the human studies, it’s obviously very difficult. Weonly chose tamoxifen because it’s available for humansand happens to be a PKC inhibitor, even though it isused for its estrogen receptor blockade. We’re seeing justas good an effect in men as in women, but that doesn’tanswer your original question. With tamoxifen, we’retalking about estrogen receptor blockade, not circulatingestrogen, thus it doesn’t necessarily tell you that it’s not anestrogen receptor effect. Most of our studies suggest thatthis antimanic effect is, in fact, due to PKC inhibition,but you’ll only know for sure when you give one of thecleaner PKC inhibitors. We’re working with the NationalCancer Institute on this right now. Anecdotally, we’ve

heard about many womenwith bipolar disorder whodevelop breast cancer, go ontamoxifen, and that tends todestabilize the bipolar illness.

Dr Kaye: Do any of ourcurrent drugs affect GSK-3and are there any differ-ences between currentlyavailable agents?

Dr Manji: The only drugthat’s a direct GSK-3inhibitor is lithium. That’sone of the reasons it may betried as a modifier of diseaseprogression in Alzheimer’sdisease. GSK-3 inhibitorsand lithium have a dramaticeffect on Alzheimer’s diseasepathophysiology in animalmodels of Alzheimer’s disease.Lithium is the only directGSK-3 inhibitor, but it’slooking like many other com-pounds can affect GSK-3,such as valproate. Recentstudies have suggested thatatypical antipsychotics mayalso have some effect onGSK-3, but overall antipsy-chotics have not been as wellstudied in this area.62,63 Insome of our studies, clozap-ine behaves more like lithiumthan olanzapine or risperi-done, or other antipsychotics.

Figure 5. Novel Therapeutic Targets for the Treatment of Severe Mood Disorders

Of the 11 potential targets shown here, 7 are currently being investigated as potential therapeutic targets. They are asfollows:1. Phosphodiesterase inhibitors2. MAPK modulators3. mGluR II/III receptor agonists4. Riluzole5. –6. NMDA antagonists (memantine, felbamate, and ketamine)7. –8. CRH antagonists (antalarmin)9. Glucocorticoid antagonists (RU486)10. Agents that upregulate bcl-2 (eg, pramipexole)11. –CRH = corticotropin-releasing hormone; MAPK = mitogen-activated protein kinases; NMDA = N-methyl-D-aspartate.Reprinted with permission from Charney, Manji. Sci STKE. 2004;225:re5.4

NE

α2ARLocusCoeruleus

5-HT

Glu

StressStress

Glia

Stress

Enhancement of cellularplasticity and resilience

Stress

Stress

1

11

3

4

8

76

5

9

2

Glu reuptaketransporter

5-HT reuptaketransporter

Dorsal Raphe5-HT 1A

Antidepressant

NE reuptaketransporter

cAMP

↑PKA

BDNF

TrkB

Bcl-2

↑CREBP

GSK-3

AKT

LithiumH

H

G5

AC

BDNF

CA2+-dependent orMAPK cascades

Gi orGq

NMDAR

AMPAR

CRH

GC

GC

GR

GC

GR

↑

↑10

S426 Vol. 6 (6A) n June 2006

PROCEEDINGS

Dr Leibenluft: One of the big trends in treatmentof bipolar disorder has been greater use of atypicalantipsychotics. Do these drugs show any of the sameGSK-3 inhibitory effects as lithium and valproate?

Dr Manji: It has some of the same effects, but notall. I am somewhat hesitant to say, as others are saying,that any antipsychotic is more than just antimanic.The data suggest that some of them have more than anantimanic effect, and we still need to see those data.Olanzapine does appear to have a modest antidepres-sant effect, but is it more than just an antimanic effect?We’re starting to do some studies with quetiapine,which has so far stood out, in our opinion, as possiblyhaving more than just antimanic effects. It may also beuseful as monotherapy for depression.

Dr Leibenluft: There’s also the issue about pro-phylaxis. To be truly a mood stabilizer, it would haveto prevent mood episodes. Are you saying that per-haps there’s been a bit too much labeling of atypicalantipsychotics as mood stabilizers? Do you think thatjumps ahead of where the treatment data really are?And, from a mechanistic perspective, what are yourthoughts on atypical antipsychotics with regard tothe different pathophysiologic pathways you’vetalked about?

Dr Manji: We can certainly show many effects.PKC inhibition does appear to be a common effect ofmany antipsychotics, typical or atypical. You can cate-gorize compounds as to which are antimanic andwhich are promanic, and they separate along thoselines in their effects on PKC. In some of these neu-rotrophic signaling cascades, we don’t see as robust adistinction. Our approach hasn’t been to try and cata-log every drug with these effects. The flip side wouldbe that not every drug has to work on regulating plas-ticity. Its effect may be due to a common downstreammechanism. You can get there multiple ways.

PATHOLOGICAL MECHANISMS

Dr Adams: You had mentioned that when patientsstop taking their antidepressant, the relapse is notimmediate; it’s delayed. I’ve probably seen that at least100 times where the patient has stopped the antide-pressant and said, “I was okay, and thought I didn’tneed it anymore,” and they relapse. And, it’s not justbecause the half-life of their medication was long.

Dr Manji: That’s exactly what we intend to show.This is, in fact, a cascade. There is a lag period; if thepatient discontinues the medication, the cascade has to

unravel before they get worse. In fact, when they dis-continue medication, often they feel better becausethey don’t have side effects. But, then the cascadeunravels and they relapse. That’s one of the biggestchallenges we face with compliance: patients linkingmedication cessation and worsening of symptoms. It’snot like missing the insulin dose. The whole cascadehas to unravel.

I don’t want to oversell some of these neurotrophiceffects because we need many more studies, but someof these treatments may be providing neuroprotectiveeffects. We may not know until we have the 20-yearlongitudinal data to show that the drugs are providingsome protection against the ravages of the illness.Perhaps we can use that information to help increasecompliance, to explain to patients that although themedications have side effects, they need to keep takingthem to avoid some of the other long-term complica-tions, as we have found with treating diabetes.

Dr Ostacher: I use the analogy of going to thegym. When you go to the gym to lift weights, thefirst day you do it, you’re just tired, not stronger. But,if you do it regularly for a month, you’re muchstronger at the end of that month, and just becauseyou didn’t lift weights one day doesn’t mean you’renot going to be strong that day. Patients can get theirheads around that without understanding the molec-ular biology of it. It often helps people to understandthat stopping the drug will lead to diminishing ofthis effect over time.

Dr Treisman: On the other hand, sometimes ifyou make an analogy like that people will ignore you,but if you use the actual scientific terms, such as glu-tamate, dopamine, acetylcholine, and PKC, peoplewill pay attention.

Dr Leibenluft: The literature on lithium andmemory is murky. Do you not believe the literaturethat lithium has an adverse impact on memory? Is thatbased on your findings, which argue the reverse? Or isit because there are many different kinds of memory?

Dr Manji: I think some patients do experiencesome of this memory deficit; I view it as a side effectthat is occurring separate from the beneficial effects.PKC in the hippocampus has certainly been implicat-ed in learning and memory. The memory problemsoccurring here and now may potentially have nothingto do with the long-term neurotrophic effects.

We’ve also attempted to find out whether you needthe full antimanic dose to have the neurotrophic

Johns Hopkins Advanced Studies in Medicine n S427

PROCEEDINGS

effects. In the Alzheimer’s disease study and in theschizophrenia study, they’re actually using low doses.We find that, in animals, plasma levels that are about50% to 33% of what we traditionally think of as ther-apeutic levels are enough to upregulate some of thesegrowth factor cascades and bring about some of theselong-term changes. Therefore, it may well be that ifyou’re using it mainly for neurotrophic benefit (andnot immediate treatment of mania), you can get awaywith using lower doses and not have some of the sideeffects. That’s why they’re looking at lower doses in theclinical studies in other disorders, as a way of makingthese drugs more tolerable.

COMORBIDITIES

Dr Adams: You were talking about the intracellu-lar signaling cascades, and the fact that there are sharedpathways between different illnesses. We often wonderwhy we can use an antidepressant to treat migraines,chronic pain, and depression. That may be a way thatwe could explain it to patients, if that was borne out.

Dr Manji: I think that will ultimately be the case;that the comorbidities we see are not by accident.There may be a shared pathophysiology and the samemay be true of the treatments. In fact, with thesedrugs, you’re targeting some of these intracellular cas-cades, which play a role in multiple illnesses, and that’show you’re getting some of the benefits, not just as anonspecific sedating agent. We are also trying to gofurther with the genetic studies, with patients whohave both bipolar disorder and migraines. We’re look-ing to see if they have white matter hypointensities onMRI. Are they a subpopulation that has some com-mon pathophysiology? Researchers are now askingthose kinds of questions to see if they can tease apartpathways that may be shared. That’s become more rel-evant in the area of drug abuse (ie, that there is somereward pathway disturbance that predisposes to devel-oping bipolar disorder and drug abuse), and theAMPA receptor trafficking is one of the areas that’slooking quite interesting.

We are working with Jules Angst, MD, to see ifnaturalistic lithium treatment for 30 years has anyeffect on the likelihood of developing Alzheimer’s dis-ease. It’s a “dirty study.” People are treated with lithi-um and with many other types of drugs (eg,antipsychotics and antidepressants), but when youcontrol for all of those other factors, the lithium-treat-ed group tends to have a lower incidence of

Alzheimer’s disease. And, it’s not just restricted to thelithium-responsive patients.

Dr Angst has data looking at all-cause mortalitywith lithium treatment in patients with bipolar disor-der. We see in patients with unipolar depression areduction in suicides but not in all-cause mortality.

Dr Ostacher: In the Swedish study, the excess mor-tality in unipolar depression was almost all due tounnatural causes (ie, accidents and suicides), and inbipolar depression it was almost all due, except in thefirst few years, to natural causes.64

Dr Manji: As we are using atypical antipsychoticsmore as mood stabilizers, we are seeing the deleteriouseffects (dyslipidemia and metabolic abnormalities), butthere’s some reason to believe that some of our agents,such as lithium, may also reduce all-cause mortality.They do have cardioprotective effects. We don’t want toovergeneralize, but because we are seeing this protec-tion, we’re asking is it simply because there is reducedstress or are these drugs targeting some fundamentalprocess that has beneficial effects beyond the brain.

REFERENCES

1. DePaulo JR Jr. Genetics of bipolar disorder: where do westand? Am J Psychiatry. 2004;161:595-597.

2. Levinson DF, Levinson MD, Segurado R, Lewis CM.Genome scan meta-analysis of schizophrenia and bipolardisorder, part I: Methods and power analysis. Am J HumGenet. 2003;73:17-33.

3. McQueen MB, Devlin B, Faraone SV, et al. Combinedanalysis from eleven linkage studies of bipolar disorder pro-vides strong evidence of susceptibility loci on chromosomes6q and 8q. Am J Hum Genet. 2005;77:582-595.

4. Charney DS, Manji HK. Life stress, genes, and depression:multiple pathways lead to increased risk and new opportu-nities for intervention. Sci STKE. 2004;225:re5.

5. Hasler G, Drevets WC, Manji HK, Charney DS.Discovering endophenotypes for major depression.Neuropsychopharmacology. 2004;29:1765-1781.

6. Craddock N, O’Donovan MC, Owen MJ. The genetics ofschizophrenia and bipolar disorder: dissecting psychosis. JMed Genet. 2005;42:193-204.

7. Angst J. Historical aspects of the dichotomy between manic-depressive disorders and schizophrenia. Schizophr Res.2002;57:5-13.

8. Maziade M, Roy MA, Chagnon YC, et al. Shared andspecific susceptibility loci for schizophrenia and bipolar dis-order: a dense genome scan in Eastern Quebec families.Mol Psychiatry. 2004;10:486-499.

9. Hasler G, Drevets WC, Gould TD, et al. Toward construct-ing an endophenotype strategy for bipolar disorders. BiolPsychiatry. 2006; e-publication.

S428 Vol. 6 (6A) n June 2006

PROCEEDINGS

10. Gould TD, Manji HK. The molecular medicine revolutionand psychiatry: bridging the gap between basic neuro-science research and clinical psychiatry. J Clin Psychiatry.2004;65:598-604.

11. Kors E, Haan J, Ferrari M. Migraine genetics. CurrHeadache Rep. 2003;7:212-217.

12. Gottesman II, Shields J. Genetic theorizing and schizophre-nia. Br J Psychiatry. 1973;122:15-30.

13. Gottesman II, Gould TD. The endophenotype concept inpsychiatry: etymology and strategic intentions. Am JPsychiatry. 2003;160:636-645.

14. Gould TD, Quiroz JA, Singh J, et al. Emerging experimentaltherapeutics for bipolar disorder: insights from the molecularand cellular actions of current mood stabilizers. MolPsychiatry. 2004;9:734-755.

15. Staehler M, Rohrmann K, Haseke N, et al. Targeted agentsfor the treatment of advanced renal cell carcinoma. CurrDrug Targets. 2005;6:835-846.

16. Silvestri GA, Rivera MP. Targeted therapy for the treatmentof advanced non-small cell lung cancer: a review of theepidermal growth factor receptor antagonists. Chest.2005;128:3975-3984.

17. Cheng JQ, Lindsley CW, Cheng GZ, et al. The Akt/PKBpathway: molecular target for cancer drug discovery.Oncogene. 2005;24:7482-7492.

18. Farese RV, Sajan MP, Standaert ML. Insulin-sensitive proteinkinases (atypical protein kinase C and protein kinaseB/Akt): actions and defects in obesity and type II diabetes.Exp Biol Med. 2005;230:593-605.

19. Joy SV, Scates AC, Bearelly S, et al. Ruboxistaurin, a pro-tein kinase C beta inhibitor, as an emerging treatment fordiabetes microvascular complications. Ann Pharmacother.2005;39:1693-1699.

20. Asaoka Y, Yoshida K, Oka M, et al. The signal-inducedphospholipid degradation cascade and protein kinase Cactivation. Ciba Found Symp. 1992;164:50-59.

21. Asaoka Y, Yoshida K, Oka M, et al. The family of proteinkinase C in transmembrane signaling for cellular regulation.J Nutr Sci Vitaminology. 1992:7-12.

22. Friedman E, Wang HY, Levinson D, et al. Altered plateletprotein kinase C activity in bipolar affective disorder, manicepisode. Biol Psychiatry. 1993;33:520–525.

23. Wang HY, Friedman E. Enhanced protein kinase C activityand translocation in bipolar affective disorder brains. BiolPsychiatry. 1996;40:568-575.

24. Wang HY, Johnson GP, Friedman E. Lithium treatmentinhibits protein kinase C translocation in rat brain cortex.Psychopharmacology. 2001;158:80-86.

25. Wang HY, Friedman E. Lithium inhibition of protein kinaseC activation-induced serotonin release.Psychopharmacology. 1989;99:213-218.

26. Wang HY, Friedman E. Protein kinase C translocation inhuman blood platelets. Life Sci. 1990;47:1419-1425.

27. Manji HK, Chen G, Hsiao JK, et al. Regulation of signaltransduction pathways by mood-stabilizing agents: implica-tions for the delayed onset of therapeutic efficacy. J ClinPsychiatry. 1996;57(suppl 13):34-46.

28. Hahn CG, Friedman E. Abnormalities in protein kinase Csignaling and the pathophysiology of bipolar disorder.

Bipolar Disord. 1999;1:81-86.29. Lenox RH, Hahn CG. Overview of the mechanism of action

of lithium in the brain: fifty-year update. J Clin Psychiatry.2000;61(suppl 9):5-15.

30. Birnbaum SG, Yuan PX, Wang M, et al. Protein kinase Coveractivity impairs prefrontal cortical regulation of workingmemory. Science. 2004;306:882-884.

31. Hahn CG, Umapathy, Wang HY, et al. Lithium and val-proic acid treatments reduce PKC activation and receptor-Gprotein coupling in platelets of bipolar manic patients. JPsychiatr Res. 2005;39:355-363.

32. Einat H, Manji HK. Cellular plasticity cascades: genes-to-behavior pathways in animal models of bipolar disorder.Biol Psychiatry. In press.

33. Kulkarni J, Garland KA, Scaffidi A, et al. A pilot study ofhormone modulation as a new treatment for mania inwomen with bipolar affective disorder.Pscyhoneuroendocrinology. 2006; e-publication.

34. Gould TD, Zarate CA, Manji HK. Glycogen synthasekinase-3: a target for novel bipolar disorder treatments. JClin Psychiatry. 2004;65:10-21.

35. Saal D, Don Y, Bonci A, Malenka RC. Drugs of abuse andstress trigger a common synaptic adaptation in dopamineneurons. Neuron. 2003;37:577-582.

36. Carlezon WA Jr, Nestler EJ. Elevated levels of GluR1 in themidbrain: a trigger for sensitization to drugs of abuse?Trends Neurosci. 2002;25:610-615.

37. Borges K, Dingledine R. AMPA receptors: molecular andfunctional diversity. Prog Brain Res. 1998;116:153-170.

38.Bleakman D, Lodge D. Neuropharmacology of AMPAand kainite receptors. Neuropharmacology.1998;37:1187-1204.

39. Knapp RJ, Goldenberg R, Shuck C, et al. Antidepressantactivity of memory-enhancing drugs in the reduction of submis-sive behavior model. Eur J Pharmacol. 2002;440:27-35.

40. Li X, Tizzano JP, Griffey K, et al. Antidepressant-like actionsof an AMPA receptor potentiator (LY392098).Neuropharmacology. 2001;40:1028-1033.

41. Bai F, Bergeron M, Nelson DL. Chronic AMPA receptorpotentiator (LY451646) treatment increases cell proliferationin adult rat hippocampus. Neuropharmacology.2003;44:1013-1021.

42. Du J, Gray NA, Falke CA, et al. Modulation of synapticplasticity by antimanic agents: the role of AMPA glutamatereceptor subunit 1 synaptic expression. J Neurosci.2004;24:6578-6589.

43. Berman RM, Cappiello A, Anand A, et al. Antidepressanteffects of ketamine in depressed patients. Biol Psychiatry.2000;47:351-354.

44. Zarate CA Jr, Singh JB, Carlson PJ, et al. A randomized trialof an NMDA antagonist in treatment-resistant major depres-sion. Arch Gen Psychiatry. In press.

45. Zarate CA Jr, Quiroz JA, Singh JB, et al. An open-label trialof the glutamate-modulating agent riluzole in combinationwith lithium for the treatment of bipolar depression. BiolPsychiatry. 2005;57:430-432.

46. McElroy SL, Zarate CA, Cookson J, et al. A 52-week, open-label continuation study of lamotrigine in the treatment ofbipolar depression. J Clin Psychiatry. 2004;65:204-210.

Johns Hopkins Advanced Studies in Medicine n S429

PROCEEDINGS

47. Calabrese JR, Bowden CL, Sachs G, et al. A placebo-con-trolled 18-month trial of lamotrigine and lithium maintenancetreatment in recently depressed patients with bipolar I disor-der. J Clin Psychiatry. 2003;64:1013-1024.

48. Calabrese JR, Suppes T, Bowden CL, et al. A double-blind,placebo-controlled, prophylaxis study of lamotrigine inrapid-cycling bipolar disorder. Lamictal 614 Study Group. JClin Psychiatry. 2000;61:841-850.

49. Bowden CL, Calabrese JR, Sachs G, et al. A placebo-con-trolled 18-month trial of lamotrigine and lithium maintenancetreatment in recently manic or hypomanic patients with bipo-lar I disorder. Arch Gen Psychiatry. 2003;60:392-400.

50. Drevets WC, Price JL, Simpson JR Jr, et al. Subgenual pre-frontal cortex abnormalities in mood disorders. Nature.1997;386:824-847.

51. Rajkowska G. Cell pathology in bipolar disorder. BipolarDisord. 2002;4:105-116.

52. Carlson PJ, Singh JB, Zarate CA Jr, et al. Neural circuitryand neuroplasticity in mood disorders: insights for noveltherapeutic targets. Neurotherapeutics. 2006;3:22-41.

53. Nestler EJ, Barrot M, DiLeone RJ, et al. Neurobiology ofdepression. Neuron. 2002;34:13-25.

54. Dickstein DP, Milham MP, Nugent AC, et al. Frontotemporalalterations in pediatric bipolar disorder: results of a voxel-based morphometry study. Arch Gen Psychiatry.2005;62:734-741.

55. MacQueen GM, Campbell S, McEwen BS, et al. Courseof illness, hippocampal function, and hippocampal volumein major depression. Proc Natl Acad Sci U S A.2003;100:1387-1392.

56. Sheline YI, Sanghavi M, Mintun MA, Gado MH.Depression duration but not age predicts hippocampal vol-

ume loss in medically healthy women with recurrent majordepression. J Neurosci. 1999;19:5034-5043.

57. Nugent AC, Milham MP, Bain EE, et al. Cortical abnormal-ities in bipolar disorder investigated with MRI and voxel-based morphometry. Neuroimage. 2005; e-publication.

58. Moore GJ, Bebchuk JM, Wilds IB, et al. Lithium-inducedincreases in human brain gray matter. Lancet.2000;356:1241-1242.

59. David DC, Hoerndli F, Gotz J. Functional genomics meetsneurodegenerative disorders. Part I: transcriptomic and pro-teomic technology. Prog Neurobiol. 2005;76:153-168.

60. Fava M, Rush AJ, Trivedi MH, et al. Background and ratio-nale for the sequenced treatment alternatives to relievedepression (STAR*D) study. Psychiatr Clin North Am.2003;26:457-494.

61. McMahon FJ, Burnevich S, Charney DS, et al. Variation inthe gene encoding the serotonin 2a receptor is associatedwith outcome of antidepressant treatment. Am J Hum Gen.In press.

62. Alimohamad H, Sutton L, Mouyal J, et al. The effects ofantipsychotics on beta-catenin, glycogen synthase kinase-3and dishevelled in the ventral midbrain of rats. JNeurochem. 2005;95:513-525.

63. Kozlovsky N, Amar S, Belmaker RH, Agam G. Psychotropicdrugs affect Ser9-phosphorylated GSK-3beta protein levelsin rodent frontal cortex. Int J Neuropsychopharmacol.2005;28:1-6.

64. Osby U, Brandt L, Correia N, et al. Excess mortality inbipolar and unipolar disorder in Sweden. Arch GenPsychiatry. 2001;58:844-850.