Animal Models in Bipolar Animal Models in Bipolar DisorderDisorderBy Jakob Avi ShimshoniBy Jakob Avi Shimshoni

I.I. IntroductionIntroduction

Bipolar Disorder (BD) is common, affecting Bipolar Disorder (BD) is common, affecting approximately 4 million persons in the USA.approximately 4 million persons in the USA.

BD is characterized by unpredictable swings in BD is characterized by unpredictable swings in mood from mania to depressionmood from mania to depression

BD can be divided into bipolar manic-depression BD can be divided into bipolar manic-depression and unipolar disorderand unipolar disorder

Clinical FeaturesClinical Features

Depressed, miserableElevated,labileMood

Retardation or agitation, poverty of movements and expression

Disinhibition, hypersexuality, excessive spending

Behavior

Fatigue. Loss of libidoInsomnia, weight lossPhysical

Guilt, unworthinessGrandiose, self confidentIdeation

Lacking, apatheticExcessive, increased psychomotor activity, distractable

Energy

Slow, monotonousFast, flight of ideasTalk

DepressionMania

Manic episodes emerge graduallyManic episodes emerge gradually

They can last as long as several months to years They can last as long as several months to years when untreatedwhen untreated

Frequent age of onset: 20-30 yearsFrequent age of onset: 20-30 years

I.I. PathophysiologyPathophysiology

Alterations in Brain StructureAlterations in Brain Structure

- - Ventricular enlargementVentricular enlargement

- Glial density reduction in - Glial density reduction in prefrontal cortexprefrontal cortex

- MRI of BD patients reveal - MRI of BD patients reveal vascular diseasesvascular diseases

Alteration in Brain functionAlteration in Brain function

- - Lower cortical metabolism in depressed BDLower cortical metabolism in depressed BD

- Decreased metabolism and blood flow in prefrontal - Decreased metabolism and blood flow in prefrontal cortex in depressed BD cortex in depressed BD

Alterations in Signal TransductionAlterations in Signal Transduction

cAMP/PK-A systemcAMP/PK-A system

- - increased Adenylyl cyclase activityincreased Adenylyl cyclase activity

- higher Gs levels- higher Gs levels - increased PK-A activity- increased PK-A activity

PIP2-PathwayPIP2-Pathway

- - increased platelet PIP2 concentration increased platelet PIP2 concentration

- increased PK-C membrane/cytosol partitioning- increased PK-C membrane/cytosol partitioning - increased PK-C activity- increased PK-C activity - Li decreases inositol levels in brain tissue- Li decreases inositol levels in brain tissue

Abnormalities of Ca 2+ signaling - Higher Ca2+ levels in platelets, neutrophils and

lymphocytes

- Increased PK-C activity may be responsible for abnormal Ca2+ levels

Na +/K +-ATPase hypothesis - Reduced Na+/K+-ATPase activity

- Lower Na+/K+-ATPase protein α2 subunit

- Intraventricular Quabain administration to animals, increases motor activity

NeurotransmittersNeurotransmitters

The Dopamine-HypothesisThe Dopamine-HypothesisIs based on the following

findings: Dysfunctional neural circuitry

of reward and incentive motivation

Decreased CSF Homovanilic acid in depressed compared to

manic patients Antidepressants enhance the

activity of the mesolimbic DA-system

Acute administration of psychostimulants like amphetamine

produce effects similar to mania Acute mania is treated with

neuroleptic drugs

Norepinephrine-SystemNorepinephrine-System

Increased NE turnover Increased NE turnover Higher urinary MHPG (methoxy-4-hydroxyphenylglycol) Higher urinary MHPG (methoxy-4-hydroxyphenylglycol)

in mania as compared to depressionin mania as compared to depression

5-HT-System5-HT-System

Decreased 5-Hydroxyindolacetic acid levels in frontal and parietal cortex

Reduced 5-HT transportes in frontal cortex

Hypothalamic-Pituitary-Adrenocortical AxisHypothalamic-Pituitary-Adrenocortical Axis

Increased HPA activity in depressed and mixed BD patientsIncreased HPA activity in depressed and mixed BD patients

Chronic hypercortisolemia is toxic to hippocampal neuronsChronic hypercortisolemia is toxic to hippocampal neurons

The Circadian RhythmThe Circadian Rhythm

Sleep-wake cycle is determined by external and internal rhythms

The internal rhythm is coordinated by a master clock in the supra-chiasmatic nucleus

Two transcriptional factors determine its function:

CLOCK and BMAL1

In BD patients subtle disturbances in circadian rhythms precede the full-scale shift in mood state

I.I. PharmacotherapyPharmacotherapy

Newer Antiepileptic drugs in BDNewer Antiepileptic drugs in BD LamotrigineLamotrigine :: Dosage: 100-300mg/day Lamotrigine shows significant benefit as monotherapy

or as adjunctive agent in bipolar depression as well as rapid cycling. Efficacy in acute mania is unclear.

Gabapentin Dosage: 900-3600mg/day Gabapentin is often prescribed for patients with BD as

adjunctive, in spite of negative results in clinical trials. Topiramate Dosage: 200-600mg/day Open-label studies suggest improvement in the manic

phase of BD patients.

Mechanism of action of mood stabilizersMechanism of action of mood stabilizers

LiLi ++:: LiLi++ reduces cAMP fluctuations, by G-protein inhibition reduces cAMP fluctuations, by G-protein inhibition Attenuation of Receptor coupled PIP2-signalling, by Attenuation of Receptor coupled PIP2-signalling, by

inhibition of IMP (Ki=0.8mM)inhibition of IMP (Ki=0.8mM) Down-regulation of PK-C isozymes and reduction in PK-C Down-regulation of PK-C isozymes and reduction in PK-C

bound to plasma-membranebound to plasma-membrane NeuroprotectionNeuroprotection Increase growth cone area of neuronsIncrease growth cone area of neurons Decreased adrenergic and dopaminergic super sensitivityDecreased adrenergic and dopaminergic super sensitivity

CBZCBZ Increase growth cone areaIncrease growth cone area Reduces basal levels of cAMP as well as levels after Reduces basal levels of cAMP as well as levels after

Adenylyl-cyclase activationAdenylyl-cyclase activation Stabilization of NaStabilization of Na++-channels-channels

VPAVPA Decrease inositol biosynthesis, by MIP-synthase Decrease inositol biosynthesis, by MIP-synthase

inhibitioninhibition Increase growth cone areaIncrease growth cone area NeuroprotectionNeuroprotection PK-C isozymes down regulation and reduced PK-C PK-C isozymes down regulation and reduced PK-C

bonding to plasma membranebonding to plasma membrane Block of voltage-dependent NaBlock of voltage-dependent Na++-channels-channels

Mechanism of Inositol Biosynthesis

NeuroprotectionNeuroprotection

Increased Bcl-2 and BDNF

Reduction in p53 proapoptotic protein

Inhibition of GSK-3β results in an increased gene transcription

Rational for the use of AED’s in BDRational for the use of AED’s in BD Gradual progression in intensity and frequency of Gradual progression in intensity and frequency of

epileptic seizuresepileptic seizures and manic-depressive episodes.and manic-depressive episodes.

Manic-depressive episodes are neither random nor Manic-depressive episodes are neither random nor predictable; epileptic seizures occurs randomly.predictable; epileptic seizures occurs randomly.

Kindling –HypothesisKindling –Hypothesis: Phenomenon whereby repeated : Phenomenon whereby repeated administration of an initially sub-convulsive electrical administration of an initially sub-convulsive electrical stimulus results in progressive intensification of seizure stimulus results in progressive intensification of seizure activity, culminating in spontaneous seizures.activity, culminating in spontaneous seizures.

Both disorders respond to several identical drugs.Both disorders respond to several identical drugs.

I.I. Animal ModelsAnimal Models IntroductionIntroduction

Animal models in psychiatry are controversial, due to the Animal models in psychiatry are controversial, due to the difficulties of trying to mimic higher cognitive functioning difficulties of trying to mimic higher cognitive functioning in animalsin animals

We classify behavioral changes in:We classify behavioral changes in: - Subjective changes: fear, euphoria, dysphoria, - Subjective changes: fear, euphoria, dysphoria,

depressed mooddepressed mood

- Objective changes: locomotor activity, circadian rhythm- Objective changes: locomotor activity, circadian rhythm

Validity of Animal Models

Face-Validity: Refers to similarity in behavior between the model and

the disorder Construct-Validity: Evaluates the correlation of the model with molecular

changes described in illness pathophysiology Predictive-Validity: Evaluates the responsiveness of the model to the drugs

used to treat the illness modeled

Animal Models of ManiaAnimal Models of Mania

There are 4 major features of mania that could be There are 4 major features of mania that could be modeled:modeled:

HyperactivityHyperactivity ElationElation IrritabilityIrritability InsomniaInsomnia

A viable model will have to demonstrate positive effects A viable model will have to demonstrate positive effects of Liof Li++ and no effect of drugs ineffective in mania and no effect of drugs ineffective in mania

Operational approach for evaluating elation:Operational approach for evaluating elation:Intracranial self stimulationIntracranial self stimulation

Sleep Deprivation Model

The rat is kept on a small platform surrounded by water for 72h

At the end of the period, it is returns to its home cage and doesn’t readily fall asleep

Symptoms presented during this time: insomnia, hyperactivity, aggressiveness, hypersexuality

Benefits: effective, economic, no invasive manipulation

Drug-screening-test In this test the latency to sleep and motor activity are measured.Example:

Acute Psychomotorstimulant Model

Hyperactivity is induced by a mixture of amphetamine (1.2mg/kg) and chlordiazepoxide (12.5mg/kg) injected s.c. 35min before the test.

Activity was assessed by recording the number of entries into the arms of a maze during a 5min period.

Brain lesion modelBrain lesion model Lesions in hippocampus and amygdala during the first

weeks after birth induce increased locomotor activity, decreased investigatory behavior and decreased seizure threshold

Induction: Injection of cholera toxin

Social Isolation Isolation of rats in the neonatal period produces

syndrome in which hyperactivity is a major component Additional changes: increased aggressiveness,

stereotypy

Animal Models of DepressionAnimal Models of Depression

IntroductionIntroduction

The largest animal models of depression is based on the The largest animal models of depression is based on the application of stressapplication of stress

Stressors used: foot shock, restraint, water immersionStressors used: foot shock, restraint, water immersion

Depressed behavior is characterized by: reduced Depressed behavior is characterized by: reduced locomotor activity and reward behaviorlocomotor activity and reward behavior

Basic requirements for validated animal modelBasic requirements for validated animal model Predictive validityPredictive validity Responsiveness to Tricyclics and MAO-A inhibitors

Face validity The model must demonstrate resemblance to clinically

defined core symptoms of the disorder Core symptoms are: loss of pleasure (anhedonia) depressed mood Construct validity limited evaluation because of lack of basic knowledge

Chronic mild stress model (CMS)

Rats are exposed sequentially to a variety of mild stressors over a period of weeks

This procedure causes a decrease in sensitivity to reward

CMS induced behavioral deficits may be maintained for several months

Normal behavior is restored by chronic treatment with Tricyclics during continual CMS

CMS-Procedure: CMS-Procedure:

Results:Results:

Forced Swim testForced Swim test

Developed by Porsolt in 1978 as a Developed by Porsolt in 1978 as a screening test for antidepressants screening test for antidepressants in rodentsin rodents

The rats are placed in a water tank The rats are placed in a water tank that doesn’t permit escapethat doesn’t permit escape

After a short struggle time they After a short struggle time they display an immobile posture display an immobile posture reflecting a state of behavioral reflecting a state of behavioral despair (helplessness) despair (helplessness)

Effect of reboxetine treatment on immobility and Effect of reboxetine treatment on immobility and defecation defecation

Reserpine ModelReserpine Model

Pharmacologically induced model, which is most frequently used for drug screening

Reserpine causes depletion of catecholamines

In humans it causes depression, sedation, parkinsonism and in animals hypoactivity

Animals are treated with the test drug prior to Reserpine injection(0.25mg/kg)

Rats are tested for activity levels and immobility time

Genetic model: Flinders sensitive line of rats (FSL)

Selective breed for increased sensitivity of muscarinic agents

FSL rats show reduced locomotor activity, reduced weight, increased REM sleep and anhedonia

High responsiveness to Tricyclics, SSRI’s, but not to psychostimmulants

Good face, construct and predictive validity

I. Conclusions

No validated model incorporate both components of bipolar disorder

The animal models for mania and depression are used currently in research as well as in the industry for drug screening

In the last few years a new in-vitro model is developed in neural cells, as a research tool as well as a screening test

I. In-Vitro Screening Approach for new ant-bipolar drugs

Dictyostelium Discoideum Model Dictyostelium D. is a soil living amoeba It is used to measure the effects of drugs on inositol

levels, using a direct IP3-binding protein assay

Dorsal root ganglion Are used to measure the effect of drugs on the growth

cone area. This effect is mediated through inositol depletion.

VPA-induced teratogenicity

• Congenital malformations – 11.1%• Neural tube defects – 2%

Structural requirements to teratogenic molecule:

1. Presence of α-hydrogen2. Presence of carboxylic function3. Branching on α-carbon with two side chains containing three carbon atoms each CH3CH2CH2

CHCH3CH2CH2

COOH

I. Promising VPA-Derivatives as Anti-BD drugs

VPA-Derivatives depleting IP3 more strongly than Li+

Not teratogenic(>3mM)

76.5%

Not teratogenic (>3mM)

78%

At 2-3mM25-60%

62%

At 0.25-1mM40-60%

88%

Teratogenicity%IP3 reductionat 0.5mM

Drug

Maximal Electroshock Seizures (MES)

(60Hz, 50mA, 0.2s)

Animal Models of Epilepsy

sc Metrazole (scMet)

Kindled rat model

Identifies drugs effective against tonic\clonic seizures seizure spread inhibition

Identifies drugs effective against absence seizures increase in seizure threshold

Identifies drugs effective against partial and seconarily generalized

seizures

Chemical Structure of Chemical Structure of Cyclic Analogs of VPA

CONH2

CONHCH3

CONHCONH2

CONHOCH3

M-TMCD

TMCU

Methoxy-TMCD

CONH

N N

S

SO2NH2

TMC-thiadiazole

TMCD

Antiepileptic activity and toxicity of cyclic analogs of VPA

333mg: 6.5% resorption 0% alformation

538171NT9229TMC-urea

660mg: 54% resorption 34%malformation330mg: 27% malformation

>500NTNT>5009TMC-Thiadiazole(ip)

300mg: 8% resorption 1% malformation

38185>100;C52>250TMCD

300mg: 15% resorption 1% alformation

1634140;h4582MTMCD

NT333150NT35108Methoxy-TMCD

600mg:8% resorption 41% alformation

78426980<646485VPA

TeratogenicityTD50mgkg

Chungmg/kg

Kindlingmg/kg

scMETmg/kg

MES mg/kg

Drug

NT>500Inactive<160NT>25082TMC-glycinamide

NT>500NTNT>25041N-Acetyl-tmc-urea

TeratogenicityTD50-Chungmg/kg

Kindlingmg/kg

scMETmg/kg

MES mg/kg

Drug

CONHCONHOCCH3

N-Acetyl-tmc-urea

CONHCH2CONH2

TMC-glycinamide

Structure of VPA-Derivatives

CONH2

COOH

CONHCH3

CONH2

COOHCONH2

CONHCH2CONH2

VPD

M-VPD

VGD

VCA VCD

DIADID

CONHCONH2

Isovaleroylurea

COOH

VPA

CONHCH2COOH

VGA

Antiepileptic activity and Toxicity of VPA-Derivatives

600mg: 10.4% resorbtion 0% malformation

63NTNT3716R-PID

NT500NTNT83>250Isovaleroylurea

600mg: 6.3%resorbtion 0%malformation

68NTNT3731PID (racemic)

not teratogenic87NTNT5932Valpromide

not teratogenic>1000226161;C300;h

>75073Valrocemide (VGD)

TeratogenicityTD50-Chungmg/kg

Kindlingmg/kg

scMETmg/kg

MES mg/kg

Drug

Results of antiepileptic activity and toxicity of drugs given ip to mice

188225200TMCA

279240269Valnoctic acid

813258Valnoctamide

633731PID

574187DID

303156238DIA

TD50scMET mg/kg

MES mg/kg

Drug

Thank you very much for your attentionThank you very much for your attention , , hopefully I didn’t induce a manic episode upon hopefully I didn’t induce a manic episode upon you !you !