ELLS seminar - Monterotondo Oct 2008

Presentazione sul tema: "ELLS seminar - Monterotondo Oct 2008"— Transcript della presentazione:

1 ELLS seminar - Monterotondo Oct 2008
Ansia e Sviluppo I though it could be useful for you to Where and how far scientyists are going in the study of anxiety disorders which would give you also an example of how animal models are ucritical for investigate psychiatric disordr In addition, I’d like to talk about the research we’re pursuing in cornelius lab where I work, investig the importance of postnatal development and the role of serotonin in the generation of ad Analysis of critical mechanisms that during postnatal development can have an impact on adult anxiety Il ruolo della serotonina nella “programmazione” dell’ansia durante lo sviluppo Luisa Lo Iacono ELLS seminar - Monterotondo Oct 2008

2 Ansia Disordini d’Ansia
- L’ansia e’ uno stato mentale che si manifesta in risposta ad una minaccia o ad un potenziale pericolo. Si accompagna a risposte fisiologiche come attivita’ autonomica, tensione muscolare e tremori, stato vigile, sovraeccitazione. L’ansia si distingue dalla paura (risposta cognitiva vs risposta emotiva). L’ansia e’ necessaria ed essenziale per la vita umana Risposta emozionale eccessiva verso una minaccia. Tendenza ad interpretare situazioni ambigue come pericolose. Disordini d’Ansia Ansia innata Ansia condizionata Piu’ di 40 milioni di americani adulti affetti ogni anno (NIMH) Prevalenza del 25% (Kessler et al., 1994) Few definitions Sensation of anx.= normal part of human experience The anxious response to environmental threatening situation help us sharpening our sensitivity and assure us the tension that is necessary to protect us from danger. , but excessive or inappopriate can become a disease and severely interfere with normal life Innate: need of no previous experience to occur Conditioned: result of experiencing certain aversive events in the past Life prevalence of 25%

3 Disordini d’ansia In the 4th edition of the diagnostic and statistical manual of menthal disorders, classified in 6 dis In its pathological form, anx. Has been classified into six disorders: gen…..each character. By specific symptoms and that togheter affect over 20% of the population at sime point in life. Most common treatments are the BDZ which are now been replaced as first line treatm. By the inhibitor of reuptake. Etiology of anxiety is still unclear but there are evidence of an involvement of the serotonergic system The first… Diagnostic and Statistical Manual of the American Psychiatric Association, Vol.4

4 Risposta ansiosa MINACCIA SOCIALE FISICA Inibizione Eccitazione
Circuiti ansia Sistemi sensoriali Comandi Motori e autonomici Elaborazione segnale Inibizione comportamentale Avoidance Risk assessment Freezing Eccitazione Stato vigile Attenzione Tachicardia MINACCIA SOCIALE FISICA As scientists like to do, in order to study complex phenomenom, We have to synthetize: in a scheme the anxiety response How is it produced? How does it work When AD gthere must be an abnormalities

5 Risposta ansiosa Dove sono I circuiti dell’ansia nel cervello?
MINACCIA SOCIALE FISICA Sistema sensoriale CIRCUITI DELL’ANSIA Elaborazione del segnale When A response becomes excessive and pathological, we assume an unappropriate functioning of anx circuits is present Questions for scientists are: If we had all answer we would notg bne hedre Dove sono I circuiti dell’ansia nel cervello? Quali anormalita’ caratterizzano la patologia? Perche’ e quando si generano?

6 1) DOVE? Neuroanatomia dell’ansia Corteccia Talamo Midbrain Amygdala
Anxiety is a complex emotion, resulting from the interaction of between many interconnected brain regions. The circuitry underlying was identified with the one underlying the fear response, and thwe main structure is the amygdala, a small almond-shaped structure in the temporal lobe. It functions as an interface, afferents fromn thal and cortex, outputs to varius networks that control the aggressive or defensive reactions. Among connections, hippocampus, a structure required for memory and learning. In 1982 the first Gray proposal of hippocapmus as struct for anxiety, and amygdala for fear; neural substrate as two separate entities. Vfear is an immediate emotional response, whereas anx requires a cognitive elaboration Supported by further studies with lesions of hippocampus and no anxiety Amygdala Ipotalamo Ippocampo Tronco encefalico Cervelletto

7 Risposta ansiosa Dove sono I circuiti dell’ansia nel cervello?
MINACCIA SOCIALE FISICA Sistema sensoriale CIRCUITI DELL’ANSIA Elaborazione del segnale When A response becomes excessive and pathological, we assume an unappropriate functioning of anx circuits is present Questions for scientists are: If we had all answer we would notg bne hedre Dove sono I circuiti dell’ansia nel cervello? Quali anormalita’ caratterizzano la patologia? Perche’ e quando si genera?

8 Eziologia dell’ansia GENI X AMBIENTE Fattori ambientali?
Si’, accesa dalle esperienze di vita, es PTSD MA I tratti ansiosi si osservano gia’ nell’infanzia Alta cronicita’ della patologia Ansia del bambino Fattori genetici? Si’, natura familiare dell’ansia Da studi su gemelli: % fattore di rischio MA The answer should be ina combination of the two.were genes would give a susceptibility and environment triggers In this context, I would like to give you a couple of example on studies which demnostrate how critical for the development of anxiety is the early life and the envirnoment experienced GENI X AMBIENTE

9 Le cure materne regolano lo sviluppo dei circuiti neurali che guidano la risposta ansiosa
Open Field Demonstrated a correlation bet level of maternal care received and level of adult anxiety in rats. In particular.. Caldji et al., 1998

10 Influenza di stress infantili sulla depressione: moderazione a carico di una mutazione genetica
5HTT: trasportatore serotonina s/s: forma corta s/l: forma media l/l: forma lunga Study on the interaction between environmental factors in childhood and genetic factora Result of a regression analyssi of a group of birth cohort in New Zwaland, 5estimating the association between cvhildhood maltreatm (3-11 years) and adult depression (after 18) They first cluster people in 3 groups genetically, then within each they divide in 3 based on infancy. Susceptibility of first group, decreaseing These two example show that early environm is critical and support the idea of the dev programming of anxiety Caspi et al., Science 2003 The developmental programming of anxiety Meccanismi biologici durante lo sviluppo sotto il controllo di fattori genetici ed ambientali possono “programmare” la predisposizione di un individuo a manifestare disordini d’ansia, durante tutta la vita

11 Risposta ansiosa Dove sono I circuiti dell’ansia nel cervello?
MINACCIA SOCIALE FISICA Sistema sensoriale CIRCUITI DELL’ANSIA Elaborazione del segnale When A response becomes excessive and pathological, we assume an unappropriate functioning of anx circuits is present Questions for scientists are: If we had all answer we would notg bne hedre Dove sono I circuiti dell’ansia nel cervello? Quali anormalita’ caratterizzano la patologia? Perche’ e quando si generano?

12 Farmacologia dell’ansia
Serotonin Specific Re-uptake Inhibitors (SSRIs): first line treatment for anxiety Genetic polymorphism in serotonin Transporter (5-HTT) gene linked to increased in anxiety From the study of their mech of actionwe know that systems involved are Serotonergico Gabaergico Circuiti neurali coinvolti

13 Il sistema serotonergico
SS is composed by The neurons that produce serotonin. They are located in a restricted zone of the brainstem, mainly in the raphe nuclei. The total number of ser neurons is small, around 20000, compared to the total n.o of neurons in the brain, but thanks to an extensive collateralization of thei axons they provide a dense innervation to all the brain areas, Simplistically the provide a general inhibitionof excitability of structures were they project

14 La sinapsi serotonergica
SSRI Buspirone I farmaci ansiolitici potenziano il segnale serotonergico Andiamo a vedere piu’ da vicino un neurone serotonergico e cerchiamo di capire come agiscono I farmaci ansiolitici. Sembra che un effetto di potyenziamento del recettore 1A sia favorevole. Sara’ coinvolto questo recettore? 5HT1A

15 Serotonin 1AR-KO: il topo “ansioso”
I’d like to tell you now the story of our mouse model, the serotonin receptor 1A KO, where the rec 1A has been genetically deleted. This is a model that helps us to study what causes anxiety and what are mechanisms involved to answer to previous questions Possono difetti nei circuiti serotonergici essere coinvolti nell’ansia?

16 OPEN FIELD Misure: Tempo passato nel centro
Durata: 60 min Sistema Video tracking Tempo passato nel centro % Distanza percorsa nel centro

17 ELEVATED-PLUS MAZE Misure: N. di ingressi nelle braccia aperte
Durata: 6 min Sistema Video tracking Misure: N. di ingressi nelle braccia aperte Tempo passato nelle braccia aperte % Distanza percorsa nelle braccia aperte

18 Il comportamento del 5-HT1A KO mouse
Open Field 10 20 30 40 50 60 % entries in open arms * WT KO Elevated-Plus Maze 1 2 1 8 6 % path in the center * * * 4 2 W T K O

19 Quando si sviluppa il fenotipo d’ansia?
Tecnica: Mutazione genetica condizionale con Doxicicline-repressible transactivation system Doxicicline = no receptor P0 P21 BIRTH MATURITY P60 Normal anxiety Increased anxiety P0 P21 Anxiety Behavioral Test ADULT BIRTH MATURITY P60 The animal is genetically mutated so to be sensitive to the treatment with a drug, called doxiciclibne. This drug is able to turn off the receptor express I’m showing you two experiments that led to important results: this is a time line representing the life of our mice. It was found that by treating with dox during early life, that means that the animal does not express the receptor durinf dev but itn does in the adult The experiment we performed further in this lab, was to rty to narrow down this window and understand which time-window is actually critical. This is important to understand what kind of developmental mechanisms are involved since in postnatal development, the nervos system is still developing following a sort of time line.

20 Trattamento cronico con osmotic mini pumps
Quando si sviluppa il fenotipo d’ansia? Tecnica: Blocco farmacologico del 5-HT1Acon l’antagonista WAY100635 Trattamento cronico con osmotic mini pumps WAY = no receptor activity BIRTH MATURITY ADULT Anxiety? P0 P21 P60 Increased anxiety P0 BIRTH MATURITY P60 P35 P14 * 100 200 300 400 500 600 Time in center We could not used the transgenic technique, which does not allow a rapid switc, so we we did was not to block the genetic expressionn of the receptor, but to block its activity by an antagonist drug. The idea was to block duting different time windows and test the behavioral consequence of these blockade to understand which period was critical for the anxiety As result we demonstrated that blocking the r from P14 to 35 was sufficient to induce anx for all the life. I’d like know to draw your attenction on the important consequence of this: meaning that a chronic treatment with a drug having effects on the serotonergic system during childhood could alter permanetly the behavior of an individual. It’s difficult to translate the age of the animal to the humans, but the corrispondent time in term on neuronal dev should be an early postnatal time. Anxiety Behavioral Test

21 5-HT1AR signaling Sinapsi Apprendimento, memoria, plasticita’ nervosa
neuronal bouton 5-HT1AR puo’ ridurre l’attivita’ dell’enzima CamKII Cai et al., J.Biol.Chem., 2002 Sinapsi Serotonina 5-HT1AR ai bg P CaMKII What happens in a 1AKO that results in anxiety? What signals in the brain are altered? It was shown in a cell culture study that 1A reduces the act of CamKII amKII is a special enzyme composed of 12 similar subunits linked together to form a big proitein which represnet the most abundant at the level of synapses \second it has the ability to modify its activity in response to the environmental inputs, or called sensory experience, translated at molecular level in neuronal stimuli, which reguilate its level of P The level of P could be compared to a battery charge, the more it is the longer its activity lasts; CActivity of CamKII can be measured by the level of P groups linked at the surface of each subunit. P Espressione genica P sinaptogenesi Potenza sinaptica Sviluppo dendritico Apprendimento, memoria, plasticita’ nervosa

22 Levels of P-CaMKII in hippocampus of 5-HT1A KO mice
WT KO Tecnica: western blot detection con anticorpo contro Phospho-CaMKII P WT KO WT KO % p-CaMKII No Stress Stress ** 50 100 150 200 % p-CaMKII 50 100 150 200 WT KO Stress Camkii is alteredd during the critical wind but not in the adult, which support further its role in the developmen of anxiety BIRTH MATURITY ADULT P0 P14 P35 P60

23 Sinapsi in 5-HT1A KO mouse
Il topo 5-HT1AR KO ha un’aumentata attivita’ di CaMKII neuronal bouton Sinapsi in 5-HT1A KO mouse Serotonina 5-HT1AR ai bg P CaMKII Now we can reflect on this phenotype, since CaMKMII is a very special enzyme for neuroscientists. Its peculiarity is in the fact first that it is extremely abundant compared to others as probably you’ve heard yesterday from Liliana’s talk it’s required for learning and memory Also it has been shown to be important at early life for the development of the synapses, and dendrites in the growing neurons. So if itb is alteredhyper functioning in KO we can hypothesize an overgrowth of dendrites Gene expression Synaptogenesis Synaptic strength Dendritic development Apprendimento, memoria, plasticita’ nervosa

24 Dendriti prossimali apicali
Aumentata arborizzazione dendritica nell’ippocampo di topi 5-HT1A KO Dendriti prossimali apicali analysis of the morphology of 1aKO CA1 neurons, where… So our hypothesis which we are currently trying to demonstrate is that a defects in the mecanisms of dendritic developmemnt in the hippocampus during a restricted time in postnatal life is able to predispose an individual to exhibit anxiety in the adulthood J.P. Horning, University of Lausanne unpublished data Un difetto nei meccanismi di sviluppo dendritico nell’ippocampo nei primi anni di vita puo’ predisporre un individuo a manifestare ansia in eta’ adulta La nostra ipotesi:

25 5-HT1AR e lo sviluppo post-natale del cervello
CONCEPTION BIRTH MATURITY ADULT BEHAVIOR Nervous system development Sensory experience Migrazione Estensione assoni Proliferazione Differenziamento Primi contatti sinaptici Maturita’ Crescita dendritica Sinaptogenesi Maturazione e rifinitura Plasticita’ “experience-dependent” Before concluding my talk, just few consideration on the postnatal time. So our attenction moved into the post-natal period of development of thy SNC, which is a critical period, very complex but fascinating at same time. While in the early stage of dev brain circuits are formed following genetic programming of neuronal prolif., migr to teir final dest, differantiations and extensions of axons and first synaptic contacts Indeed, after birth the final shaping of neural circuits occurs, guided by the interaction between the genetic programming and the environmental factors. It is characterized therefore by an intense synaptogenesis and growth of dendrites which allow the refinement and maturation of the circuits. Environm inputs in the brain, driven after birth by sensory experience are translated into neuronal activity, that drives anatomical changes such as synaptic remodeling or dendritic refinements and shape the final connections that subserve the adult brain function. This is defined as experience-dependent plasticity, endows the brain with an ongoing ability to accommodate to dinamically changing inputs during developm and is essential for the survival of the individuals in a constantly changing environment. CaMKII is involved in this plasticity, sinbce it responds to the enviornment. This form of plasticity decrease througout life, having critical time period in which it is highly presnt which differe between structures. I’ll give you the example of the visual cortx We do think such critical p of pl exists also in anxiety circuits, meaningg that the environment during childhood is much more critical that somebody could expect Kid in a war or kid in a nice family. We believe that the 1A has a role during post-natal dev, which is mediated by the CamKII in mediating neuronal changes in response to the environment, and contributing to the final maturation of circuits and the formation of adult behavior..

26 EMBL-Phenotyping Facility
Thank you! Cornelius Gross Enrica Audero Valeria Carola Tiago Ferreira Thodoris Tsetsenis Olga Ermakova Giovanni Frazzetto Olivier Mirabeau Apar Jain Raffaella Bosurgi Tiziana Rossetti Rosa Paolicelli EMBL-Animal Facility Francesca Zonfrillo Antonella Vecchio EMBL-Phenotyping Facility Mumna Al Banchaabouchi Dominika Farley