VULNERABILITY OF EXPRESSION REDUCED SERT MICE TO LEARNED HELPLESSNESS

1. VULNERABILITY OF EXPRESSION REDUCED SERT MICE TO LEARNED HELPLESSNESS By Jane Belyavskaya Mentor: Jeff Muller Affiliation: Columbia University (Sackler Institute of Neurobiological Sciences

2. Introduction • Serotonin: - key modulatory transmitter located in the Central Nervous System - Implicated in the regulation of certain developmental, behavioral, and physiological processes • Many receptor types that mediate the effects of serotonin • The activation of these receptors is regulated almost completely by serotonin transporters (SERTs)

3. SERT: protein that acts to clear serotonin from extracellular regions of the brain

4. Present Research and Use in Everyday Life • Behavioral Changes in Serotonin Transporter Deficient Mice suggest that SERTs are necessary in the development of the brain – lack of them causes greater vulnerability to serious depression and anxiety, as well as greater prevalence of risk- taking • Modulation of SERT function can have important effects on neuropsychiatric functioning - SSRI’s = serotonin selective reuptake inhibitors  enhance serontenergic transmissions and decreases depression and anxiet

5. Taking it a step further… • There are different genetic variations of the SERT promoter gene - s-allele:  suggested in the expression of less SERT’s, and therefore more serotonin left in the extracellular regions of the brain  also suggested in the prevalence of depression and anxiety among those who possess this gene (at odds with SSRI’s)  has let do the conclusion that serotonin reuptake is necessary during development as shown but fundamental KO experiment - l-allele: suggested in the expression of less SERT

6. G x E (Interaction Between the Gene And Environment) • Critical to normal development and mature function, including mental faculties and health • Search for causes of mental health problems has recognized the importance of such interaction • A human variation in the serotonin transporter gene has been shown to be associated with increased depression only when accompanied by a history of significant stressful life events, such as job loss, divorce, etc.

7. Relationship With the Midbrain • Amygdala: - Arousal - Controls automatic responses associated with fear - Emotional Responses - Hormonal Secretions • Hippocampus: - Consolidation of New Memories - Emotions - Navigation - Spatial Orientation • Prefrontal Cortex  Responsible for the executive functions which include: - mediating conflicting thoughts - making choices between right and wrong - predicting future events - governing social control

8. People with at least one short SERT gene end up with smaller amygdalae, and even more dramatically, smaller cingulate cortices as well. Therefore there is a prevalent different relationship between the structures.

9. Control Loop For Fear

10. Genotypes In Mice Mice do not have distinguishable promoter regions and therefore no s and l alleles, so complete testing among these alleles is not really possible The next best thing: - WT mice (with two functioning SERT genes) act as the representation of the l-allele - HEZ [heterozygous] mice (with one functioning and one KO SERT gene) act as the s- allele

11. Problem Part 1: Establish a standard for how s-allele humans react in the presence of stress as compared to l-allele patients Part 2: How this aforementioned G x E interaction affects neuronal activity among previously implicated parts of the brain

12. Part 1: In the presence of stress, low expression of serotonin transporters within the brain by a promoter region causes greater susceptibility towards it impacts Part 2: Neuronal firing within the brain is seriously affected by the amount of SERT expression is noted, especially in those areas most affected by stressful events (i.e. the amygdala, prefrontal cortex, and hippocampus) Hypothesis

13. The Stressor : Inescapable Shock/Learned Helplessness • Stressor established within 3 sessions: - each consisting of 70 shocks per mouse - each on average 15 seconds apart - on average 3 seconds in duration - Measure of current = 6 milliamps - No where to escape • Then, moved into a container with a door and shocked again – those who had been properly stressed and were in a condition of learned helplessness had a greater latency of escape • Cause the mice to feel completely powerless to change his or her circumstances for the better. • The result of learned helplessness is often severe depression and extremely low self-esteem.

14. Adult Inescapable Shock Stress Design

15. Materials

16. Behavioral Testing • Novelty Induced Hypophagia • Novelty Suppressed Feeding : showed a “trend” that suggests that there would have been more evident results if the sample size had been larger • Forced Swim Test

17. Mice were placed into Plexiglas activity chambers equipped with infrared beams to detect horizontal activity and vertical activity Activity of mice was recorded for 30 min. Inescapable shock reduced total locomotion of the mice I.S. stress increased the proportion of total locomotion spent in the center region of the open field Open Field

18. Shock Escape Mice place into box w/ two compartments separated by a guillotine At the beginning: subject experience slight shock The amount of time it took the mouse to escape to the other chamber was measured. Real story : expected and warranted results achieved The I.S. HET mice took longer to escape to another chamber than did the I.S. HET Expected significant different between the Controlled WT/HET and the stressed WT/HET

19. Protein Staining to Indicate Active Neurons Within the Brain • All brains were sliced, stained, and mounted on slides in the course of the past few months • The Stain : FOS-B • Using a high-end microscope supplied with a camera, we will be either counting manually or with an automated program on the computer • The amygdala, prefrontal cortex, and hippocampus are the main focus of these ventures

20. Expected Results • Considering that control loop for fear, the expected outcome of presence of immediate early gene of FOS-B • There should be less firing in the prefrontal cortex in HET mice than WT in the presence of stress • On the other hand, there should be more firing in the amygdala of HET mice than WT in the presence of stress since the prefrontal cortex has failed to calm down the “anxiety” within that region

21. Discussion: Why is this important? • This is the first time that the interaction between serotonin transporter expression levels and adult stress can be modeled in mice. • If it is proven that the amygdala in s-allele humans fires more in the presence of stress then in l-allele, then it would lay more groundwork for the study of how the alleles themselves influence the amygdala. • How does less serotonin reuptake cause more neuron firing in the amygdala?

22. Further Research • Learned Helplessness is only one of the many psychological situations found within humans and simulated by Inescapable Shock • Though this is common, other stressors can also be experimented with in order to find out how different psychiatric disorders affect behavior and neuronal activity within the brain, or rather how genotypes influence environmental interaction • Ex. Social Defeat, Prenatal Separation

23. Willeit M, Stastny J, Pirker W, Praschak-Rieder N, Neumeister A, Asenbaum S et al. No evidence for in vivo regulation of midbrain serotonin transporter availability by serotonin transporter promoter gene polymorphism. Biol Psychiatry 2001; 50: 8–12. Caspi A, Moffitt TE. Gene–environment interactions in psychiatry: joining forces with neuroscience. Nat Rev Neurosci 2006; 7: 583–590. Ansorge MS, Zhou M, Lira A, Hen R, Gingrich JA. Early-life blockade of the 5-HT transporter alters emotional behavior in adult mice. Science 2004; 306: 879–881. http://en.wikipedia.org/wiki/Prefrontal_cortex http://biology.about.com/library/organs/brain/blamygdala.htm http://biology.about.com/library/organs/brain/blhippocam.htm Williams RB, Marchuk DA, Gadde KM, Barefoot JC, Grichnik K, Helms MJ et al. Serotonin-related gene polymorphisms and central nervous system serotonin function. Neuropsychopharmacology 2003; 28: 533–541. Citations