After months of deliberation, Dana staff and guest judge Eric Chudler have selected the winner of the Dana Foundation’s Design a Brain Experiment Competition. The challenge asked United States high school students to design an original brain-related experiment. Students did not complete their experiments. Instead, the competition encouraged students to use their knowledge of the brain to come up with creative ideas and hypotheses.The winning experiment, designed by Michaela Ennis, a senior at the Pingry School in New Jersey, proposes an examination of the effects of social defeat on anxious behavior, pinpointing the molecular mechanisms for that behavior. Ennis will be attending MIT next year, has participated in summer research and scholar programs at Rutgers and Rockefeller University, and according to her teacher, Deirdre O’Mara, is a superstar in science.
To examine the effect of social defeat on anxious behavior in both the short- and long-term, and then to pinpoint the molecular mechanisms for this behavior.
The human brain is by far the most poorly understood organ, and the next generation of science will be defined by discoveries of how the brain creates the psychological mind. By determining the underlying molecular mechanisms of different psychological conditions, not only may the condition be treated, but the mechanism for normal function of the brain may also be elucidated. Chronic anxiety is an issue common to many neurological conditions, and social anxiety can be seen in numerous disorders, including autism. A mouse model of chronic social defeat has been used to replicate bullying and induce chronic anxiety. It has been shown that different strains of lab mice will react with varying severities of anxiety following SD, pointing to a genetic component. By determining the mechanism of the anxiety created by social defeat, a target can likely be found for many different types of anxiety that effect a wide range of people .
In one study, mice were socially defeated by larger mice for 10 days, and then presented with a social situation involving a mouse their own size. The social defeat (SD) caused the mice to display anxious behavior including increased fear and decreased inquisitiveness. Potential physiological causes of this were mediated by vasopressin and oxytocin. Levels of activated vasopressinergic (AVP) neurons were increased in socially defeated mice, and levels of one type of vasopressin receptor (V1BR) and the oxytocin receptor (OTR) were increased in the medial amygdala following social defeat. Blocking of the V1BR partially attenuated some of the anxious behaviors, as well as lowered levels of firing AVP neurons in socially defeated mice. However, in normal mice, blocking V1BR actually slightly increased anxious behavior .
Other studies have shown V1BR and OTR playing a pro-social role in normal mice, and increase in these receptors’ levels can increase sociality in non-SD mice. This evidence suggests that the increase in receptor levels due to SD may be sensitizing the system rather than the actual cause of anxiety .
Another gene that may play a role is brain-derived neurotrophic factor (BDNF), which was found to be increased in the medial amygdala both 2 hours and 28 days after social defeat (although this study did not test behavior after SD, the fact that BDNF levels remain elevated suggests that the effects of SD can be long term. This was then confirmed by other studies, which showed depression-like behaviors and anxiety lasting up to 7 weeks after SD). BDNF helps to maintain existing neurons, create synapses, and differentiate new neurons. BDNF is incredibly important to long term memory, and this suggests that changes in BDNF levels after SD help to create other neurological changes [4, 5].
Epigenetic changes have recently been shown to be incredibly important in learning and memory. Changes in DNA methylation patterns or histone acetylation can severely alter learning capabilities. Evidence suggests that traumatic experiences can create unique epigenetic changes. For example, abused children were found to have unique methylation of the GR gene. Methylation of this gene can also be found in rat pups that were given poor quality care by their mothers. Because epigenetic changes have been shown to be strongly implicated in brain function, unique epigenetic changes due to SD are likely. One study found an increase in histone H3 acetylation 30 minutes after SD in rats, providing more evidence for this theory. However, much more research must be done, specifically in what genes these potential epigenetic changes may be targeting [5–7].
Forced firing of vasopressinergic neurons will produce pro-social effects in normal mice, but will produce the same anxious effects as social interaction does in the SD mice. Examination of epigenetic changes in the medial amygdala and paraventricular nucleus of SD mice as compared to normal control mice will uncover the genes involved with social anxiety. Targeting these genes through knockdown or over-expression mouse models will greatly affect the degree of anxiety and depression displayed after SD. Changes in expression levels of these genes may also be seen in other models of anxiety.
Chronic social defeat can be created by placing young (8-10 weeks) mice into the housing of older (14-16 weeks) mice. The younger intruder mice will be consistently attacked by the older mice. This continues for 10 days. The mice are then allowed one day to rest, and the following day the first round of mice are tested. Another set of the mice will be kept for a period of a few months before testing in order to examine long-term changes after SD [2, 8].
Anxious behavior can be tested through frequency of freezing, risk assessment position, and grooming. An increase in the frequency of all three of these behaviors is associated with anxiety. Depression-like behavior can be examined using a forced swim test and sucrose preference test. Depressed mice spend less time attempting to swim in the forced swim test, and have a decreased preference for sweetness [2, 5].
Optogenetics has been used to control the behavior of mouse models in the past, and can be used to target specific neuron types. Transgenic mice expressing channelrhodopsin-2 under cell-type specific promoters have been used to target neurons expressing specific neurotransmitters in the past. Recently, optogenetics was used to remove the potentiation created by cocaine use, which essentially abolished symptoms of addiction. Through this method, vasopressinergic neurons can be forced to fire, even when the mice are not presented with a social situation [9, 10].
To examine DNA methylation patterns in the paraventricular nucleus and medial amygdala, these regions of the brain can be removed, and bisulfite sequencing can be performed on the isolated DNA. To determine levels of acetylation, qPCR can be done to look at expression levels of a number of histone acetyltransferases (HAT) and histone deacetylases (HDAC) [5–7].
If vasopressinergic neurons are the beginning of the cascade in the social response, then forced firing of vasopressinergic neurons would produce pro-social behavior in normal mice and anxious behavior in SD mice (with no social stimulus; despite this, pro-social and anxious behavior can still be identified in behavioral tests through locomotor activity). This is due to the as yet unknown change downstream of vasopressin in this response. If firing of vasopressinergic neurons produces no real behavioral response, then it is likely that these neurons are not sufficient for activation of a social response. This means that the potential change between SD and normal mice may not be downstream of vasopressinergic neurons in this response, but rather part of the activation of the response. Although vasopressin is still necessary for a social response in this scenario, it would not be sufficient. If firing of these neurons produces anxiety in both the normal and SD mice, then prior evidence was misleading and vasopressin is the main component in the anxious response after SD [2,3,9].
If changes in DNA methylation are detected, this will allow the targeting of specific genes in which expression levels are changed after SD. First, the genes revealed to be implicated in SD should be examined in the context of other potential functions. Then, the genes can be knocked down (if found to be over-expressed in SD mice) or over expressed (if found to be under-expressed in SD mice) in a mouse model to determine if targeting these genes is a potential treatment strategy for anxiety related to SD. Finally, expression levels of these genes can be studied in other models of anxiety. If levels of HATs or HDACs are found to be significantly different before and after SD, this will not reveal which genes are being regulated, and further research will have to be done. Despite this, it will expand on the prior study of HAT3 in SD and provide even more solid evidence that epigenetic changes play a role in the molecular mechanism behind the anxiety and depression resulting from SD [5-7].
In conclusion, social defeat produces anxiety and depression in mice both directly after the SD, and in the long term. The molecular mechanism for this anxiety and depression resulting from chronic social defeat is currently unknown. It is likely that firing of vasopressinergic neurons triggers a social response, and that another change downstream of this is what determines if that response is anxiety or pro-social behavior. Up-regulation of BDNF after SD may play a role in helping to generate the other changes necessary for the anxious response to social stimuli. Evidence suggests that epigenetic changes are implicated in learning and memory, and determining the effect that SD has on epigenetic changes may very well elucidate the unknown molecular mechanism. By determining what causes the behavior that results from SD, other anxiety disorders may be treated, and the normal function of the human brain may be better understood.
 Savignac HM, Finger BC, Pizzo RC. Increased sensitivity to the effects of chronic social defeat stress in an innately anxious mouse strain. Neuroscience 2011; 192: 524-36.
 Litvin Y, Murakami G, Pfaff DW. Effects of chronic social defeat on behavioral and neural correlates of sociality: Vasopressin, oxytocin, and the vasopressinergic V1b receptor. Physiology & Behavior 2011; 103: 393-403.
 Keverne EB, Curley JP. Vasopressin, oxytocin and social behaviour. Current Opinion in Neurobiology 2004; 14: 777-83.
 Fanous S, Hammer RP Jr, Nikulina EM. Short- and long-term effects of intermittent social defeat stress on brain-derived neurotrophic factor expression in mesocorticolimbic brain regions. Neuroscience 2010; 167(3): 598-607.
 Hollis F, Wang H, Dietz D. The effects of repeated social defeat on long-term depressive-like behavior and short-term histone modifications in the hippocampus in male Sprague-Dawley rats. Psychopharmacology 2010; 211(1): 69-77.
 Puckett RE, Lubin FD. Epigenetic mechanisms in experience-driven memory formation and behavior. Epigenomics 2011; 3(5): 649-64.
 Sweatt DJ. Experience-dependent epigenetic modifications in the central nervous system. Biological Psychiatry 2009; 65(3): 191-97.
 Golden SA, Covington HE 3rd, Berton O, Russo SJ. A standardized protocol for repeated social defeat stress in mice. Nature Protocols 2011; 6(8): 1183-91.
 Zhao S, Ting JT, Atallah HE, et al. Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nature Methods 2011; 8(9):745-52.
 Pascoli V, Turiault M, Luscher C. Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour. Nature 2012; 481: 71-75.