Example: If a child is diagnosed with ASD, and then they are diagnosed with Tuberous Sclerosis, the child will still have ASD, but now the cause of the ASD is identified (i.e. a mutation in the TSC1 or TSC2 gene). 

Mutations in the genes listed above can cause a disruption in cell function in the nucleus, cytoplasm or membrane level LEADING to neuronal structural differences, such as dendritic spine variances. (18, 20)

Changing one's diet to supplement specific microbiota in the gut or the removal of all bacteria may influence a stress response, as seen in an experiment done to mice

GOAL: To further investigate the link of maternal obesity on social interaction, oxytocin levels and VTA plasticity of offspring and isolate a strain of bacteria that can possibly reverse these effects

Buffington et. al. 2016. Figure 1B-D, F-G. 

Buffington et. al. 2016. Figure 1J. 

Buffington et. al. 2016. Figure 2C-F. 

Buffington et al. 2016. Figure 3G-J. 

            

Previously, Sullivan et al. (2014) have shown that maternal diet influences the neural circuitry of offspring through shaping of the intrauterine environment, thereby affecting offspring behaviour in the long term (21). Furthermore, many studies, including Galley et al. (2014) article have shown that maternal obesity alters microbiome in human toddlers, as well (15). In addition to microbiota remodeling, other physiology affects in offspring have been observed, including impaired insulin resistance in offspring as a result of maternal gestational diabetes and obesity as shown in an article by Maftei et al (15). In the present Buffington, et al. (2016) experiment, L. reuteri was shown to increase in oxytocin levels in Paraventricular nuclei (PVN) of hypothalamus, which projects to the Ventral tegmental area (VTA). The VTA and nucleus accumbens (NAc) are associated with the reward system and modulate social behaviours. In socially impaired mice, LTP in the VTA was affected. Previous and current studies have focused on the effect of maternal-high fat diet induced inflammation in the offspring brain. A study by White et al. (2009) high fat diet was associated with nitric oxide synthase, as well as IL-6 and Iba-1 and GFAP in offspring, indicating increased inflammatory signalling (24). 

Bruce-Keller et al. (2009). Figure 3C-D, G-H.

*This reduction was not due to a decrease in PVN neurons – as they remained the same with the NeuN staining

NEVERTHELESS...

 

This shows that maternal obesity can cause a reduction of oxytocin neurons, which can be restored with the introduction of L. reuteri. 

 

This implies that oxytocin and dopaminergic system must work together to promote LTP for the processing of social relevant cues 

 

(2) Bresnahan M, et al. 2015. Association of maternal report of infant and toddler gastrointestinal symptoms with autism: evidence from a prospective birth cohort. JAMA Psychiatry. 72(5):466-474

 

(3) Bruce-Keller AJ, Fernandez-Kim S-O, Townsend RL, Kruger C, Carmouche R, Newman S, et al. 2017. Maternal obese-type gut microbiota differentially impact cognition, anxiety and compulsive behavior in male and female offspring in mice. PLoS ONE 12(4): e0175577. 

 

(4) Caplin, Maya. “Lactobacillus reuteri.” ProBiotics America, 31 Oct 2016, https://probioticsamerica.com/lactobacillus-reuteri/ 
 
(5) Connolly N, Anixt J, Manning P, Ping DL, Marsolo KA, Bowers
K. 2016. Maternal metabolic risk factors for autism spectrum disorder - An analysis of electronic medical records and linked birth data. Autism Res. 
 
(6) Friedman JE. 2016. The maternal microbiome: cause or consequence of obesity risk in the next generation? Obesity. 25(3):497-498
 
(7) Fuchs F, Senat MV, Rey E, Balayla J, Chaillet N, Bouyer J, Audibert F. 2017. Impact of maternal obesity on the incidence of pregnancy complications in France and Canada. Science Reports. 7:10859
 
(8) Galley JD, Bailey M, Dush CK, Sullivan SS, Christian LM. 2014. Maternal obesity is associated with alterations in the gut microbiome in toddlers. PLoS. 1(9).

 

(9) Gareau MG, et al. 2011. Bacterial infection causes stress-induced memory dysfunction in mice. Gut. 60(3):307-317
 
(10) Hamptom T. 2016. Maternal obesity affects sociality of offspring by altering gut bacteria. Journal of American Medical Association. 316(5):484.

 

(11) Kang DW, et al. 2017. Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study. Microbiome 5(10): https://doi.org/10.1186/s40168-016-0225-7
 
(12) Krakowiak P, Walker CK, Bremer AA, Baker AS, Ozonoff S, Hansen RL, Hertz-Picciotto I. 2012. Maternal metabolic conditions and risk for autism and other neurodevelopmental disorders. Pediatrics. 129(5)
 
(13) Lieshout RJ, Taylor VH, Boyle MH. 2011. Pre-pregnancy and pregnancy obesity and neurodevelopmental outcomes in offspring: a systematic review. Obesity Reviews. 12(501):548-559
 
(14) Ma J, Prince AL, Bader D, Hu M, Ganu R, Baquero K, Blundell P, Harris RA, Frias AE, Grove KL, Aagaard KM. 2014. High-fat maternal diet during pregnancy persistently alters the offspring microbiome in a primate model. Nat. Commun. 5: 3889

 

(15) Maftei O, Whitrow MJ, Davies MJ, Giles LC, Owens JA, Moore VM. 2015. Maternal body size prior to pregnancy, gestational diabetes and weight gain: associations with insulin resistance in children at 9-10 years. Diabetes Med 32(2):174-80
 
(16) Mehta SH, Kerver JM, Sokol RJ, Keating DP, Paneth N. 2014. The association between maternal obesity and neurodevelopmental outcomes of offspring. Journal of Pediatrics. 165(5):891-896
 
(17) Parashar A, Udayabanu M. 2016. Gut microbiota regulates key modulators of social behaviour. European Neuropsychopharmacology. 26(1):78-91.
 
(18) Penagarikano O, Abrahams BS, Herman EI, Winden KC, Gdalyahu A, Dong H, Sonnenblick L, Gruver R, Almajano J, Bragin A, Golshani P, Trachtenberg JT, Peles E, Geschwind DH. 2011. Absence of CNTNAP2 leads to epilepsy, neuronal migration abnormalities and core autism-related deficits.  National Institute of Health. 147(1):235-246
 
(19) Reynolds LC, Inder TE, Neil JJ, Pineda RG, Rogers CE. 2014. Maternal obesity and increased risk for autism and developmental delay among very preterm infants. Journal of Perinatology. 34:688-692
 
(20) Selimbeyoglu A, Kim CK, Inoue M, Lee SY, Hong ASO, Kauvar I, Ramakrishnan C, Fenno LE, Davidson TJ, Wright M, Deisseroth K. 2017. Modulation of prefrontal cortex excitation/inhibition balance rescues social behaviour in CNTNAP2-deficient mice. Science Translational Medicine 9:1-10

 

(21) Sullivan EL, Nousen EK, CHamlou KA. 2014. Maternal high fat diet consumption during the perinatal period programs offspring behavior. Physiology & Behaviour 123: 236-242
 
(22) Vuong HE, Hsiao EY. 2017. Emerging Roles for the Gut Microbiome in Autism Spectrum Disorder. Biological Psychiatry. 81:35-37 

 

(23) Wang X, Wang BR, Zhang XJ, Xu Z, Ding YQ, Yu G. 2002. Evidences for vagus nerve in maintenance of immune balance and transmission of immune information from gut to brain in STM-infected rats. World J Gastroenterol. 8(3):540-505

 

(24) White CL, et al. 2009. Effects of high fat diet on Morris maze performance, oxidative stress, and inflammation in rats: contributions of maternal diet. Neurobiol Dis. 35(1):3-13

  

(25) White CL, Purpera MN, Morrison CD. 2009. Maternal obesity is necessary for programming effect of high-fat diet on offspring. Am J Physiol Regul Integr Comp Physiol. 296(5):464-72

Youtube Video Links
(1) 16 S rRNA Gene Sequencing Service by LC Sciences
https://www.youtube.com/watch?v=c6w1uZL0HjE

(2) Poop Transplants by MinuteEarth
https://www.youtube.com/watch?v=Dim7YXYlRm0

(3) Shotgun Sequencing Simple Animation by Lorna Daly
https://www.youtube.com/watch?v=B_ebMtsGSb0

(4) The Party: A Visual Experience of Autism - 360 Film
https://www.youtube.com/watch?v=OtwOz1GVkDg

The two conditions in this experiment will be 1) MHFD offspring + cut vagus nerve (experimental) and 2) MHFD offspring + intact vagus nerve (control). Both mice will be administered oxytocin intranasally and then placed in both a reciprocal social interaction and a three-chamber test for sociability and social novelty after 30 min to assess social behaviours. One day later, the VTA will be tested for evidence of LTP by 1) inducing an EPSP and 2) measuring AMPAR/NMDAR ratios. If, as expected, both conditions show LTP after oxytocin administration and the subsequent social interaction, then the results Buffington et al. (2016) reported are bolstered with stronger evidence. It would support the hypothesis that the role of L. reuteri is simply to increase oxytocin levels in the PVN, paving the way for it to reverse characteristic deficits after a social interaction. As the vagus nerve is cut in these mice, there is no known way that the gut bacteria could have influenced this process, meaning that only oxytocin and social interaction are necessary for social deficits to be reversed in MHFD offspring.

If these results are not observed, however, and MHFD offspring without a functioning vagus nerve still exhibit social deficits after oxytocin treatment, then the authors’ hypothesis about oxytocin is rejected and furthermore, it complicates their overall claims. A possible reason for such results is that communication between the brain and the gut through the vagus nerve was a confounding factor in their experiment. In this situation, introducing oxytocin intranasally may have normally induced changes in the brain that triggered communication with the gut in order for social interaction to successfully induce LTP. As the control condition has an intact vagus nerve, this back-and-forth signaling would have allowed for the observed reversal of social deficits, which would indicate that the methodology in the Buffington et al. (2016) paper led to inaccurate results. Moreover, these findings would contradict their proposed sequential mechanism of L. reuteri increasing oxytocin, leading to LTP occurring in the VTA after social interaction. Instead, it would appear that L. reuteri is only necessary to increase oxytocin levels initially and that any composition of microbiota is sufficient to carry out the process beyond that point. Additionally, it would show that the process is not entirely linear, with oxytocin feeding back to the gut, and thereby requiring both components working in unison for LTP to occur in social interactions and reverse deficits.