1. Maternal Thyroid Hormone Insufficiency Affects Short-Term Object Recognition Memory in Adult Offspring of Long-Evans Rats
Megan L. Sieg University of Illinois at Urbana-Champaign
College of Veterinary Medicine
Department of Comparative Biosciences
Neuroscience Program
Animal Behavior Conference 2017
Indiana University at Bloomington
April 8, 2017

2. Thyroid hormone is essential for brain development
Humans
Thyroid hormone is essential for normal brain development
Until the fetus is able to produce enough of it’s own TH, it relies on it’s mother’s TH
Able to begin producing it’s own TH by gestational week 12 in humans
Humans born with fully developed and functional TH axis
Howdeshell, Environmental Health Perspectives

3. Maternal hypothyroidism impairs cognition in children
Lower scores on IQ tests than peers born to euthyroid mothers
Learning disabilities
Intellectual and developmental disabilities in extreme cases
Previous studies have shown that untreated maternal hypothyroidism during pregnancy can result in children
Scoring lower on IQ tests than their peers born to euthyroid mothers
Having learning disabilities
And, in extreme cases of maternal hypothyroidism, intellectual and developmental disabilities
However, most cases of overt maternal hypothyroidism are diagnosed and treated

4. How does maternal subclinical hypothyroidism affect children?
SCH often goes undetected and untreated
Estimated prevalence of %
Little known of effects of maternal SCH on children
Few human studies with conflicting results
What is of greater concern is the effect of maternal subclinical hypothyroidism on children
SCH is “subtle” hypothyroidism that is often asymptomatic and therefore is often untreated
The estimated prevalence for maternal SCH is % of pregnant women
Little research on effects of maternal SCH on children
Human work: few studies and conflicting results
Children born to mothers who were SCH during the 2nd trimester scored lower on IQ tests at age 7-9 (Haddow et al 1999)
Children who were born preterm with transient hypothyroidism showed neurodevelopmental deficits in several areas, including memory, at 5.5 years of age (Delahunty et al 2010)
Children born to mothers who suffered from SCH tested similarly on IQ tests as children born to euthyroid mothers and those born to hypothyroid mothers treated for hypothyroidism at ages 4-15 (Behrooz et al 2011)
Children born to SCH mothers scored similarly to children born to euthyroid mothers on developmental assessments at 2 years of age (Chen et al 2015)

5. A rodent model of maternal SCH
Developed by Sui & Gilbert (2003)
Dams dosed with propylthiouracil (PTU) via drinking water
Doses as low as 3 ppm
Reduced LTP of hippocampal EPSPs
Impaired spatial learning in Morris water maze
Altered expression of many genes associated with development and synaptic function in cortex and hippocampus
Rodent studies
Model developed by Sui & Gilbert (2003) consists of dosing dams during both gestation and the early postnatal period with the anti-thyroid agent, propylthiouracil (PTU) via drinking water at does ranging from 3 ppm to 10 ppm
Prenatal exposure roughly equivalent to the first half of human development during gestation
Postnatal exposure roughly equivalent to the second half of human development during gestation
With this model, they have seen effects on the long-term potentiation of hippocampal EPSPs,
impaired spatial learning,
reduced response suppression in fear conditioning,
altered expression of genes associated with development and synaptic function in the cortex and hippocampus,

6. Studying learning & memory: The Novel object recognition task
Hippocampus and lateral perirhinal cortex involved in object recognition memory (Hammond et al 2004)
NOR task takes advantage of preference for novelty
No reinforcement
Minimal training required
Used to study learning and memory
Previous work from collaborator indicated that in this model of maternal SCH, learning and memory are affected in the offspring
Perirhinal cortex and hippocampus involved in object recognition memory
Many previous studies have found impairments in recognition memory (often using NOR task) with perirhinal cortex lesions
In 2004, Hammond et al showed that hippocampal lesions led to impairments in NOR task after 24 hr retention interval, but not 5 min retention interval
Hippocampus involved, but perirhinal cortex and other parahippocampal structures are sufficient for shorter retention intervals—but how short?
NOR is a behavioral assessment that requires no external reinforcements (punishments or rewards) and only a little training
Takes advantage of rodents’ tendency to approach and explore a novel object more than a familiar object
Thought that these episodes may be more vulnerable than those paired with a reward or stimulus
Used to study learning and memory, type depends on retention interval used
Diagram
HF = hippocampus
AMY = amygdala
PER = postrhinal cortex
EC = entorhinal cortex
POR = postrhinal cortex

7. Treatment Groups CON-CON: Never exposed to PTU
Propylthiouracil (PTU)
CON-CON: Never exposed to PTU
CON-PTU: Postnatal exposure
PTU-CON: Prenatal exposure
PTU-PTU: Perinatal exposure
GD 6 – PND 14
Males
Females
0 ppm PTU
GD6 – PN14
CON-CON 16
3 ppm PTU PN2 – PN14
CON-PTU 10 11
3 ppm PTU
0 ppm PTU PN2 – PN14
PTU-CON 9
PTU-PTU 12 14
Propylthiouracil (PTU) = chemical used to decrease TH production
Inhibits TPO (thyroid peroxidase, enzyme)  inhibits oxidation of iodine (I-  I0)  decreased TH synthesis
Inhibits dehydrogenases at target tissues  decreased conversion of T4 (less active) to T3 (more active)
Is able to cross placenta
2 groups of dams given 0 ppm PTU in drinking water GD 6-PND 14
2 groups of dams given 3 ppm PTU in drinking water GD 6-PND 14
Littermates switched at PND 2 to create 4 treatment groups

8. Treatment Groups CON-CON: Never exposed to PTU
Propylthiouracil (PTU)
CON-CON: Never exposed to PTU
CON-PTU: Postnatal exposure
PTU-CON: Prenatal exposure
PTU-PTU: Perinatal exposure
Cross-foster at PN Day 2
GD 6 – PND 14
Males
Females
0 ppm PTU
GD6 – PN14
CON-CON 16
3 ppm PTU PN2 – PN14
CON-PTU 10 11
3 ppm PTU
0 ppm PTU PN2 – PN14
PTU-CON 9
PTU-PTU 12 14
Littermates switched at PND 2 to create 4 treatment groups

9. Treatment Groups CON-CON: Never exposed to PTU
Propylthiouracil (PTU)
CON-CON: Never exposed to PTU
CON-PTU: Postnatal exposure
PTU-CON: Prenatal exposure
PTU-PTU: Perinatal exposure
Cross-foster at PN Day 2
Males
Females
0 ppm PTU
GD6 – PN14
CON-CON 16
0 ppm PTU GD6
3 ppm PTU PN2 – PN14
CON-PTU 10 11
3 ppm PTU GD6
0 ppm PTU PN2 – PN14
PTU-CON 9
3 ppm PTU
PTU-PTU 12 14
TH axis less developed at birth in rats compared to humans, actually ~beginning of 2nd half of pregnancy in humans

10. Treatment Groups CON-CON: Never exposed to PTU
Propylthiouracil (PTU)
CON-CON: Never exposed to PTU
CON-PTU: Postnatal exposure
PTU-CON: Prenatal exposure
PTU-PTU: Perinatal exposure
Cross-foster at PN Day 2
Males
Females
0 ppm PTU
GD6 – PN14
CON-CON 16
0 ppm PTU GD6
3 ppm PTU PN2 – PN14
CON-PTU 10 11
3 ppm PTU GD6
0 ppm PTU PN2 – PN14
PTU-CON 9
3 ppm PTU
PTU-PTU 12 14
99 animals: 49 males, 50 females

11. Novel object recognition training & task
Habituation
Rat placed in empty chamber
5 min
Not recorded
Familiarization
Following day, rat placed in chamber with two of the same objects
3 min
Recorded
Rat placed facing away from objects
Chose to do 2 familiarization trials prior to test day because of pilot data
Indicated that 1 trial was not enough for them to remember the cupcake
No differences in exploration time or entries to objects after 1 familiarization trial prior to test day
Test
Rat placed back into chamber with one of the original objects and one new object after a delay
In preliminary analyses, we found that minute 1 always significantly differed from minutes 2 and 3 while 2 and 3 never differed from one another, so our focus here is on the results of analyzing minute 1
Day 1 Habituation
Day 2 Familiarization 1 4 hour delay Familiarization 2
Day 3 Familiarization 3 1 hour delay Test

12. Prenatal exposure reduces time spent exploring the novel object
We chose to analyze the data a little differently
Instead of comparing treatment groups, we compared animals that were exposed prenatally and not, and postnatally and not
Prenatal = PTU-CON + PTU-PTU
Prenatal none = CON-PTU + CON-CON
Postnatal = CON-PTU + PTU-PTU
Postnatal none = PTU-CON + CON-CON
Box & whisker plots show
Mean for each group = line in box
Confidence intervals = whiskers
Box represents range from highest values to lowest values
Animals exposed prenatally explored the novel object less than animals not exposed prenatally
No effect of postnatal exposure
No prenatal x postnatal interaction

13. Prenatal exposure reduces percentage of trial spent exploring the novel object
Converted raw data to percents to compare percent of the trial each group spent exploring the novel object
Converting the data to percents confirmed what we saw with the raw data:
Animals exposed prenatally spent less time exploring the novel object
Animals exposed postnatally did not spend less time exploring the novel object
No prenatal x postnatal interaction

14. Prenatal exposure reduces the number of entries made to the novel object
Animals exposed prenatally also made fewer entries to the novel object than animals not exposed prenatally
No effect of postnatal exposure
No prenatal x postnatal interaction

15. Conclusions
Exposure during early gestation affects short-term recognition memory
Prenatal exposure to PTU affects time exploring the novel object
Prenatal exposure to PTU affects number of entries to the novel object
Additional evidence that maternal SCH affects offspring learning and memory
Maternal SCH may affect learning and memory in humans
Early exposure
Additional insight for future human studies
In rodents, early exposure to this model of maternal SCH affects short-term recognition memory
Prenatal exposure to PTU affects time exploring the novel object
Prenatal exposure to PTU affects number of entries to the novel object
Maternal SCH may affect learning and memory in humans
These results provide evidence that exposure to maternal SCH during the first half of pregnancy may be more detrimental to learning and memory than exposure later on
Provide additional insight for future directions for human studies

16. Acknowledgements Questions?
UIUC Dr. Paul Eubig Dr. Rebecca Smith Dr. Justin Rhodes Dr. Alison Bell Environmental Protection Agency Dr. Mary Gilbert Department of Comparative Biosciences
Eubig Lab
Catherine Townes
Rekha Balachandran
Megi Aliaj
Questions?

17. References
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Brown, Malcolm W., & Aggleton, John P. (2001). Recognition memory: What are the roles of the perirhinal cortex and hippocampus? Nature Reviews Neuroscience, 2, 51-61
Gilbert, M. E. & Sui, Li. (2006). Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Research, 1096, 10-22
Gilbert, Mary E. (2011). Impact of low-level thyroid hormone disruption induced by propylthiouracil on brain development and function. Toxicological Sciences, 124(2),
Hammond, Rebecca S., Tull, Laura E., & Stackman, Robert W. (2004). On the delay-dependent involvement of the hippocampus in object recognition memory. Neurobiology of Learning and Memory, 82, 26-34
Howdeshell, Kembra L. (2002). A model of the developmet of the brain as a construct of the thyroid system. Environmental Health Perspectives, 110(3),
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