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Along with genetic risk factors there also are perinatal environmental factors that contribute to the risk of developing Autism Spectrum Disorder. These environmental factors are known to activate the immune system, such that dysregulation of the immune system early in life may have negative consequences on neural function and the development of appropriate social behavior. Our experiment is designed with the intent of modeling the multitude of social behavior deficits seen in many neurodevelopmental disorders. Microglia release inflammatory cytokines in response to an immune challenge throughout which in effect “primes” them. The exaggerated cytokine release from the microglia, as well as a second “hit” later in life, alters neuronal function and leads to behavioral disorders. Our model introduces an immune challenge to rats on day 4 of life (P4) and then again on day 25 (P25), known as a “two-hit model of neuroinflammation”. Play behaviors such as pouncing, pinning, and chasing were observed and analyzed in juvenile male and female test rats. With this data we can observe the differences in the participation vs initiation in a play bout with our experimental animals compared to controls. Additionally, we are interested in the impact our model has on dopamine receptors as dopamine plays a crucial role in reward and is important for the expression of social play. Dopamine receptor expression was quantified by examining the relative gene expression of D1R and D2R within the amygdala and mPFC at 4, 24, and 72 hours post P25 injection. Results are showing a main effect of sex in the amygdala of D2R and a sex x P4 injection interaction of D1R of amygdala and mPFC. By looking at this data we can further investigate the differences present between the sexes. Funding: NIH R01MH16553 to JMS.

Delaware Neuroscience Symposium 2021

The effect of neuroimmune dysregulation on dopamine receptors and social play behaviors of male and female juvenile rats

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This annual event showcases and celebrates the neuroscience research in Delaware.  Posters and talks highlight neuroscience research carried out by graduate students, undergraduate students, and research staff at all of Delaware’s research and education institutions. The Lounge section of the symposium allows real-time interactions and networking among attendees.  New this year is an expo of research core facilities in Delaware.  Check them out in the Research Cores section of our poster session, or see the logos below.

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Evidence suggests that microglia, the immune cells of the brain, have an important role in the development of brain circuits. As a result, activation of the immune system early in life significantly increases the risk of various neurodevelopmental disorders, including Autism Spectrum Disorder. Perinatal exposure to infectious pathogens is suggested to be an environmental risk factor that has a role in activating the developing immune system, and the subsequent onset of neurodevelopmental diseases. Furthermore, the “two-hit hypothesis of immune activation” suggests that multiple exposures to immune activation further increases the risk of neurodevelopmental disorders. Immune activation during a “sensitive period” of neurodevelopment may result in an exaggerated response to subsequent immune challenges and the onset of behavioral disorders during the juvenile period. Additionally, microglia number and activation profiles differ between males and females during early development. Thus, males might be more susceptible to the consequences of immune activation as a result of the two-hit model. In order to better understand the two-hit model of neuroinflammation and its effect on the expression of social behaviors in males and females, we tested the impact of neonatal bacterial infection (E.coli) and a subsequent juvenile immune challenge (lipopolysaccharide, LPS) on two tests of rodent social behavior - social interaction and social recognition. The results from hand-scoring the time spent interacting in each test revealed that female control animals were not performing the test above chance. Therefore, we were interested in measuring the overall locomotive activity of test rats. Using an open-source ImageJ plugin, MouBeAt, locomotive behavior parameters were examined. Preliminary analysis of the data thus far indicates that the timing (neonatal or juvenile) and nature (E.coli or lipopolysaccharide) of the neuroimmune activation have a different effect on the expression of locomotive activity in male and female juvenile rats during a social behavior test.

Locomotive activity and sociability in male and female juvenile rats following mild immune activation

Acetylcholine (ACh) released from motor neurons activates postsynaptic ionotropic ACh receptors (AChRs), resulting in an electrical signal that leads to muscle contraction. Formation and maintenance of the neuromuscular junction is required for proper ACh signaling. Conventionally, HMTs act in the nucleus to methylate histones. Surprisingly, we found that C. elegans tropomyosin, which acts in the cytoplasm to regulate muscle contraction and stabilize actin filaments, was methylated by a HMT. In C. elegans, actin rich muscle arms extend from the body-wall muscles to the nerve cord, allowing the muscle cell to be innervated. This led us to hypothesize that cytoplasmic HMT activity regulates actin filaments and neuromuscular junction structure. Previously, tropomyosin mutants were shown to have fewer and wider muscle arms compared to wildtype. We performed extensive imaging of muscle arms in HMT mutants using confocal microscopy and observed that the HMT mutant exhibited significantly wider muscle arms compared to wild type but had no difference in muscle arm number per muscle. We then used tropomyosin RNA interference (RNAi) to knock down tropomyosin in the HMT mutant to determine if the loss of HMT and tropomyosin has an additive effect on muscle arm phenotype. After extensive muscle arm imaging and analysis, it appears that the HMT mutant grown on tropomyosin RNAi has significantly wider and fewer muscle arms per muscle compared to wildtype. This indicates that HMT and tropomyosin function together as the muscle arm phenotype observed is the same as the tropomyosin knockdown. In conclusion, our results suggest that a HMT plays a novel cytoplasmic role to control muscle development and function.

A Cytoplasmic Role for a Histone Methyltransferase in C. elegans Muscle Development

Microglia, the resident immune cells of the brain, interact with neurons to shape cognitive development. Microglial activation during the early postnatal period has been linked to long-term effects on cognition and learning in rats. We seek to examine the role of microglia in hippocampal learning by establishing a method to selectively deplete microglia in the dorsal hippocampus during a unique juvenile period development in rats. Liposome-encapsulated clodronate (LEC) is a drug that is selectively ingested by and results in the death of phagocytic cells. We infused 3 microliters LEC bilaterally into the dorsal hippocampus (dHP) of male and female rats (Sprague-Dawley) at P16 to determine whether LEC selectively depletes microglia during a period of hippocampal development. We utilized immunohistochemical (IHC) staining and RT-PCR to examine changes in microglia density and gene expression in the CA1, CA3, and dentate gyrus (DG) subregions of dorsal and ventral hippocampus (vHP) on P21, P24, P27, P30, and P40. Iba1 stain for microglia revealed that LEC significantly decreased the percent area of microglia density and the number of microglia cells per area of microglia in subregions CA1 and DG of dHP from P21-P30. LEC did not significantly deplete microglia in any vHP subregions, nor the CA3 subregion of dHP, which indicate that the spread of LEC does not go beyond the intended target of dHP CA1 and DG subregions. Future research will use this data to establish a timeline of microglial depletion during the juvenile period in order to investigate the role of microglia in hippocampal learning deficits following immune activation.

Quantification of microglia in the dorsal hippocampus following temporary intra-hippocampal microglia depletion during development

Prenatal alcohol exposure (PAE) causes a spectrum of physical, cognitive, and behavioral deficits in offspring and these disorders are categorized under the umbrella term Fetal Alcohol Spectrum Disorders (FASD). FASD affect at least 2-5% of births in the United States, making it the primary cause of preventable developmental abnormalities (May et al., 2009). PAE prevents the proper development of the corpus callosum (CC), the largest white matter tract in the brain, in adolescence which impairs cognition and behavior (Wozniak et al., 2019; Riley et al., 1995). Axonal myelination of CC, beginning in the third trimester of pregnancy and terminating in young adulthood, is particularly vulnerable to the teratogenicity of PAE. Using a rodent model of third trimester equivalent AE, our study examines longitudinal alterations to CC myelination in the adolescent AE brain. These findings will be used to assess the efficacy of a targeted adolescent exercise intervention on restoring CC myelination in our rodent model of FASD. Aerobic exercise is a known stimulator of myelinogenesis. Male and female Long-Evans rat pups were either administered alcohol via intragastric intubation in a third trimester binge model of FASD [5.25 g/kg/day on postnatal days (PD) days 4-9] or were sham-intubated as controls. On PD 30, half of all rats were housed for twelve days in modified cages equipped with a metal running wheel. All rats, including homecage controls, remained socially-housed during the entirety of the experiment. Diffusion Tensor Imaging (DTI) scans were acquired on PD 30 and 42 for rats in all groups. Preliminary analysis of DTI scans reveals that third trimester AE delays CC myelination in adolescence, supporting the clinical literature. Neither exercise intervention nor sex had a main effect on CC myelination. Immunocytochemical and electron microscopic analysis of brain tissue samples will validate these findings.

Volume and Myelination of the Corpus Callosum in a Third Trimester Model of Fetal Alcohol Spectrum Disorders: Does Voluntary Aerobic Exercise Matter?

Over recent years there has been a great surge in interest, knowledge, and technology surrounding genetics and genetic services. As a result, genetic counseling has become a rapidly growing field of medicine. In 1988, Harris’s pioneering manuscript “Genetic Counselling and the New Genetics” envisioned the future of genetic counseling as one “confirming clinical diagnoses, recognizing genetic heterogeneity, assessing risk, and providing a link between laboratories, physicians, and patients.” Today, genetic testing is a powerful tool that can be used to help direct precision medicine. Diseases and disorders that are caused by abnormalities in an individual's genome are currently classified into four categories: single-gene, multifactorial, chromosomal, and mitochondrial. This state-of-the-art review focuses on how diseases and disorders caused by genomic abnormalities are linked to neurobiological differences. Further, the review aims to provide insights into how to link laboratories, physicians, and patients to bridge the gap between genetics and neurobiology.

Genes, Brains, and Behavior: Genetic counselors’ role in precision medicine

Early life experiences shape neurodevelopmental, epigenetic, and future behavioral landscapes of individuals. While both animal models and human twin studies suggest that there is a predisposition for certain stimuli to be innately fear-inducing, molecular mechanisms remain poorly understood. Prior work in our laboratory has demonstrated that administering TMT (2,5-dihydro-2,4,5-trimethylthaizonline), a synthetic odorant from fox feces, induces robust freezing in naïve rodents without any prior learning. However, experiencing predator odor during early development alters the response later in life, suggesting a change in perception of the innate fear-inducing odor. Administering TMT to dams and pups during development (PN1-21) inside the home-cage alters the quality of maternal care, pup ultrasonic-vocalizations, and lowers freezing (PN30) upon re-exposure comparable to control animals. To extend these findings to other predator odorants and examine how maternal behavior may alter later innate fear responses, pups with or without their dam were exposed to either a noxious control (butyric acid) or a bobcat urine for 20 minutes in their home-cage for the first 21 days of life. Concurrent with odor exposures, video and USV recordings were captured to quantify maternal care and pup behavior, respectively. On PN30, rodents were exposed to bobcat urine for 10 minutes and freezing behavior was recorded. Finally, on PN35, to examine if fear behaviors were generalized to other paradigms, an open-field test was administered. Preliminary results suggest that developmental exposure to bobcat urine lowered the quality of maternal care and increased freezing upon re-exposure on PN30. There were no differences in open-field behavior or pup ultrasonic vocalizations. These results support the complex relationship of early exposure to predator stimuli, effects of maternal behavior, and the expression of innate fear later in life. Future research will examine epigenetic alterations linked to long lasting changes in fear responses from early experience with predator odors.

Impact of developmental exposure to predator urine on maternal behavior and the expression of innate fear

The pain of Black Americans is historically underdiagnosed and undertreated (Anderson, Green, & Payne, 2009). While many structural and individual level factors associated with a history of, and ongoing medical racism contribute to these disparities (Gee & Ford, 2011; Feagin & Bennefield, 2014) recent work has demonstrated that these disparities are also evident at the level of face and emotion perception and contribute to disparities in care (Mende-Siedlecki, 2019). The present work examines whether perceiver deficits in pain recognition are reflected in divergent neural activity in regions associated with face processing, pain, and empathy. Replicating previous work, we find that participants had higher thresholds for perceiving pain on Black male vs White male faces, such that more pain needed to be expressed on Black male faces to be seen as equivalent to White male pain. In turn, we find that Black males were prescribed less pain-relieving cream than White males overall. Moreover, we find that perceptual bias continued to facilitate the effect of target race on treatment when status/strength stereotypes and explicit racial bias were included as competing within-subjects mediators. Finally, we find differential activity in posterior STS and ATL associated with increasing pain on White vs Black faces, which overlap with face selective regions identified by a face localizer as well as the broader literature.

Neural Underpinnings of Racial Bias in Pain Perception & Treatment

Several studies have shown that receiving speech or language services for expressive language delays positively impacts academic indices of success (e.g., Del Tufo, Earle, & Cutting, 2019). However, beyond expressive language delays we do not know if receiving services before the age of 5 has a positive impact on academic performance. Expressive language refers to the production of spoken language, and the milestones of expressive language, such as the onset of babbling, production of first words, and putting several words together, are early indicators of normal language development. We analyzed a national sample of data (National Survey of Children’s Health, 2011/2012) of children 0-17 years of age, through cross-sectional, telephone-based interviews to determine the (1) factors that impact whether or not a child will receive speech and/or language services before the age of 5, and (2) the impact of receiving speech and/or language services on the indices of academic success. To answer the aforementioned questions we implemented hierarchical logistic regression and linear regression, respectively. The results indicate that several factors beyond expressive language impact children’s receipt of speech and/and language services before age 5. These factors include sex of the child, socioeconomic status, period of gestation, and parent’s concerns regarding receptive language. These factors together predicted if a child would receive speech and/or language, which, in turn, had an impact on academic performance.

Which factors beyond expressive language delays lead to the receipt of speech and/or language services before age 5?

Alcohol exposure (AE) during pregnancy is known to affect fetal medial prefrontal cortex (mPFC) and hippocampus (HC) development. Previous work has demonstrated that these two structures are vulnerable to AE during the brain growth spurt, which occurs during 3rd trimester of human pregnancy and the first two postnatal weeks in rodents. Our lab has shown that thalamic nucleus reuniens (Re) is damaged in a rodent model of human 3rd trimester AE. We have also shown that Re is critical for mPFC–HC oscillatory synchrony and spatial working memory (SWM). The current study investigated the effects of early AE on SWM ability in adulthood in rats. We predicted that AE would lead to SWM impairment that worsened as working memory demand increased. We administered 5.25 g/kg/day of ethanol in milk formula via intragastric intubations in a binge manner on PD 4-9 (AE group) or performed intubations without liquid (sham intubated (SI) group). On PD90, rats were trained on a continuous alternation (CA) T-maze task. Upon reaching a choice accuracy criterion of 80% for two consecutive days on CA, we introduced a delay between trials (DA) in order to increase SWM demand. The rats underwent 6 DA sessions, consisting of 12 each of 10-second (s), 30 s, and 60 s delay trials given in a pseudorandom sequence. Our results show that animals in both AE and SI groups learned the CA task at similar rates. However, on the DA task, the SI group showed consistent improvement across sessions on the 30-s delay trials, whereas the AE group did not (postnatal - treatment x day interaction: F(5,195)=2.70, p = .02). For the 10 s delay trials, both AE and SI groups showed improvement across days (main effect of day: F(5,195)=7.93 , p < .001), whereas for the 60 s delay trials, both groups performed uniformly poorly across days. These findings support the hypothesis that binge AE during the brain growth spurt produces SWM deficits that depend on SWM demand. This study will lead to a better understanding of the impact of HC-Re-mPFC circuitry damage due to developmental AE on cognitive function later in life.

The effect of neuroimmune dysregulation on dopamine receptors and social play behaviors of male and female juvenile rats

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