Behavior, Immunology, and Neuroscience Research at Duke UniversityRahul Joseph, Centre College ’14

Duke University Mechanisms of Behavior REU ProgramBrown Fellows Summer Enrichment Project 2012

ABSTRACTThis summer, I participated in a 10-week, intensive research experience in behavior, immunology,

and neuroscience at Duke University, facilitated through the Mechanisms of Behavior REU (Research Experiences for Undergraduates) program as approved by the National Science Foundation. Working under the guidance of Dr. Staci Bilbo, I investigated the effects of combined prenatal environmental stressors on adult function of microglia, the primary immune cells of the brain. Though no significant difference in neuroimmune gene expression was found between the treatment groups, we showed that microglia are the predominant producers of cytokines and other neuroimmune proteins in the brain, reinforcing their significance as a connection between the nervous and immune systems. This summer enrichment experience provided an in-depth focus on a specific area of health research. It also helped me establish lasting relationships with professors and students interested in health sciences and cultivated my knowledge of the research that sustains medical practices.

EXPERIMENTAL CONCLUSIONSFetal programming is associated with

microglial priming

Combined stressors yield marked changes in contextual memory and anxiety, as well as brain cytokine protein levels

Microglia are the primary producers of cytokines/other proteins associated with neuroimmune function

Gene expression levels (level of RNA) do not differ, yet significant differences were found at the level of protein — Post-translational modifications must be involved

is the inflammasome (a post-translational protein complex) the culprit?

PERSONAL OBJECTIVETo participate in an intensive, competitive research experience in neuroscience in order to 1) gain

expertise in a specific area of the biological sciences through university-level research, 2) develop a broader and more accurate paradigm of medical sciences and health professions, and 3) build lasting relationships with other students and professionals involved in medical and health science careers.

ROLE OF MICROGLIA Microglia are the primary immune cell of brain

— Make up 20% of total glial brain cells

Microglia can induce an inflammatory immune response

— Release cytokines, chemokines cell signaling

Microglia are critical in the bidirectional interactions among the nervous, immune, and endocrine systems

Like other macrophages, microglia are antigen-presenting

— Can recognize antigens after initial response to launch faster, stronger immune responses

WHAT IS FETAL (PERINATAL) PROGRAMMING?

Fetal programming is a phenomenon in which early life events influence development and adult function

— These events may include exposure to environmental stress, pathogens, and toxins

Microglial priming has been linked to fetal programming of later-life behavior

— This priming is the result of overactivation of microglial cells early in development

Could microglial proteins (immune system) be affecting nervous system processes?

BEHAVIORAL AND MOLECULAR EXPERIMENTAL METHODS

As an undergraduate researcher, I gained hands-on experience in the laboratory with both behavioral tests and molecular analytical techniques. Working with both live animals and isolated cell tissue samples gave me the full experience of neuroscience research. Understanding the methods used to answer the questions of health science research will be key to my ability to evaluate and apply medical advances to either research or patients in the future.

Behavioral tests

— Elevated Zero Maze Test

— Open Field Test

— Porsolt Swim Test

Molecular analytical techniques

— Saline perfusion

— Cell isolation (using CD11b as microglial marker)

— RNA isolation

— cDNA synthesis

— RT-PCR analysisGenome Sciences Research Building II at Duke University is the home of the Psychology and Neuroscience department and the Duke Mechanisms of Behavior REU program.

Panel C is a timeline of prenatal environmental stressor exposure in the mouse model. Panel D shows cell isolation of two separate cell populations, CD11b positive and CD11b negative, using the MACS® magnetic beads and column, taken from MACS® Miltenyi Biotec. Panel E shows the apparatus used in the Elevated Zero Maze Test for anxiety. Panel F is a photograph of the apparatus for the Open Field Test for anxiety used in the Bilbo lab.

C D

E F

Panel A is a diagram of microglial activation taken from Bilbo & Schwarz, 2009. Panel B shows an immunohistochemical stain of microglia using the protein Iba1 (ionized calcium binding adaptor molecule 1) as a microglia marker.

A B

EXPERIMENTAL OBJECTIVE & APPROACHTo determine if two separate environmental stressor exposures during gestation will synergize to enhance priming of

adult neuroimmune function. Our approach was to apply diesel exhaust exposure and resource restriction in the mouse model through Diesel Exhaust Particle (DEP) instillations into the maternal lungs and Nest Restriction (removal of one third of nesting material and elevation of cage floor over bedding via wire mesh, adopted from Rice et al., 2008) during the latter half of gestation. We therefore tested four experimental groups: DEP alone, Vehicle (VEH; a control for DEP without polluting particles), DEP with Nest Restriction, and VEH with Nest Restriction.

BEHAVIORAL RESULTSFrom previous behavioral tests:

Only DEP/Nest Restriction males showed impaired contextual memory in adults after fear conditioning.

DEP/Nest Restriction males showed increases in anxiety.

No significant increases in anxiety or contextual memory deficit were found in females or in other male treatment groups.

MOLECULAR RESULTS We did not show a significant difference in neuroimmune gene expression between the treatment groups.

We did show that the CD11b positive brain cell population (containing microglia exclusively) expressed neuroimmune genes at a significantly higher level than the CD11b negative population.

— This result has been greatly debated in the neurobiological community

G

JI

H

Panel G is a bar chart displaying relative expression of the toll-like receptor (TLR)4 gene, expressed by microglia and other cells in the brain, in the CD11b positive and negative cell populations for males and females of each treatment groups. Panel H displays relative expression of pro-inflammatory cytokine interleukin-1 beta (IL-1β). Panel I displays relative expression of anti-inflammatory cytokine interleukin-10 (IL-10). Panel J displays relative expression of caspase-1, a regulator of IL-1β levels. Each bar chart shows that the CD11b positive cells (microglia) are the predominant producers of these neuroimmune proteins. We do not see significant differences between treatment groups.

PERSONAL CONCLUSIONSThrough this enrichment experience I have:

Gained expert knowledge regarding microglia, which are still not yet fully understood

Reinforced my view of the close connection between health science research and medical practice

Networked with several professionals and students involved with health science careers

Realized my passion for application of scientific findings, particularly in the medical sphere

Narrowed my future enrichment experience options to more direct involvement with medical practice and patient interactions