Prenatal Alcohol Exposure and Transient Systemic Hypoxia-Ischemia Result in Subtle Alterations in Dendritic Complexity in Medial Frontal Cortical Neurons in Juvenile and Young Adult Rat Offspring in a Pilot Study

Abstract

Prenatal alcohol exposure (PAE) is associated with long-term neurodevelopmental deficits resulting in impaired executive functioning and motor control. Intriguingly, PAE has been linked with an increased risk of transient systemic hypoxia-ischemia (TSHI), which alone results in suboptimal fetal growth and neurodevelopmental consequences. Here, using two translationally relevant preclinical models, we investigated the short-term and lasting effects of PAE and TSHI on the morphology of the medial prefrontal cortex (mPFC), a region important in executive function, and tested whether PAE interacts with TSHI to produce a distinct pattern of injury relative to either condition alone. The four experimental groups included sham (saccharin water, no TSHI), PAE (5% alcohol, no TSHI), TSHI (saccharin water, TSHI), and PAE+TSHI (5% alcohol, TSHI). Brains were extracted for Golgi-Cox staining at Postnatal Day 35 (P35) or P100 and processed for 3D Sholl analysis. The analysis of the mPFC at P35 showed no significant differences in the number of branches or dendritic length overall, although the impact of TSHI compared to alcohol was significant for both. There were no significant differences in the number of Sholl intersections overall at P35, although a sex difference was noted in PAE offspring. At P100, analysis of filament dendritic length and branching number was also significantly impacted by TSHI compared to alcohol. Interestingly, sex was also a significant factor when assessing the impact of alcohol. PAE and TSHI both had an insignificantly increased number of Sholl intersections at P100 compared to the control. The observed changes to dendritic complexity at P100 demonstrate altered neuronal morphology in the mPFC that endure into adulthood. Given the importance of the mPFC in executive functioning, these pilot data provide insight into morphological changes that may contribute to the neurobehavioral deficits observed following exposure to PAE and TSHI and highlight the need for additional investigations into this area.

Keywords: dendrites; placental insufficiency; prefrontal cortex; prenatal alcohol exposure; pyramidal neuron.

Figure 1

The overall experimental design. Rat dams voluntarily drank 0% alcohol for the first two days, 2.5% alcohol on days 3 and 4, and then 5% alcohol thereafter, for 4 h daily for 2 weeks prior to breeding to ensure intake was within expected levels. Rat dams consuming within 1 standard deviation of expected intake were then randomized to consume 5% alcohol or saccharin water during pregnancy. Prenatal alcohol exposure (PAE) occurred from Embryonic Day (E)0 to E18, whereby rat dams drank 5% alcohol or saccharin water for 4 h daily. Transient systemic hypoxia–ischemia (TSHI) or a sham procedure was performed on E19. Births occurred normally on E22 and pups were weaned on Postnatal Day (P)23. On P35 or P100, brain tissue was collected from offspring and stained with Golgi–Cox. Microscopy z-stack images were obtained and Imaris software was used to create a 3-dimensional rendering of the pyramidal neurons in the region of interest, with a Sholl analysis of dendritic complexity conducted.

Figure 2

Isolation of dendritic arbors. After the z-stack imaging was obtained from the medial frontal cortex (noted on the left, shaded), the Imaris software was used to create 3D renderings. Pyramidal neurons were isolated with the “filaments function”, with the soma (blue) and apical dendrite (light gray) specifically noted.

Figure 3

Dendritic morphology at P35. The total filament length was analyzed between treatment group and sex, and treatment group overall. The female TSHI group had the longest total dendritic length (A) as well as the highest amount of total number of branches (B). Tukey’s multiple comparison tests showed a difference between the TSHI and PAE groups for both dendritic length and the total number of branches. * p < 0.05.

Figure 4

Dendritic morphology at P100. The total filament length was analyzed between treatment group and sex, and treatment group overall. The male TSHI group had the longest total dendritic length (A) as well as the highest amount of total number of branches (B). There was a significant difference between the PAE and TSHI groups in the dendritic length and the total number of branches. Sex had a significant impact on the dendritic length and the total number of branches for the PAE group. * p < 0.05.

Figure 5

Sholl analysis in the medial frontal cortex at P35. The Sholl analysis was carried out in the medial frontal cortex region at P35. (A) Sholl analysis was completed on 6 pyramidal neurons per offspring with the number of intersections observed at each specific distance recorded per each treatment group. (B) Representative traces of neurons illustrate differences in branching complexity between treatment groups (scale bar = 50 μm). Although the TSHI group had longer dendritic length and more overall branching, there were no statistical differences found between the four treatment groups.

Figure 6

Sholl analysis in the medial frontal cortex at P100. The Sholl analysis was carried out in the medial frontal cortex (blue) region at P100. (A) Sholl analysis was completed on 6 pyramidal neurons per offspring with the number of intersections observed at each specific distance recorded per each treatment group. (B) Representative traces of neurons illustrate differences in branching complexity between treatment groups (scale bar = 50 μm). There were no statistical differences found between the four treatment groups.