Single-day Postnatal Alcohol Exposure Induces Apoptotic Cell Death and Causes long-term Neuron Loss in Rodent Thalamic Nucleus Reuniens

Abstract

Fetal alcohol spectrum disorders (FASD) constitute a prevalent, yet preventable, developmental disorder worldwide. While a wealth of research demonstrates that altered function of hippocampus (HPC) and prefrontal cortex may underlie behavioral impairments in FASD, only one published paper to date has examined the impact of developmental alcohol exposure (AE) on the region responsible for coordinated prefrontal-hippocampal activity: thalamic nucleus reuniens (Re). In the current study, we used a rodent model of human third trimester AE to examine both the acute and lasting impact of a single-day AE on Re. We administered 5.25 g/kg of ethanol to male and female Long Evans rat pups on postnatal day (PD) 7. We used unbiased stereological estimation to evaluate cell death or cell loss at three time points: 12 h after alcohol administration; 4 days after alcohol administration (i.e., PD11); in adulthood (i.e.,PD 72). AE on PD7 increased apoptotic cell death in Re on PD7, and caused short-term cell loss on PD11. This relationship between short-term cell death versus cell number suggests that alcohol-related cell loss is driven by induction of apoptosis. In adulthood, alcohol-exposed animals displayed permanent cell loss (mediating volume loss in the Re), which included a reduction in neuron number (relative to procedural controls). Both procedural controls and alcohol exposed animals displayed a deficit in non-neuronal cell number relative to typically-developing controls, suggesting that Re cell populations may be vulnerable to early life stress as well as AE in an insult- and cell type-dependent manner.

Keywords: fetal alcohol spectrum disorders; histology; immunocytochemistry; neuroanatomy; stereology.

Figure 1:. Experimental timeline of all animals and tissue generated.

Rat pups for all time points were injected with trace amounts of black India ink for identification on postnatal day (PD) 3. Alcohol exposed pups (AE) and sham intubated (SI) control pups had blood drawn on PD7, 90 minutes after the second dose of ethanol. Suckle control (SC) pups were weighed on PD7, then left undisturbed until either tissue extraction (PD7 and PD11) or weaning (PD72). All pups for sacrifice at PD72 were weaned on PD23.

Figure 2:. Alcohol exposure (AE) on postnatal day 7 increases apoptotic cell death in thalamic nucleus reuniens.

A representative outline of thalamic nucleus reuniens (Re) can be found in panel 2A (3V indicates the third ventricle; scale bar = 1mm). Representative photomicrographs from a sham intubated (SI) and an alcohol-exposed (AE) animal (panels 2B and 2C, respectively) demonstrate representative quantification of apoptotic cells based on clustering of apoptotic bodies within tissue (scale bars = 10 μm). Each dotted contour would have been identified as 1 apoptotic cell by the experimenters Only AE females (solid bars) had altered volume of Re relative to any other group (panel 2D; males represented by striped bars). SI did not alter apoptotic cell death, while AE increased apoptosis approximately 30-fold (panel 2E). All data in this figure are separated by sex due to the sex-by-treatment interaction observed in panel 2E. Graphs present raw data points (in grey) with black bars representing mean ± SEM for that group. Groups with similar letters over them indicate that those groups did not significantly differ.

Figure 3:. Alcohol exposure (AE) on postnatal day 7 causes cell loss in thalamic nucleus reuniens in postnatal day 11 rat brain.

A representative image of cresyl violet-stained tissue (panel 3A; scale bar = 1mm) demonstrates a typical contour of reuniens. Due to the absence of sex differences or sex-based interactions (p’s > 0.480), data in 3B are collapsed across sex. AE caused a significant reduction in cell number at 4 days post-AE (p = 0.011). There was no significant impact of SI (p = 0.714). Graphs present raw data points (in grey) with black bars representing mean ± SEM for that group. Groups with similar letters over them indicate that those groups did not significantly differ.

Figure 4:. Alcohol exposure (AE) on postnatal day 7 results in permanent neuron loss in thalamic nucleus reuniens, while intubation independently reduces non-neuronal cell number relative to typically developing control animals.

A representative image of cresyl violet-stained tissue (panel 4A; scale bar = 1mm) demonstrates a typical contour of reuniens. Due to the absence of sex differences or sex-based interactions (p’s > 0.131), data are collapsed across sex. Significant volume and cell loss was observed in Re of AE animals (panel 4B). Less severe cell loss was also observed in sham intubated (SI) animals (4B, right). Follow-up analysis with NeuN-labeled tissue (panel 4C; counterstained with Pyronin Y; scale bar = 1mm) revealed that alcohol uniquely reduced neuron number in Re (panel 4D, left) while intubation reduced non-neuronal cell number in both the SI and AE groups (panel 4D, right). Graphs present raw data points (in grey) with black bars representing mean ± SEM for that group. Groups with similar letters over them indicate that those groups did not significantly differ.

Figure 5:. Alcohol exposure on Postnatal day 7 decreases volume of thalamic nucleus reuniens (Re) through cell loss.

Raw data points for volume and total cell number estimates for each animal (5A). Red circles indicate SC animals, green triangles indicate SI animals, and hollow blue boxes indicate AE animals. Mediation analysis revealed that AE was associated with a significant reduction in Re volume (5B; p = 0.007). This reduction in Re volume was mediated by a reduction in cell number (p < 0.001), rather than a direct influence of AE on volume (p = 0.464, value in parentheses in diagram). Values in 5B are raw (non-standardized) coefficient values from mediation analysis, and are measured in either total cell number (for “Re Cell #”) or mm³ (for “Re Volume”), † not significant, ** p < 0.010, *** p < 0.001