Acute Ethanol Exposure during Synaptogenesis Rapidly Alters Medium Spiny Neuron Morphology and Synaptic Protein Expression in the Dorsal Striatum

Abstract

Fetal alcohol spectrum disorders are caused by the disruption of normal brain development in utero. The severity and range of symptoms is dictated by both the dosage and timing of ethanol administration, and the resulting developmental processes that are impacted. In order to investigate the effects of an acute, high-dose intoxication event on the development of medium spiny neurons (MSNs) in the striatum, mice were injected with ethanol on P6, and neuronal morphology was assessed after 24 h, or at 1 month or 5 months of age. Data indicate an immediate increase in MSN dendritic length and branching, a rapid decrease in spine number, and increased levels of the synaptic protein PSD-95 as a consequence of this neonatal exposure to ethanol, but these differences do not persist into adulthood. These results demonstrate a rapid neuronal response to ethanol exposure and characterize the dynamic nature of neuronal architecture in the MSNs. Although differences in neuronal branching and spine density induced by ethanol resolve with time, early changes in the caudate/putamen region have a potential impact on the execution of complex motor skills, as well as aspects of long-term learning and addictive behavior.

Keywords: cognitive deficits; dendritic morphology; environmental chemicals; fetal alcohol spectrum disorder; maternal exposure; striatum.

Figure 1

Golgi-stained MSNs in the caudate/putamen. Representative Neurolucida tracing of medium spiny neurons from a saline-treated animal (A) and an ethanol-treated animal (B), demonstrating the difference in observed branch patterns at 24 h. The scale bar in the bottom right corner of the image represents 10 μm.

Figure 2

Ethanol treatment during synaptogenesis significantly increases MSN dendritic length and number of nodes after 24 h. (A–C). Dendritic number was significantly greater in ethanol-treated animals 1 month after exposure (B); p = 0.002), and it trended towards significance at 24 h (A); p = 0.065). There was not enough statistical power to conclude that ethanol did not affect dendrite number at 5 months of age (C); Power: 0.717). (D–F). Dendritic length was significantly greater in ethanol-treated animals 24 h after exposure ((D); p = 0.027), there was not enough statistical power to determine the effects at 1 month (E); Power: 0.3805), and there was no significant difference at 5 months of age (F); p = 0.384). (G–I). Dendritic node number was significantly greater in ethanol-treated animals 24 h after exposure (G); p = 0.018), but the number of nodes was not significantly different at 1 month (H); p = 0.868). There was not enough statistical power to conclude there was no significant node difference at 5 months of age (I); Power: 0.6472). (J–L). The small sample and effect size provides insufficient statistical power to determine if there is an effect of ethanol on soma size. For all experiments, between 6 and 8 medium spiny neurons in the caudate-putamen region of the striatum were traced in 3–5 animals at each treatment level (n = 4 saline and n = 4 ethanol at 24 h; n = 3 saline and 3 ethanol at 1 month; n = 3 saline and n = 5 ethanol at 5 months). For all panels, error bars represent SEM values. *: significant at p < 0.05.

Figure 3

Acute high-dose ethanol administration during synaptogenesis significantly affects dendritic spine number. (A–C). Twenty-four hours post-ethanol administration: (A). number of dendritic spines was significantly decreased in ethanol-treated animals 24 h after exposure (p = 0.002), but there was not sufficient statistical power to determine if there was an effect on spine length (B); Power: 0.744) or spine type (C); power: 0.7003). (D–F). One-month post-ethanol administration: (D). number of dendritic spines was significantly increased in ethanol-treated animals 1 month after exposure (p = 0.030), but there was no significant effect of treatment on spine length (E); p = 0.944). There was not sufficient statistical power to determine if there was an effect on spine type (F); Power: 0.4577). (G–I). Five months post-ethanol administration: No significant differences were detected between ethanol and saline groups 5 months after treatment for spine number (G), spine length (H), or spine type (I), but there was insufficient statistical power to determine the impact of ethanol treatment (power: 0.4496, 0.4577, and 0.5183, respectively). For spine analysis, 3–5 animals were used in each treatment group (n = 5 saline and 4 ethanol at 24 h; n = 3 saline and n = 3 ethanol at 1 month; n = 3 saline and n = 4 ethanol at 5 months). *: significant at p < 0.05.

Figure 4

Protein analysis in the striatum of mice 24 h following saline or ethanol administration. (A). Fractalkine levels are not affected by ethanol treatment (p = 0.477). (B). Hsp-70 is increased in response to ethanol treatment, but this difference is not significant (p = 0.084). (C). Beta-tubulin levels are not affected by ethanol treatment (p = 0.281). (D). PSD-95 is increased in response to ethanol-treatment (p = 0.033). (E). Western blot analysis of PSD-95 protein in mouse striatum, normalized with beta-tubulin (n = 6 mice for both ethanol and saline samples). (F). Representative Western blot of PSD-95 and tubulin proteins from mouse striatal tissue. Each lane represents a striatal sample from a different mouse. For all panels, error bars represent SEM values. *: p < 0.05.

Figure 5

Ethanol treatment increases the level of PSD-95 found in the striatum 24 h after exposure. (A–F). Immunofluorescence images taken at 400× magnification; images left to right of DAPI, PSD-95, and FoxP1 staining. DAPI labels nuclei, and FoxP1 is a marker for MSNs. (A–C) depict ethanol-treated animals, while D–F depict saline-treated animals (Scale bar = 50 µm). (G–H) depict examples of PSD-95 immunofluorescence images used for counting puncta. (I). Quantification of PSD-95 puncta in immunofluorescence images (461.46 ± 58.38 saline, 984.17 ± 10.0 ethanol, *: p = 0.010).