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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 2  |  Issue : 2  |  Page : 60-66

Postsynaptic density-95 expression is increased following neonatal ethanol exposure in wild-type but not adenylyl cyclase 1 and 8 knockout mice


John D. Dingell VA Medical Center; Department of Neurosurgery, Wayne State University School of Medicine, Detroit, MI, USA

Date of Submission23-Dec-2016
Date of Acceptance16-May-2017
Date of Web Publication30-Jun-2017

Correspondence Address:
Alana C Conti
John D. Dingell VA Medical Center, B4250 (11R), 4646 John R St., Detroit, MI 48201
USA
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ed.ed_26_16

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  Abstract 

Context: Fetal alcohol spectrum disorders are a continuum of defects including cognitive and behavioral impairments. Ethanol exposure causes increased apoptosis in striatum of wild-type (WT) mice within 4 h of exposure, an effect that is exacerbated in mice lacking adenylyl cyclases 1/8 (double knockout [DKO]).
Aims: To further understand the effects of neonatal ethanol exposure on striatal neurons, the current study focused on the acute expression of postsynaptic density-95 (PSD-95) and synaptophysin.
Subjects and Methods: WT and DKO mice were treated with a single dose of saline or 2.5 g/kg ethanol at postnatal days 5–7. At various time points after ethanol exposure, striatal tissues were collected for protein and mRNA analysis.
Statistical Analysis: Independent two-way ANOVAs for each time point were performed using SigmaPlot 12.
Results: Genetic deletion of AC1/8 alone significantly increased PSD-95 expression at all time points analyzed compared to saline-treated WT controls. Neonatal ethanol increased PSD-95 protein expression in WT mice 2–6 h after exposure, with no effect in DKO mice. By 24 and 48 h, ethanol exposure had no effect on PSD-95 protein expression in WT mice but resulted in a significant reduction in DKO mice. Neither PSD-95 mRNA nor synaptophysin protein expression was affected by ethanol and/or AC1/8 knockout.
Conclusions: Acute ethanol exposure in WT mice elicits a postsynaptic effect which may be designed to combat the detrimental effects of ethanol exposure. The lack of this acute increase in PSD-95 protein expression in DKO mice may reflect the increased striatal neurodegeneration reported in these mice 4 h after neonatal ethanol treatment compared to WT mice, further evidenced by the delayed reduction in PSD-95 levels 24–48 h postexposure.

Keywords: Adenylyl cyclase, fetal alcohol spectrum disorder, postsynaptic density-95, striatum, synaptophysin


How to cite this article:
Susick LL, Knouna KM, Minwalla AJ, Conti AC. Postsynaptic density-95 expression is increased following neonatal ethanol exposure in wild-type but not adenylyl cyclase 1 and 8 knockout mice. Environ Dis 2017;2:60-6

How to cite this URL:
Susick LL, Knouna KM, Minwalla AJ, Conti AC. Postsynaptic density-95 expression is increased following neonatal ethanol exposure in wild-type but not adenylyl cyclase 1 and 8 knockout mice. Environ Dis [serial online] 2017 [cited 2022 Oct 3];2:60-6. Available from: http://www.environmentmed.org/text.asp?2017/2/2/60/209263


  Introduction Top


Exposure of a developing fetus to alcohol can result in fetal alcohol spectrum disorders (FASDs), a continuum of defects ranging from facial anomalies to cognitive and behavioral impairments, including deficits in executive functioning. The severity of these deficits depends on multiple factors including dose and timing of exposure. The neurotoxic effects of ethanol arise, in part, from the activation of gamma-aminobutyric acid A receptors and inhibition of N-methyl-D-aspartate (NMDA) receptors, (reviewed in the study by Costa et al.)[1] and result in increased cell death and changes in neuron morphology. This has been demonstrated in humans, with many brain regions, including the basal ganglia and caudate, being dramatically reduced in children exposed in utero to alcohol.[2],[3] Similarly, using a rodent model of FASD, exposure to ethanol during the third-trimester equivalent has been shown to increase apoptosis in the striatum of wild-type (WT) mice within 4 h of ethanol injection.[4],[5] In addition, mice lacking adenylyl cyclases (AC) 1/8 (double knockout [DKO]), the only calcium/calmodulin-stimulated AC isoforms in the brain, were more sensitive to the neurodegenerative effects of neonatal ethanol exposure as demonstrated by a greater increase in apoptotic cells found in the striatum compared to WT mice.[4]

Besides an increase in cell death, a reduction in brain volume could also be attributed to a reduction in cell size, which has been demonstrated previously with the finding of simplified dendritic complexity of medium spiny neurons in the caudate putamen of P30 mice [6] and the nucleus accumbens shell of adult rats [7] following neonatal ethanol exposure. However, no significant long-term effects of neonatal ethanol exposure on spine density have been found in the striatum.[6],[7],[8]

In addition to changes in neuron number and morphology, neonatal ethanol exposure may also modulate synaptic function through changes in proteins such as postsynaptic density-95 (PSD-95) and synaptophysin. PSD-95 is a membrane-associated scaffolding protein that regulates NMDA receptor trafficking and clustering and has been associated with synapse strength and spine maturation.[9],[10],[11],[12],[13] Synaptophysin is a synaptic vesicle protein and can play a role in synapse formation and stabilization.[14] To build upon previous studies that investigated dendritic changes following acute neonatal exposure to ethanol,[6] the current study aims to examine the effects of neonatal ethanol exposure on the expression of PSD-95 and synaptophysin in WT and AC1/8 knockout mice. The postnatal injection paradigm used in the previous and current studies was developed to model third-trimester neonatal exposure.[15],[16],[17] Using this model, blood ethanol concentrations reached 276 ± 5 mg/dL and 262 ± 9 mg/dL 15 min following injection, in WT and DKO pups, respectively.[6]


  Subjects And Methods Top


Animals

All mice were backcrossed a minimum of ten generations to WT C57BL/6J mice from the Jackson Laboratory (Bar Harbor, ME). To generate mice for these experiments, homozygous mutants, DKO, and WT mice were bred in our colony. Mice were maintained on a 12 h light/dark schedule with ad libitum access to food and water. All experiments were performed using male mice 5–7 days after birth (P5–P7) weighing 2.5–3.0 g.[4],[5],[6],[18],[19] Only litters with an even number of male pups in this weight range were used to ensure that a littermate was available for use as a saline-treated control. Only two pups from a litter, one saline and one ethanol exposed, were assigned to a time point, i.e. if four males from a single litter were available for treatment, they were used for two different time points. Pups were randomly assigned to either saline or ethanol treatment groups and any pups that did not survive to the selected time point were excluded from the analysis. A minimum of three animals per genotype per treatment for immunoblotting was determined to be required to achieve a sample size for >88% power (minimum power function = 0.88 and α=0.05) with significance level set at P ≤ 0.05, based on a power analysis (SigmaPlot 12) using a minimum detectable difference of 0.7, which was obtained from preliminary data conducted in the Conti laboratory and pertinent published literature for comparable studies in which desired effect sizes were shown to be significant. All mouse protocols were in accordance with the National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee at Wayne State University. Animals were assigned a numerical code at the time of treatment; however, samples were decoded before loading samples into the electrophoresis gels.

Ethanol treatment

WT and DKO pups were injected subcutaneously [4],[5],[6],[18],[19] with a single dose of ethanol (2.5 g/kg) prepared as a 20% solution using 100% ethanol (Decon Laboratories, King of Prussia, PA, USA) in normal saline at P5–P7. Littermate male pups were injected with corresponding volumes of saline as controls. Both ethanol- and saline-treated pups were placed on a heating pad at 31°C [4],[5],[6],[18],[19] away from dams until the ethanol pups regained consciousness, at which time all pups were returned to their dams. Time away from dams was limited to <3 h to minimize maternal separation and nutritional effects. Pups were euthanized 2, 4, 6, 24, or 48 h after injection and brains were removed. The striatum was removed, flash frozen in liquid nitrogen, and stored at −80°C until use.

Reverse transcription polymerase chain reaction

Striatal tissues were processed using the GenElute Mammalian total RNA Miniprep kit (Sigma–Aldrich, St. Louis, MO, USA), by homogenizing the cells with an electric pestle in the provided Lysis Solution with 2-mercaptoethanol. The homogenized tissue was filtered using the provided columns and centrifuged at 12,000 × g. Next, 500 μL of 70% ethanol was mixed with the lysate and this mixture was added to the GenElute binding column and centrifuged at 12,000 × g for 15 s. The column was washed with 500 μL of wash solution 1 and centrifuged at 12,000 × g for 15 s. For the second wash, 500 μL of wash solution 2, diluted with ethanol, was added to the column and centrifuged at 12,000 × g for 15 s. An additional 500 μL of wash solution 2 was added to the column, and it was centrifuged at 12,000 × g for 2 min. RNA was eluted by adding 50 μL of elution solution into the binding column and centrifuging for 1 min at 12,000 × g. Eluted RNA was stored at −80°C until use. Quantification of RNA was determined by measuring the absorbance of 2 μL of RNA in 98 μL of water at 260 nm and 280 nm in quartz cuvettes. Following reconstitution of the RNA, first-strand cDNA was produced using the SuperScript III First-Strand Synthesis System (Life Technologies, Grand Island, NY, USA). In brief, 2.5 μM oligo DTs, 2 μg sample RNA, and 0.5 μM dNTPs were incubated at 65°C for 5 min to allow annealing. cDNA synthesis mix was prepared as per the manufacturer's instructions and was added to the RNA, which was then incubated at 50°C for 50 min, followed by incubation at 85°C to terminate the reaction. This cDNA was used for reverse transcription polymerase chain reaction (RT-PCR) using SYBR green (Life Technologies, Grand Island, NY, USA) with primers for PSD-95 (forward 5'-GGCACCGACTACCCCACAG-3', reverse 5'-AACACCATTGACCGACAGGA-3') or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (forward 5'-CCAGTGAGCTTCCCGTTCA, reverse 5'-GAACATCATCCCTGCATCCA-3').[20] The PCR was performed in an ABI7500 Prism (Applied Biosystems) quantitative real-time-PCR cycler, which yielded optimal conditions of one cycle at 95°C for 10 min followed by forty cycles of 95°C for 15 s and then 60°C for 1 min. Primer concentrations were optimized and used at the following concentrations: 0.9 μM PSD-95 forward, 0.9 μM PSD-95 reverse; 0.2 μM GAPDH Forward, 0.2 μM GAPDH reverse. A dissociation curve was generated and PSD-95 mRNA expression was normalized to GAPDH mRNA for each sample run in triplicate. Data were analyzed using the comparative cycle threshold (Ct) method, as previously described.[21] For each replicate, Ct, defined as the fractional cycle number at which the fluorescence passes a fixed threshold (based on the baseline data collected in the first 15 cycles), was calculated. Therefore, for example, for each sample, an average Ct for PSD-95 (CtPSD-95) and GAPDH (CtGAPDH) was obtained. Samples were normalized against GAPDH using the following equation: CtGAPDH-PSD-95 = mean (CtGAPDH) − mean (CtPSD-95).

ΔCtGAPDH-PSD-95 was calculated for each experimental group, and differences between groups were calculated using the following equation as an example:

CtPSD-95 = control (CtGAPDH-PSD-95) − experimental group (CtGAPDH-PSD-95), where ΔΔCtPSD-95 represents the difference in PSD-95 mRNA expression between WT saline (control) and WT ethanol, DKO saline, or DKO ethanol. The magnitude of difference in PSD-95 gene expression is equal to 2 ΔΔCtPSD-95. Relative PSD-95 mRNA levels were normalized to GAPDH and plotted relative to expression in control mice, which equals 1.

Immunoblotting

Whole cell extracts from striatal tissues were prepared by homogenization in lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid [EDTA], 1% Triton X-100, 10% glycerol with protease and phosphatase inhibitors) and centrifuged (10,000 × g) for 10 min at 4°C. Protein (10 μg) was separated on a 4%–12% Bis-Tris gel using MOPS running buffer (Invitrogen, Grand Island, NY, USA). Proteins were transferred to a nitrocellulose membrane using a semi-dry transfer apparatus with NUPAGE transfer buffer at 15V for 40 min (Invitrogen, Grand Island, NY, USA) and exposed to antibodies against PSD-95 (9272; Cell Signal, Danvers, MA), synaptophysin (5461; Cell Signal, Danvers, MA), or actin (A5060 Sigma-Aldrich) at 1:5000 in blocking buffer, 5% dry milk in Tris-buffered saline with 0.1% Tween 20, overnight at 4°C. Membranes were incubated in secondary antibodies against mouse (1:5000; PSD-95) or rabbit (1:5000; Synaptophysin, 1:4000; actin) for 1 h following washing in Tris-buffered saline with 0.1% Tween 20. Immunoblot signals of PSD-95, synaptophysin, and actin were detected using SuperSignal West Dura Chemiluminescent substrate (ThermoScientific, Rockford, IL, USA) were quantified using densitometric analysis with Image J software (NIH [22]) and are presented as a ratio of PSD-95/actin or synaptophysin/actin averaged within groups. The membrane was stripped between PSD-95, synaptophysin, or actin analysis; therefore, protein bands were cut from separate images and lined up for figure preparation only.

Statistical analysis

Five independent two-way ANOVAs, one for each time point, were performed for each protein or mRNA ratio using SigmaPlot 12 (Systat Software, Inc, San Jose, CA, USA) with Genotype and Treatment as the between-subjects factors for immunoblot data of PSD-95/actin and synaptophysin/actin or 2 ΔΔCtPSD-95 values for PSD-95 mRNA expression. Tukey's post hoc tests were performed where appropriate.


  Results Top


Considering the rapid striatal cell loss and delayed simplification of dendritic morphology observed following acute ethanol exposure,[4],[6] studies to quantify the synaptic proteins, PSD-95, and synaptophysin were conducted. PSD-95 is a postsynaptic protein that anchors NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors to the postsynaptic membrane and influences spine maturation and synapse strength.[9],[10],[11],[12],[13] Synaptophysin is a synaptic vesicle protein that is involved in synapse formation and synaptic transmission.[14],[23] Analysis of striatal tissue from saline or ethanol-treated WT and DKO pups for PSD-95 expression using immunoblotting revealed a significant interaction of Genotype and Treatment at all time points examined (2 h [F (1, 11) = 11.02, P = 0.011], 4 h [F (1, 11) = 6.17, P = 0.038], 6 h [F (1, 12) = 13.39, P = 0.005], 24 h [F (1, 11) = 8.26, P = 0.021], and 48 h [F (1, 11) = 16.92, P = 0.003]). Post hoc analyses demonstrated a significant increase in PSD-95 expression in saline-treated DKO mice compared to saline-treated WT mice at all time points examined [Figure 1] and [Figure 2]. PSD-95 expression was also significantly increased 2, 4, and 6 h after neonatal ethanol exposure in WT mice compared to saline-treated WT mice [Figure 1]. Interestingly, 24 and 48 h after ethanol exposure, no significant effect of ethanol treatment was found in the WT mice, but a significant reduction in PSD-95 expression was demonstrated in the DKO animals [Figure 2]. Significant effects of Genotype at 4 and 24 h were found with DKO mice having higher PSD-95 expression when both treatment groups were combined (4 h [F (1, 11) = 13.42, P = 0.006]; 24 h [F (1, 11) = 9.28, P = 0.016]). At 24 h, this increase is driven by the saline-treated DKO group being significantly elevated. In addition, significant effects of treatment at 2, 24, and 48 h were also found, revealing a significant increase in PSD-95 expression in ethanol-exposed mice compared to saline-treated mice when both genotypes were combined (24 h [F (1, 11) = 6.95, P = 0.030]; 48 h [F (1, 11) = 16.92, P = 0.010]). Both of these differences were also driven by the significant elevation in PSD-95 expression found in the saline-treated DKO mice. No significant effects of genotype or treatment were found at any other time point (genotype 2 h [F (1, 11) = 2.78, P = 0.134]; 6 h [F (1, 12) = 3.29, P = 0.103]; 48 h [F (1, 11) = 1.46, P = 0.261]; treatment 2 h [F (1, 11) = 5.14, P = 0.053]; 4 h [F (1, 11) = 3.30, P = 0.107]; 6 h [F (1, 12) = 2.48, P = 0.150]).
Figure 1: Postnatal ethanol exposure acutely increased postsynaptic density-95 expression in wild-type but not double-knockout mice. (a) Immunoblot images and (b) quantification of postsynaptic density-95 and actin demonstrated that postsynaptic density-95 expression was increased 2, 4, and 6 h after neonatal ethanol exposure in wild-type mice compared to saline-treated wild-type mice. Postsynaptic density-95 expression was also increased in saline-treated double-knockout mice compared to saline-treated wild-type mice at the 2, 4, and 6 h time points. No effect of ethanol exposure was found in double-knockout mice compared to saline-treated double-knockout controls. Mean ± standard error of mean, n = 3 per group. *Compared to respective saline control P < 0.05, #Compared to wild type saline P < 0.05, Main effect of Genotype P < 0.05

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Figure 2: Postsynaptic density-95 expression is decreased in double-knockout mice 24 and 48 h after neonatal ethanol exposure. (a) Immunoblot images and (b) quantification revealed that genetic deletion of adenylyl cyclases 1/8 alone increased expression of postsynaptic density-95 compared to saline-treated wild-type mice at the 24 and 48 h time points. Neonatal ethanol exposure reduced postsynaptic density-95 expression in double-knockout mice 24 and 48 h after exposure compared to saline-treated double-knockout mice with no effect in wild-type mice. Mean ± standard error of mean, n = 3 per group. *Compared to respective saline P < 0.05, #Compared to wild type saline P < 0.05, Main effect of Genotype P < 0.05, ¥Main effect of treatment P < 0.05

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Striatal tissue from WT and DKO mice treated neonatally with saline or ethanol were also assayed for PSD-95 mRNA levels using quantitative RT-PCR. As shown in [Figure 3], a slight decrease in PSD-95 mRNA was found at the 2 h time point, but this failed to reach significance. A small increase in PSD-95 mRNA expression was also found at the 24 and 48 h time points in DKO animals treated with ethanol neonatally, but this also failed to reach significance [Figure 3].
Figure 3: Postsynaptic density-95 mRNA expression was not affected by either genetic deletion of adenylyl cyclases 1/8 or neonatal ethanol exposure. Quantification of postsynaptic density-95 gene expression demonstrates no changes following neonatal ethanol exposure in wild-type or double-knockout mice at any time points. Relative postsynaptic density-95 mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase and plotted relative to expression in control mice, which equals 1. Mean ± standard error of mean, n = 3 per group

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As shown in [Figure 4], no effects of neonatal ethanol exposure on synaptophysin expression were found in either genotype, as analyzed by immunoblotting. In addition, no effect of genetic deletion of AC1/8 was found at any time point examined.
Figure 4: Neither genetic deletion of adenylyl cyclases 1/8 nor neonatal ethanol exposure had an effect on the expression of synaptophysin. (a and c) Immunoblot analysis and (b and d) quantification of synaptophysin expression does not display changes following neonatal ethanol exposure in wild-type or double-knockout mice at any time points. Mean ± standard error of mean, n = 3 per group

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  Discussion Top


In the current study, we demonstrated an early increase in PSD-95 protein expression in WT mice 2, 4, and 6 h after neonatal ethanol exposure. This upregulation of PSD-95 could be a compensatory response to the inhibition of NMDA receptors by ethanol to traffic more receptors to the cell membrane and restore NMDA signaling. Alternatively, previous work in hippocampal cultures has demonstrated that NMDA receptor activation resulted in PSD-95 ubiquitination and degradation in as little as 15 min; therefore, ethanol-induced inhibition of NMDA activity could increase PSD-95 expression by attenuating the degradation of PSD-95.[24] This reduction in PSD-95 expression by NMDA activation was inhibited by forskolin, an adenylyl cyclase activator, suggesting a role for the ACs in PSD-95 regulation.[24] This hypothesis is further supported by the lack of effect of neonatal ethanol exposure found on PSD-95 gene expression at any time point tested. To our knowledge, this is the first study to measure PSD-95 mRNA following neonatal ethanol exposure, thus adding to the novelty of this study. Interestingly, an increase in PSD-95 protein expression following neonatal ethanol exposure was not observed in DKO mice at 2, 4, or 6 h, which could represent a ceiling effect, as the baseline PSD-95 levels in saline-treated DKO animals is elevated, preventing ethanol from increasing the protein levels further.

At both 24 and 48 h after ethanol exposure, PSD-95 protein expression in ethanol-exposed WT mice was similar to that observed in the saline-treated WT group. These results are comparable to previous spine density measurements which demonstrated no difference between the saline- and ethanol-exposed WT groups at P21,[8] P30[6] or in adults.[7] Interestingly, saline-treated DKO mice continue to exhibit an increase in PSD-95 expression at the 24 and 48 h time points compared to saline-exposed WT mice. This increase does not seem to impact spine density, which demonstrated a reduction in DKO animals compared to saline-treated WT controls.[6] It should be noted that this difference could be due to the differences in age as the 48 h time point in the current study corresponds to an age of P7–P9, whereas spine density was measured at P30.

A recent study by Kim et al. demonstrated an increase in PSD-95 protein expression in the cortex and hippocampus but not striatum of rats following gestation exposure of 2 or 4 g/kg ethanol via intragastric intubation from gestational day 7 to 16.[25] A likely explanation for this lack of effect of ethanol in the striatum is the timing of ethanol exposure. The period of vulnerability to ethanol for the striatum largely occurs postnatally.[26],[27] Contrary to the current findings, other studies have failed to find a significant effect of neonatal ethanol exposure on PSD-95 protein expression. For example, exposure to a 36% ethanol-derived calorie liquid diet throughout gestation had no effect on PSD-95 protein expression in the forebrain at P1 in rats.[28] Similarly, 6 g/kg ethanol administered by intragastric intubations from gestational day 7 through twenty had no effect on PSD-95 protein expression in the CA1, CA3, or dentate gyrus regions of the hippocampus at P1, P10, P30, or P60 in rats.[29] Another study also found no effect of gestational ethanol exposure using a two-bottle choice paradigm in hippocampal tissue from adult mice.[30] Each of these studies employed gestational ethanol exposure paradigms, whereas in the current study, animals were treated postnatally to model third-trimester exposure. This underscores the specificity of outcomes based on route of administration, timing of exposure, and brain region examined.

Interestingly, the acute PSD-95 protein expression levels are inversely related to a previous Sholl analysis, a measure of dendritic complexity, from medium spiny neurons in the caudate putamen at P30. In our previous study, a single injection of 2.5 g/kg of ethanol administered subcutaneously at P5–P7 decreased the number of Sholl intersections in ethanol-exposed WT mice compared to saline-treated WT mice. In addition, Sholl intersections were also decreased in saline-treated DKO mice compared to saline-treated WT mice. No further effect of ethanol was found in the DKO mice.[6]

It should be noted, however, that previous studies have failed to demonstrate an effect of neonatal ethanol exposure on NMDA receptor-mediated spontaneous excitatory postsynaptic currents in hippocampal and cortical neurons.[31],[32] This is postulated to be caused by the subunit composition of NMDA receptors, which changes during development. The GluN2B subunit has been shown to be predominantly expressed at P4–P10, with a gradual increase in the expression of GluN2A occurring from P9–P20[31] and reviewed in.[33] The GluN2A subunit is thought to confer sensitivity to ethanol since ethanol exposure resulted in the internalization of NMDA receptors containing GluN2A but not GluN2B subunits.[34]

In addition, ethanol does not directly affect AC activity;[18],[35] however, both adult and neonatal mice lacking AC1/8 are more sensitive to the effects of ethanol as demonstrated by an increase in sedation in adult animals and an increase in neurodegeneration in neonates following ethanol exposure.[4],[18],[36] Therefore, further investigation into the regulation of the ACs following neonatal ethanol exposure is necessary.

In this study, we found no changes in synaptophysin protein expression following ethanol exposure in WT mice at any time point examined. Synaptic input to the striatum largely originates in the cortex and therefore is the major source of synaptophysin in the striatum. In addition, striatal neurons in culture remain largely aspiny without co-culture with cortical neurons,[37] indicating an important role of cortical neurons in striatal synapse formation. Previous studies have demonstrated neurodegeneration in the cortex 8–24 h after neonatal ethanol exposure;[5],[15],[16],[38] therefore, we expected to find a reduction in synaptophysin expression in the striatum. However, other studies have also failed to find alterations in synaptophysin protein expression, for example, a study by Elibol-Can et al. found no effect of 6 g/kg ethanol administered by intragastric intubations from gestational day 7 through 20 on synaptophysin protein expression in the hippocampus at P1, P10, P30, or P60.[29] Similarly, a voluntary consumption paradigm with ethanol intake of over 9 g/kg/day throughout gestation failed to produce changes in synaptophysin protein levels in the frontal cortex or hippocampus of 4–5-month-old rats.[39] It should be noted that hippocampal neurons have been shown to be vulnerable to ethanol both pre- and post natally.[26] We also found no change in synaptophysin protein expression in the DKO animals, which is surprising in light of a previous study demonstrating a reduction in spine density that was seen in these mice at P30.[6] Again, the difference in age of animals used in these two studies could account for the differences in these results.

PSD-95 has been suggested to aid in the association of proteins in the PSD including NMDA receptors and downstream signaling molecules; therefore, the acute increase in PSD-95 protein expression demonstrated here could represent a synaptic response to acute neonatal ethanol exposure in WT mice designed to combat the detrimental effects of ethanol exposure. Furthermore, a lack of an acute increase in PSD-95 protein expression in DKO mice may reflect the increased neurodegeneration reported in the striatum 4 h after neonatal ethanol treatment compared to WT mice. The delayed reduction in PSD-95 protein levels 24–48 h postethanol exposure could be an exaggerated response to a reduction in cortical innervation caused by the neurodegeneration of the cortex 8–24 h after neonatal ethanol.[5],[15],[16],[38] These observed alterations in PSD-95 protein expression, paired with the lack of effect in synaptophysin, indicate a uniquely postsynaptic effect resulting from neonatal ethanol exposure. As these time points represent the acute postexposure period, future studies will explore additional postsynaptic markers that may also be affected by early-life ethanol treatment.

Financial support and sponsorship

These studies were supported with resources and the use of facilities at the John D. Dingell VA Medical Center, Detroit, MI and by funds from WSU Department of Neurosurgery (ACC) and National Institute on Alcohol Abuse and Alcoholism (NIAAA) grants F32 AA020435 (LLS) and K01 AA017683 (ACC).

Conflicts of interest

There are no conflicts of interest.

 
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