Introduction

It is proved that multifaceted consequences have been observed in early development due to the use of ethanol during pregnancy [1] and most extensive outcome of prenatal ethanol exposure causes behavioral changes [2, 3]. Several clinical investigations showed that exposure of in utero ethanol exhibits hyperlocomotion particularly in boys, which diagnosed after few years of birth and this deficit may increase in severity during the adult stage [4–6]. Similarly, animal models studies also showed that upon prenatal ethanol the behavioral abnormality was observed in developing fetus after birth [7–9]. Moreover, the prenatal ethanol exposure also affects the pre- and postsynaptic DA receptors and DA receptor-mediated behaviors [10]. Although there is a substantial amount of data concerning the morphological and behavioral effects linked with prenatal ethanol exposure [11], still the mechanisms underlying developmental defects caused by maternal ethanol consumption remain unclear.

Dopamine (DA) a major neurotransmitter in the brain involved in the controlling abnormal neuronal excitability in the kindling and in neuroplasticity linked with this phenomenon that alteration in the function of the dopaminergic system [12–14]. Previous studies on human and animal models suggested that DA plays an important role and any dysfunctions of the DA in cortical region may lead to impaired attention deficit/hyperactivity disorder [8, 15, 16].

Evidence indicates the importance of DA activation and behaviors for ethanol reinforcement and previously it was explained by a large number of groups [17], and the effects of DA are regulated by a family of G protein-coupled DA receptor subtypes, which include DA D1–D5 receptor and these are the most abundant in human cortex [18, 19]. Recently, we have studied the decreased GABAB1R expression and increased neuronal cell death in developing rat brain upon PTZ-induced seizure [20].

There are many potential mechanisms by which neurons may die after seizures and most important is the induction of apoptosis. We have also studied that maternal ethanol exposure and PTZ-induced seizure significantly decreased GABAB1R expression and increased neuronal cell death in prenatal rat brain may lead to the higher risk of developmental disturbances and malformations [21]. Moreover, ethanol exposure modulates the expression of GABABR, which may further lead to the modulation of DAD1R activitiesthat might have important influence on the underlying cause of ethanol’s effects [22].

It is well known that receptors undergo adaptive up- or downregulation following alterations in neuronal activity. The present study was designed to further characterize the role of D1R upon exposure of ethanol and PTZ in prenatal rat cortical and hippocampal neurons. For this purpose, the expressional changes in D1R were observed upon ethanol and PTZ exposure in prenatal rat neurons. Our results showed significant decreased D1R expression and increased expression of pro-apoptotic Bax, Bak and decreased anti-apoptotic protein Bcl-2 expression, further leading to the apoptosis by significant increase in expression of cleaved caspase-3 in prenatal rat neuronal cell cultures. This neuronal apoptosis and downregulation of D1R explain the possibility of higher risk of developmental disturbances and malformations during early developmental stage.

Methodology

Animal treatment

Female (n = 10) Sprague–Dawley rats 250 g were housed in a temperature-controlled environment with lights from 06:00 to 20:00 hours with food ad libitum. Timed pregnant [the day of insemination equals] to gestational days (GD) 0.5. After GD 17.5 pregnant female rat was killed by decapitation, after an i.v. injection of pentobarbital sodium (3 mg/100 g bw).

Primary cell culture and drug treatment

Cultures were prepared from the cortical and hippocampal areas from prenatal rats at GD 17.5 as previously explained by [21]. Briefly, pooled hippocampal and cortical tissues were treated with 0.25 % trypsin-EDTA for 20 min and dissociated by mechanical trituration in ice-cold calcium- and magnesium-free Hank’s balanced salt solution (pH 7.4). After 3 days, cortical and hippocampal neuronal cells were treated with normal media as control and media contain ethanol 100, PTZ 15, 100 mM ethanol plus 15 mM PTZ in different groups and combinations. All drug-treated groups were incubated for 1 h in vitro culture.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

RT-PCR analysis was performed using cDNA from control and drug-treated neuronal culture. Total RNA was isolated with Trizol reagent (Life Technologies, Rockville, MD). First strand cDNA was transcribed from 2 µg of RNA using oligo (dt) 15, M-MLV reverse transcriptase (Promega), following the protocol provided by the company. A total of 4 µl of cDNA was used for PCR amplification in presence of 1 µl Taq DNA polymerase. Thermal cycling was performed under the following conditions: 94 °C for (5 min), 30 cycles at 94 °C (1 min) D1R (53 °C, 28 cycles) for (1 min), and 72 °C for (1 min) followed by 72 °C (5 min) for the final extension. As a negative control GAPDH (58 °C, 25 cycle) was performed. PCR products were run on a 1 % agarose gel containing ethidium bromide and viewed under UV light. The primers used were the following: D1R forward 5-ATTCTTCCCTGAACCCCATT-3, D1R reverse: 5-GTGGAATGCTGTCC ACTGTG3′, GAPDH forward: 5-GCCATCAATGACCCCTTCATT-3, GAPDH reverse: 5-CGCCTGCTTCAC CACCTTCTT-3.

Western blotting

Western blot analysis was followed as previously [21]. Briefly, primary cultured cortical and hippocampal cells were homogenized in cell lysis buffer with protease inhibitor 100 mM PMSF. After ultra centrifugation (12,000g, 10 min X 2), the protein containing supernatant was separated. The protein content was measured by spectrophotometer at 595 nm using the Bio-Rad (Hercules, CA) protein assay and 30 µg proteins were applied per lane. Immunoreactions were carried out using a rabbit polyclonal IgG D1R, cleaved caspase-3, Bax, Bcl-2 antibodies (1:1,000, 24 h, 48 °C, Santa Cruz, Cell signaling) or rabbit-derived anti-rat D1R, cleaved caspase-3, Bax, Bak, Bcl-2 antibodies (1:1,000, Abcam Limited, UK). Following rinses, horseradish peroxidase conjugated goat anti-rabbit IgG-HRP (1:10,000, Bio-Rad) was added for 2 h at room temperature. Proteins were detected by chemiluminescence using an ECL-detecting reagent according to their protocol. Western blots were analyzed by densitometry using the computer-based Sigma Gel (SPSS, Chicago) system. Density values were expressed as mean ± SEM. In every case, the acceptance level for statistical significance was *P < 0.05 and **P < 0.01 versus control group.

TUNEL staining for neuronal cell death

In situ detection of apoptotic cell death was performed using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) on hippocampal neuronal cell culture as previously done [23]. TUNEL staining was performed according to supplier’s recommendations using kit (Genescript, NJ, USA). Propidium iodide (PI) was as a counter stain for 10 min at room temperature. TUNEL-positive (green) and PI-positive (red) staining patterns were acquired by the use of a confocal laser scanning microscope (Fluoview FV 1000, Olympus, Japan). TUNEL-positive cells in the different regions of each section were counted by observers blinded to the treatment conditions.

Data analysis and statistics

Western blots were scanned and analyzed by densitometry using a computer based on the Sigma Gel System (SPSS Inc., Chicago, IL, USA). Density values were expressed as mean ± SEM. Comparisons between treated groups and controls were done by Student‘s t test to determine the significance of differences between relevant treatment groups. In every case, the acceptance level for statistical significance was *P < 0.05 and **P < 0.01 versus control group.

Results

Ethanol and PTZ exposure modulates D1R expression in the prenatal rat cortical and hippocampal neurons

To study the effect of ethanol and PTZ on the expression of D1R in hippocampal and cortical cells, primary cultured neuronal cells were divided into four groups and treated as control, ethanol, PTZ, ethanol plus PTZ. RT-PCR analysis was used to examine the D1R mRNA expression level at early developmental stage. The results showed that D1R expression at mRNA level in hippocampal neurons was decreased significantly upon ethanol, PTZ and ethanol plus PTZ treated as compared to control group (Fig. 1a, b). Further, Western blot analysis was done to confirm whether the changes in D1R are similar as resulted at mRNA level. The results showed similar expression that upon exposure of ethanol, PTZ and ethanol plus PTZ on neuronal cells significantly decreased D1R protein expression in hippocampal as well as cortical neurons (Fig. 2a, b). Thus, the decreased protein level of D1R expression was as a result of decrease in mRNA level upon exposure of ethanol, PTZ ethanol plus PTZ as compared to the control groups (Fig. 2a, b).

Fig. 1
figure 1

RT-PCR analyses for the change in mRNA level of the D1R in the primary cultured hippocampal (a) and cortical (b) neuronal cells. B Density values expressed as mean ± SEM (n = 3) of the corresponding mRNA of D1R are presented. The density values on the Y-axis are expressed as arbitrary units (AU). *P < 0.05 and **P < 0.01 versus control group

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Fig. 2
figure 2

Western blot analyses of the D1R protein in the primary cultured hippocampal (a) and cortical (b) neuronal cells. B Density values expressed as mean ± SEM (n = 3) of the corresponding mRNA of D1R are presented. *P < 0.05 and **P < 0.01 versus control group

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Pro-apoptotic Bax, Bak and anti-apoptotic Bcl-2 proteins expression in neuronal cell cultures upon ethanol and PTZ exposure

In this study, we observed the change in pro-apoptotic Bax, Bak and anti-apoptotic Bcl-2 protein expression upon ethanol, PTZ and ethanol plus PTZ in primary cultures of hippocampal and cortical neurons using Western blot analysis. Our results showed that ethanol, PTZ and ethanol plus PTZ exposure significantly increased the expression of pro-apoptotic protein Bax and Bak (Fig. 3a–d) and significant reduction in the level of the anti-apoptotic protein Bcl-2 in neuronal cell cultures as shown in Fig. 3e and f. Moreover, the significant increase in Bax, Bak and decrease in Bcl-2 expression suggested that ethanol, PTZ and ethanol plus PTZ exposure in neuronal cell cultures have a significant deleterious effect which further leads to the apoptosis.

Fig. 3
figure 3

Western blot analyses of the Bax, Bak and Bcl-2 in the primary cultured hippocampal (a, c, e) and cortical (b, d, f) neuronal cells. β-actin is taken as loading control in each case. A Immunoblots of Bax, Bak and Bcl-2 of hippocampal and cortical neuronal cells under different treatment conditions. The immunoblots were labeled with an anti-Bax and Bcl-2 antibodies. B Density values were expressed as mean ± SEM (n = 4). *P < 0.05 and **P < 0.01 versus control group

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Caspase-3 protein expression increased in neuronal cell culture upon ethanol and PTZ exposure

Caspase activity is a main feature of an apoptotic pathway. To study the effect of ethanol, PTZ and ethanol plus PTZ on neuronal cells at early developmental stage, Western blot analysis was done. Our results showed that upon exposure of ethanol, PTZ and ethanol plus PTZ on primary neuronal cell culture the expression of cleaved caspase-3 was increased significantly as compared to the control group. The results suggested that ethanol, PTZ and ethanol plus PTZ exposure on cortical and hippocampal cells at early stage lead to apoptotic neuronal cell death on both part of the brain (Fig. 4a, b).

Fig. 4
figure 4

Western blot analyses of the caspase-3 in the primary cultured hippocampal (a) and cortical (b) neuronal cells. β-actin is taken as loading control in each case. A Immunoblots of caspase-3 of hippocampal and cortical neuronal cells under different treatment conditions. The immunoblots were labeled with an anti-caspase-3 antibody. B Density values were expressed as mean ± SEM (n = 4). *P < 0.05 and **P < 0.01 versus control group

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TUNEL staining showed that ethanol and PTZ exposure causes neuronal cell death

DNA break is one of the main features of apoptosis and the visualization of which is possible by TUNEL staining. TUNEL staining was used to observe the DNA damage upon ethanol and PTZ treatment that is one of the hallmarks of apoptosis. The neuronal cell deaths were analyzed and results showed that ethanol and PTZ treatment significantly increased the total number of TUNEL-positive cells within the hippocampal neuronal cell culture (Fig. 5b, c) as compared to the control group.

Fig. 5
figure 5

Representative photomicrographs of TUNEL staining showed apoptotic neuronal cells death upon ethanol administration along with PTZ. The arrows indicate ethanol-induced TUNEL stained apoptotic dead neurons counterstained with PI in the hippocampal neuronal cells. ×40 objective field, scale bar 20 μm

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Discussion

In the present study, we have observed the D1R expression and apoptotic neuronal cell death in cortical and hippocampal prenatal rat brain upon ethanol and PTZ exposure. Our results showed that ethanol and PTZ treatment significantly induce the downregulation of D1R and increased in neuronal cell death in both part of the prenatal rat brain. Prenatal rat primary neuronal cell culture was observed using RT-PCR analysis for mRNA and Western blot analysis was done for the detection of D1R protein expression, pro apoptotic proteins Bax, Bak, anti-apoptotic Bcl-2 and cleaved caspase-3 expression while, TUNEL and PI was used for detection of neurodegeneration in the prenatal brain.

Previously, it was reported that density of D1- and D2-like receptors was decreased in the hippocampal CA3, dorsal caudate-putamen and striatal regions of the brains of epileptic rats [24].

It was reported that ethanol significantly decreased dopaminergic D1 as well as D2 receptors and further biochemical and behavioral studies showed that in utero ethanol exposure produces a long-lasting effect in the development of electrophysiological and pharmacological characteristics of midbrain DA systems, in adulthood [25, 26].

Further it was also reported that prenatal exposure to 3 g/kg ethanol causes a significant decrease in the number of DA D2 binding sites within the dorsal and ventral striatum [27]. Earlier findings indicated that prenatal ethanol exposure may predominantly produce diminished reactivity of the D2 but not D1 subtypes of DA receptors or an opposite outcome [28]. Others [29] reported that prenatal ethanol exposure did not alter DA concentration or turnover and produced a transient increase in D1 but not D2 receptor binding, in mice. Furthermore, we have recently shown [30] that ethanol administered for 1 week to rats produces decreases in D1 and D2 receptor densities and no changes in dissociation constants. Reduced dopamine levels and D2 receptor numbers have been shown in brains of alcohol-preferring animals, in genetic models of alcoholism [31].

In the present study, we have observed that ethanol and PTZ exposure down regulate the D1R expression and induced apoptotic neuronal death in prenatal rat brain. Therefore, our results suggested that exposure of ethanol and PTZ in primary neuronal cell culture leads to significant decrease in the expression of D1R and further leads to apoptotic neuronal cell death by significantly increasing the expression of pro apoptotic Bax, Bak and significantly decreasing the anti-apoptotic protein Bcl-2 leading to the increase in the expression of cleaved caspase-3 protein. These results further explain the possibility of higher risk of developmental disturbances and malformations during early developmental stage.