Neuroactive steroids are endogenous neuromodulators capable of altering neuronal activity. Synthesis of neuroactive steroids occurs in the adrenal glands, gonads, and de novo in the brain. The 5α-reduced pregnane steroids, (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP or allopregnanolone) and (3α,5α)-3,21-dihydroxypregnan-20-one (3α,5α-THDOC), are positive allosteric modulators of γ-aminobutyric acid type A (GABAA) receptors. GABAA receptors are the primary inhibitory receptor family in the brain and mediate many of the behavioral effects of ethanol (EtOH). Both 3α,5α-THP and 3α,5α-THDOC enhance neuronal inhibition at a known binding site on GABAA receptor α-subunits (Hosie et al., 2006) and have corresponding behavioral effects similar to EtOH. These GABAergic neuroactive steroids are very potent positive modulators of GABAA receptors, which produce pharmacologically relevant effects at nanomolar concentrations (Morrow et al., 1987).
A large body of evidence suggests increased levels of 3α,5α-THP and 3α,5α-THDOC following EtOH administration contribute to both electrophysiological and behavioral effects of EtOH in rodents. In rats, EtOH activates the hypothalamic–pituitary–adrenal (HPA) axis (Boyd et al., 2010) leading to physiologically significant increases in 3α,5α-THP and 3α,5α-THDOC in the blood plasma, cerebral cortex, and hippocampus (Barbaccia et al., 1999; Porcu et al., 2009; VanDoren et al., 2000). Adrenalectomy or inhibition of 5α-reduced steroid synthesis with the 5α-reductase (5α-R) inhibitor finasteride reduces some of the behavioral effects of EtOH, including the hypnotic (Khisti et al., 2003), anxiolytic-like (Hirani et al., 2005), anticonvulsant (VanDoren et al., 2000), and antidepressant-like (Hirani et al., 2002) effects in rats. Furthermore, finasteride reduces some of the subjective effects of alcohol in healthy men (Pierucci-Lagha et al., 2005). Finasteride also blocks EtOH inhibition of neuron firing in the medial septum (VanDoren et al., 2000), hippocampus (Tokunaga et al., 2003), hippocampal slice (Sanna et al., 2004), and long-term potentiation (LTP) in the hippocampal slice preparation (Tokuda et al., 2011). Taken together, these findings suggest EtOH-induced elevations of 3α,5α-THP and 3α,5α-THDOC contribute to many of the physiological and behavioral effects of EtOH. However, outside of the cerebral cortex and hippocampus, it is not known whether EtOH increases levels of 3α,5α-THP in other brain regions that contribute to EtOH's myriad of pharmacological effects.
GABAergic neuroactive steroids are involved in stress responses; therefore, steroid concentrations may be altered in stress related neurocircuitry following EtOH administration. GABAergic neuroactive steroids are increased in the plasma and cerebral cortex following environmental stress (Barbaccia et al., 1996; Purdy et al., 1991), including EtOH administration. GABAergic neuroactive steroids contribute to negative feedback on the HPA axis at the level of the hypothalamus in rats (Owens et al., 1992; Patchev et al., 1994, 1996), but not in C57BL/6J mice (Sarkar et al., 2011). Furthermore, recent evidence in C57BL/6J mice suggests GABAergic neuroactive steroids are involved in initiation of the stress response (Sarkar et al., 2011). In the current study, we investigated EtOH-induced changes of 3α,5α-THP in the paraventricular nucleus (PVN) of the hypothalamus, the bed nucleus of the stria terminalis (BNST), and amygdala due to the involvement of these regions in stress, emotion, and EtOH responses (Armario, 2010; Cui et al., 2012; Koob, 2013).
EtOH and 3α,5α-THP both impair learning and memory performance in a similar manner (Matthews et al., 2002), which may be due to modulation of activity of specific cellular populations in the hippocampus (Matthews et al., 2002; Tokuda et al., 2011; Tokunaga et al., 2003). In vitro, EtOH increases 3α,5α-THP immunoreactivity in hippocampal pyramidal cells (Tokuda et al., 2011), and finasteride prevents EtOH's inhibitory effect on these cells in anesthetized rats (Tokunaga et al., 2003). Therefore, we examined the cellular layer specificity of EtOH effects on 3α,5α-THP levels in the hippocampal formation.
Systemic and intracerebroventricular administration of exogenous 3α,5α-THP or systemic administration of the longer acting 3α,5α-THP analogue, ganaxolone, alter EtOH consumption in rodents. Both 3α,5α-THP and ganaxolone have been shown to produce biphasic effects on EtOH consumption, with low doses increasing consumption and high doses decreasing consumption (Besheer et al., 2010; Ford et al., 2005, 2007; Janak et al., 1998; Morrow et al., 2001). The mechanisms by which 3α,5α-THP affects EtOH consumption are not clear, but these effects could be due to modulation of dopamine release in mesocorticolimbic circuitry (Motzo et al., 1996; Rouge-Pont et al., 2002). Therefore, we examined the effects of EtOH on 3α,5α-THP immunoreactivity in the ventral tegmental area (VTA), nucleus accumbens (NAc), striatum, and medial prefrontal cortex (mPFC).
Previous studies measuring EtOH-induced changes of 3α,5α-THP in the brain have used radioimmunoassay (RIA) or gas chromatography–mass spectroscopy (GC-MS), which lack the sensitivity to determine levels in discrete cell layers or brain regions with low levels of 3α,5α-THP. Although EtOH increases levels of 3α,5α-THP in the cerebral cortex and hippocampus, it is unknown whether EtOH alters levels of 3α,5α-THP in other brain regions. Immunohistochemistry (IHC) using an anti-3α,5α-THP primary antibody was used to examine EtOH-induced changes in cellular 3α,5α-THP levels. This technique has been used to determine the cellular distribution of 3α,5α-THP in the rat brain (Saalmann et al., 2007) and displays the sensitivity to detect changes in immunoreactivity that are associated with corresponding changes in electrophysiological measurements (Saalmann et al., 2006; Tokuda et al., 2011). We examined EtOH-induced changes in cellular 3α,5α-THP expression across multiple brain regions that are implicated in alcohol use disorders. This approach provides brain region specificity of neuroactive steroid measurements that has not previously been reported following EtOH administration and may reveal novel mechanisms of EtOH action.
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- Materials and Methods
The goal of the present study was to use IHC to examine EtOH-induced changes in cellular 3α,5α-THP expression in multiple brain regions implicated in alcohol use disorders. Our findings support previous evidence that EtOH increases 3α,5α-THP concentrations in the rat cerebral cortex and hippocampus using RIA or GC-MS. We extend previous findings by showing that EtOH increases cellular 3α,5α-THP in the mPFC, CA1 pyramidal cell layer, and polymorph cell layer of the DG but not in granule cells of the DG. Therefore, EtOH-induced increases in cellular 3α,5α-THP in the hippocampus are isolated to specific cellular populations. We also show that EtOH increases cellular 3α,5α-THP levels in the BNST and the PVN of the hypothalamus. Interestingly, acute EtOH administration reduced cellular 3α,5α-THP levels in the NAc “shore” (core-shell border) and the CeA. To our knowledge, this is the first example of acute EtOH reducing 3α,5α-THP levels in the rat brain or periphery. We also determined that EtOH does not alter 3α,5α-THP levels in the VTA, DMS, or the lateral or basolateral amygdala. Therefore, EtOH produces divergent brain region and cellular layer specific changes in 3α,5α-THP concentrations.
The IHC technique used in the present study provides brain region and cell layer specificity of EtOH-induced changes in 3α,5α-THP. A similar IHC assay was used to demonstrate that 3α,5α-THP is located primarily in GABAergic and glutamatergic projecting principal neurons, but not in interneurons or glia (Saalmann et al., 2007). The present results indicate similar relative intensity of basal staining across brain, with strong labeling of cortical and hippocampal cell layers as well as the striatum. There are some important differences between measurements of 3α,5α-THP with IHC compared to RIA measurements. IHC measures cellular levels of 3α,5α-THP but RIA measures absolute levels of neuroactive steroids. Therefore, if there are significant pools of extraneuronal 3α,5α-THP present in brain tissue, one might predict IHC measurements would differ from results obtained by RIA. Our current IHC results in the mPFC, CA1 pyramidal, and DG polymorph cell layer show that EtOH increased relative 3α,5α-THP immunoreactivity by 24 ± 6%, 32 ± 12%, and 52 ± 5%, respectively. However, RIA studies measuring EtOH-induced increases in 3α,5α-THP in whole cerebral cortex and hippocampus have found larger increases in 3α,5α-THP. For example, increases of 5- to 17-fold (Barbaccia et al., 1999), approximately 3-fold (Khisti et al., 2004; VanDoren et al., 2000), and approximately 2-fold (Boyd et al., 2010) have been observed in the cerebral cortex, while studies examining whole hippocampus have found 4- to 11-fold (Barbaccia et al., 1999) and approximately 6-fold (Khisti et al., 2004) increases in 3α,5α-THP concentrations. The discrepancies in magnitude of EtOH-induced increases in 3α,5α-THP could be due to relatively small increases in 3α,5α-THP in the mPFC when compared to the whole cortex, and similarly, small relative increases in 3α,5α-THP in the CA1 pyramidal cells and the polymorph cell layer of the DG compared to whole hippocampus. It is important to note we are using an affinity purified 3α,5α-THP antiserum that appears to be more specific in detecting 3α,5α-THP. The only other study examining EtOH's effects on 3α,5α-THP using the affinity purified 3α,5α-THP antiserum used IHC with immunofluorescent detection and found over a 200% increase in fluorescent intensity in CA1 pyramidal cells (Tokuda et al., 2011) compared to a 32 ± 12% increase in immunoreactivity in the present study using DAB detection. Although EtOH increased 3α,5α-THP using both fluorescent and DAB visualization, the magnitude of 3α,5α-THP changes may differ due to differences in IHC methodology.
The 3α,5α-THP antisera used in the present study was found to cross-react with 3α-HP (41 ± 0.14%) and 3α,5β-THP (22 ± 0.43%). This is not a major limitation as both of these steroids have similar GABAergic activities as 3α,5α-THP. Furthermore, there is no evidence that 3α-HP (Griffin and Mellon, 2001) or 3α,5β-THP are present in the brain in appreciable concentrations. In addition, EtOH does not elevate 3α,5β-THP levels in rat plasma (Porcu et al., 2010). Therefore, 3α,5α-THP is the most likely endogenous antigen immunolabeled using this approach. A potential limitation of the study is that only 1 dose of EtOH was examined. In the cerebral cortex, a threshold dose of EtOH (1.3 to 1.5 g/kg) is required to produce an increase in 3α,5α-THP and increasingly higher doses produce less of an increase in 3α,5α-THP (Boyd et al., 2010; VanDoren et al., 2000). However, since only 1 dose was used it is not known whether a similar threshold dose is needed to produce increases or decreases in 3α,5α-THP in the cortical and subcortical brain regions examined in the present study. Similarly, it is not clear whether EtOH produces a biphasic effect on 3α,5α-THP levels in these brain regions.
The diverse effects of EtOH administration on cellular 3α,5α-THP levels suggest that EtOH may alter local synthesis and/or metabolism of 3α,5α-THP in the rat brain. These results suggest EtOH-induced changes of cellular 3α,5α-THP in the brain are not due exclusively to adrenal gland derived elevations of neuroactive steroids or steroid precursors, which would be expected to increase 3α,5α-THP throughout the brain. However, we observed no change in 3α,5α-THP levels after acute EtOH administration in the granule cell layer of the DG and the DMS, which both display dense labeling of 3α,5α-THP (Saalmann et al., 2007) as well as the biosynthetic enzymes 5α-R and 3α-hydroxysteroid dehydrogenase (3α-HSD) needed for 3α,5α-THP synthesis (Agis-Balboa et al., 2006). Furthermore, there was a reduction in cellular 3α,5α-THP in the NAc and the CeA. Based on these findings, our data suggest there are unknown mechanisms contributing to EtOH's effects on 3α,5α-THP concentrations. In the cerebral cortex, EtOH-induced elevations of 3α,5α-THP are dependent on the pituitary–adrenal axis (Boyd et al., 2010; Khisti et al., 2003; O'Dell et al., 2004). In vitro, however, there is evidence for EtOH-induced brain synthesis of 3α,5α-THP in hippocampal slices (Sanna et al., 2004; Tokuda et al., 2011). Thus, it is not known whether EtOH alters brain synthesis of 3α,5α-THP in vivo independent of circulating steroids. Therefore, it will be important to determine the role of circulating steroids in the current observations, which may provide insight into this important question.
The most striking finding in the present study is that EtOH produces brain region and cellular layer specific changes in 3α,5α-THP concentrations. These data suggest EtOH alters local synthesis of 3α,5α-THP in the rat brain. A possible mechanism that may explain these findings is EtOH may change local expression and/or activity of steroidogenic enzymes. Steroidogenesis is initiated by cholesterol transport to cytochrome P450 side-chain cleavage (P450scc), resulting in conversion of cholesterol to pregnenolone. Previous work from our laboratory has shown that de novo adrenal synthesis of the cholesterol transporter, steroidogenic acute regulatory protein (StAR), is necessary for EtOH-induced increases in 3α,5α-THP in the cerebral cortex (Boyd et al., 2010). Acute EtOH administration increases StAR and P450scc mRNA in the rat frontal cortex and hypothalamus, and StAR mRNA is also increased in the hippocampus (Kim et al., 2003). The synthesis of 3α,5α-THP from progesterone is accomplished by the sequential actions of 5α-R and 3α-HSD. Acute EtOH administration increases 5α-R type 1 (5α-RI) and 3α-HSD mRNA in the frontal cortex and 3α-HSD mRNA in the hypothalamus, but no changes in transcript expression of either enzyme were detected in the hippocampus (Kim et al., 2003). Unfortunately, there are no data examining the effects of acute EtOH on expression of these enzymes in the NAc or CeA where we observed reductions in 3α,5α-THP. However, chronic intermittent EtOH administration reduces 3α,5α-THP levels in the hippocampus, which is associated with concurrent decreases in 5α-RI and 3α-HSD mRNA expression (Cagetti et al., 2004). Taken together, EtOH-induced changes in steroidogenic enzyme expression may underlie the divergent changes in 3α,5α-THP levels observed in the rat brain. Additionally, EtOH may directly or indirectly change the activity of steroidogenic enzymes, which could alter steroid concentrations. Ultimately, EtOH may alter the expression and/or activity of enzymes involved in steroid synthesis and metabolism, resulting in local changes in neurosteroid levels that are dependent on which enzymes are expressed in a particular cell.
Another possible explanation for the reduction in cellular 3α,5α-THP levels observed in the NAc and CeA is extracellular redistribution of 3α,5α-THP. Neuroactive steroids have been proposed to act on membrane bound receptors by a paracrine or autocrine mechanism (Herd et al., 2007), or by lateral diffusion through the cell membrane to access transmembrane neuroactive steroid binding sites on GABAA receptors (Akk et al., 2007). Although controversial, we cannot rule out the possibility that EtOH leads to a “release” or redistribution of 3α,5α-THP into the extracellular space, perhaps in a brain region specific manner.
Significance of Ethanol-Induced Changes in Cellular Expression of 3α,5α-THP
Evidence suggests EtOH-induced increases in 3α,5α-THP contribute to the behavioral effects of EtOH in rodents and some of the subjective effects of alcohol in humans. Contribution of 3α,5α-THP to the pharmacological effects of EtOH is likely due to potentiation of GABAA receptors across multiple brain regions. The localization of 3α,5α-THP and the biosynthetic enzymes 5α-R and 3α-HSD in principal output neurons suggests a major role of 3α,5α-THP may be to modulate neural circuitry (Agis-Balboa et al., 2006; Saalmann et al., 2007). The neurophysiological effects of 3α,5α-THP have been best characterized in hippocampal pyramidal cells. In vivo, EtOH (1.5 g/kg) reduces spontaneous activity of hippocampal pyramidal cells in anesthetized rats, which is prevented by finasteride preadministration (Tokunaga et al., 2003). In vitro, EtOH (50, 100 mM) increases 3α,5α-THP in isolated hippocampal tissue and amplitude of GABAA receptor mediated spontaneous and evoked inhibitory postsynaptic currents in CA1 pyramidal cells (Sanna et al., 2004), the latter of which is prevented by pretreatment with finasteride. EtOH (60 mM) also inhibits LTP in CA1 pyramidal cells, with concomitant increases in 3α,5α-THP immunostaining (Tokuda et al., 2011). Furthermore, pretreatment with finasteride blocks EtOH's inhibition on LTP, suggesting that EtOH's ability to inhibit LTP is mediated by 3α,5α-THP. In the present study, EtOH increased 3α,5α-THP in the polymorphic cell layer, but not the granule cell layer of the DG. Although we did not distinguish which polymorphic cells exhibit increased 3α,5α-THP, the most prominent cell type in this layer is the mossy cell. Mossy cells in the polymorphic layer project to the molecular and granule cell layers of the DG (Amaral et al., 2007), the latter of which constitutes the only projection from the DG to the hippocampus. Considering the major role the hippocampus and DG play in learning and memory, 3α,5α-THP induction by EtOH may contribute to EtOH's effects on memory and cognition.
EtOH-induced changes in brain concentrations of 3α,5α-THP may also modulate neuronal activity in other brain regions examined in the present study. In vivo evidence suggests EtOH-induced increases in 3α,5α-THP modulate neuronal activity outside of the hippocampus as well. For example, finasteride prevents EtOH (1.5 g/kg) inhibition of spontaneous firing of medial septal/diagonal band of Broca neurons (VanDoren et al., 2000). In addition to hippocampal pyramidal cells, we observed increases in cellular 3α,5α-THP in the polymorph DG, mPFC, BNST, and PVN of the hypothalamus. The physiological consequences of 3α,5α-THP increases may alter neuronal activity and synaptic plasticity in these regions and related circuitry. Clearly, studies are needed to examine how increases in 3α,5α-THP in these brain regions may contribute to the physiological and behavioral effects of EtOH.
EtOH-induced reductions in cellular 3α,5α-THP were observed in the NAc and CeA. These observations are particularly interesting because these 2 brain regions are strongly associated with EtOH reinforcement and consumption (Gonzales et al., 2004; McBride, 2002). A reduction in cellular 3α,5α-THP would presumably reduce the amount of GABAA receptor mediated neuronal inhibition, which may increase firing of mesolimbic dopamine neurons and CeA circuitry involved in EtOH reinforcement. In the NAc, reductions in 3α,5α-THP were isolated to the “shore” (core-shell border), as no changes in 3α,5α-THP were detected exclusively in the shell or core. This is an interesting finding since EtOH-associated cues and operant self-administration of EtOH both increase dopamine release in the “shore” following operant training (Howard et al., 2009), but in the core or shell, only EtOH-associated cues increase dopamine release following operant training. It is important to note, however, that acute systemic EtOH administration increases dopamine release in the NAc core and shell (Imperato and Di Chiara, 1986; Yim et al., 2000) where EtOH did not alter 3α,5α-THP levels. Administration of 3α,5α-THP produces biphasic effects on EtOH consumption (Finn et al., 2010; Ford et al., 2005, 2007; Janak et al., 1998) and dopamine release in the NAc (Motzo et al., 1996; Rouge-Pont et al., 2002). Furthermore, 3α,5α-THP modulates EtOH's effects on mPFC dopamine content (Dazzi et al., 2002). Therefore, investigating possible interactions of 3α,5α-THP and dopaminergic activity in the mesocorticolimbic system may be valuable in the effort to reduce EtOH consumption via neuroactive steroid modulation.
The current data show that EtOH produces divergent brain region and cell layer specific changes in cellular 3α,5α-THP concentrations. These divergent effects suggest that EtOH induces local regulation of 3α,5α-THP levels. These changes in 3α,5α-THP levels likely contribute to the neurophysiological and behavioral effects of EtOH in rats. Examining the mechanisms by which EtOH alters levels of 3α,5α-THP across the brain may lead to new therapeutic strategies for treating alcohol use disorders.