Alcohol promotes lasting neuroadaptive changes that may provide relief from depressive symptoms, often referred to as the self-medication hypothesis. However, the molecular/synaptic pathways that are shared by alcohol and antidepressants are unknown. In the current study, acute exposure to ethanol produced lasting antidepressant and anxiolytic behaviours. To understand the functional basis of these behaviours, we examined a molecular pathway that is activated by rapid antidepressants. Ethanol, like rapid antidepressants, alters γ-aminobutyric acid type B receptor (GABABR) expression and signalling, to increase dendritic calcium. Furthermore, new GABABRs are synthesized in response to ethanol treatment, requiring fragile-X mental retardation protein (FMRP). Ethanol-dependent changes in GABABR expression, dendritic signalling, and antidepressant efficacy are absent in Fmr1-knockout (KO) mice. These findings indicate that FMRP is an important regulator of protein synthesis following alcohol exposure, providing a molecular basis for the antidepressant efficacy of acute ethanol exposure.
The presence of major depression increases the risk of alcohol use disorders (AUDs) by ∼2-fold (and vice versa)1. The self-medication hypothesis suggests AUDs may develop when the initial antidepressant actions of alcohol are shifted to depressant allostatic states with chronic abuse2. The molecular mechanism underlying the initial antidepressant effects of alcohol is unknown.
A major advance in understanding and treating depression is the recognition that NMDA receptor (NMDAR) antagonists act as rapid and effective antidepressant drugs3. A single injection of an NMDAR antagonist or ‘rapid antidepressant’ is effective within 2 h and has sustained antidepressant efficacy for 2 weeks4. These long-lasting properties depend on the activity of mammalian target of rapamycin (mTOR)5,6, a serine/threonine kinase essential for messenger RNA translation7. Recently, we demonstrated that activation of mTOR-dependent protein synthesis by NMDAR antagonists requires a shift in GABABR signalling from opening potassium channels to facilitating an increase in dendritic calcium6,8. Interestingly, both acute ethanol and rapid antidepressants block NMDARs4,9. In light of these data, we propose that ethanol has lasting antidepressant efficacy, shares the same downstream molecular signalling events as rapid antidepressants, and requires de novo protein synthesis (Supplementary Fig. 1).
Studies suggest that antidepressant efficacy requires two phases—an induction phase and a sustained phase10,11. Notably, GABABR-mediated, mTORC1-dependent protein synthesis is required for the long-lasting sustained phase of rapid antidepressants. Our previous work indicates that both new protein synthesis and an increase in protein stability are required for the GABABR shift in function necessary to increase mTORC1 activity8. However, the mechanism that initiates such dynamic changes in protein expression by rapid antidepressants remains unclear.
FMRP is an RNA-binding protein that has been characterized as a repressor of mRNA translation. Some forms of synaptic activity trigger FMRP to release its targets, allowing them to be translated12,13. Moreover, degradation and new protein synthesis of FMRP creates a window for the translation of specific mRNAs, facilitating long-lasting changes in synaptic function14,15. Complete loss of FMRP results in Fragile-X syndrome (FXS), the single most common genetic cause of autism16. Moreover, reduced levels of FMRP, caused by a pre-mutation, lead to a higher incidence of tremors, ataxia, memory loss, and neuronal neuropathy in older men17. These findings argue that precise levels of FMRP protein and its target mRNAs are required for normal neuronal function.
Drugs of abuse promote profound changes in gene expression, mRNA translation rates and synaptic protein composition18,19. Some studies suggest that drugs and alcohol highjack the molecular mechanisms that underlie synaptic plasticity20,21. In agreement with this premise, FMRP has been implicated in cocaine addiction22. However, little is known about the mRNA targets and the signalling mechanisms involved. Here we describe a critical role for FMRP in mediating GABABR synthesis and plasticity following acute ethanol exposure, a mechanism required for antidepressant efficacy.
Antidepressant and anxiolytic effects of ethanol on behaviour
To determine if acute alcohol has antidepressant properties, as predicted by the self-medication hypothesis, we first assessed the efficacy of alcohol on antidepressant- and anxiolytic-like effects on behaviour. The forced swim test (FST) is a rodent behavioural test predictive of antidepressant activity in humans23. Rodents treated with a single injection of NMDAR antagonists or rapid antidepressants swim longer and thus have reduced immobility relative to controls. Notably, these positive effects on behaviour last long after the drug has been metabolized5,6,8,24. Therefore, we considered the possibility that ethanol, which blocks NMDARs9, could also act like an antidepressant at 24 h, well beyond the intoxication period25. To test this, C57BL/6 mice were injected with ethanol (2.5 g kg−1, intraperitoneal (i.p.)), a concentration that is achieved during self-administration in mice26. Twenty-four hours after injection, the immobility of ethanol-treated mice was reduced by ∼15% relative to controls (Fig. 1a), similar to our previous observation in mice that had been exposed to the rapid antidepressant Ro-25-6981 (refs 6, 8). These results demonstrate that acute ethanol elicits a lasting antidepressant effect on behaviour similar to that seen with rapid antidepressants8.
As another measure of antidepressant effect of ethanol on behaviour, we assessed the grooming behaviour of mice using the splash test after ethanol or saline administration. The splash test measures latency to groom and dedicated grooming time as indicators of self-care and motivational behaviour27,28. Lack of self-care is often observed in humans with depressive disorder29. We have previously shown that mice receiving a single i.p. injection of the rapid antidepressant Ro-25-6981 spend more time grooming compared with control mice8. We hypothesized that ethanol would produce similar effects on grooming behaviour. Indeed, ethanol-treated mice spent more time grooming and displayed shorter latency to initiate grooming relative to controls (Fig. 1b,c).
Ethanol is a well-known anxiolytic substance30. However, the anxiolytic effect of a single dose of ethanol 24 h after administration has not been determined. We subjected ethanol- and saline-injected mice to the open field test to assess the influence of ethanol on anxiety-like behaviours after 24 h. Mice that spend more time in the centre of the open field are scored as having reduced anxiety-like behaviour relative to mice that remain close to the perimeter31. Indeed, mice that received a single dose of ethanol (2.5 g kg−1, i.p.) had reduced anxiety-like behaviour, spending ∼40% more time in the centre relative to controls (Fig. 1d). There was no significant difference in total distance travelled or average speed between the groups (Fig. 1e,f). These data suggest that the anxiolytic effects of ethanol last up to 24 h post injection.
Acute ethanol increases GABABR2 and surface GABABRs
Both ethanol and rapid antidepressants block NMDARs in the hippocampus4,9. One of the first events triggered by NMDAR antagonism is increased dendritic GABABR2 protein expression8. GABABRs are obligate heteromultimers, consisting of GABABR1 and R2. GABABR2 is required for expression of receptors at the surface by masking an endoplasmic reticulum retention sequence on GABABR1 (ref. 32). Similarly, treatment with a rapid antidepressant leads to (1) increased dendritic expression of GABABR2 but not GABABR1 (ref. 8) and (2) a corresponding increase in surface expression of GABABR1 (refs 6, 8).
To determine if acute ethanol exposure in vivo rapidly increases the levels of GABABR1 and/or GABABR2, hippocampal synaptoneurosomes were isolated from mice that had been injected with a single dose of ethanol (2.5 g kg−1, i.p.) or saline for western blot analysis. The hippocampi were collected within the initiation phase (30 min post injection), a phase where molecular changes facilitate increased downstream mTORC1 activity11. Consistent with rapid antidepressants, acute ethanol injection increased the protein expression of GABABR2 by ∼37% in the hippocampus with no significant change in GABABR1 (Fig. 2a–c; uncropped blots, Supplementary Fig. 7a).
To further identify the subcellular localization of ethanol-induced increase in GABABR2, we examined GABABR expression in cultured hippocampal neurons. GABABR1 and R2 were immunostained and quantified in the dendrites. A concentration of 30 mM ethanol was chosen, as it has been shown to reduce NMDAR activity in hippocampal neurons and reflects that achieved in vivo following i.p. injection9,33. Acute ethanol exposure (30 mM, 2 h) increased the dendritic levels of GABABR2 by ∼47%, but did not affect GABABR1 levels (Fig. 2d–g). We did not observe a difference in the diameter of the primary dendrites between vehicle- and ethanol-treated neurons, demonstrating that ethanol does not modify dendritic calibre (Supplementary Fig. 2). These in vivo and in vitro findings establish a role for ethanol in increasing GABABR2 protein expression.
Since GABABR2 is required for the surface expression of the heteromultimeric receptor, we predicted that the ethanol-induced elevation in GABABR2 levels would increase expression of receptors at the surface. We measured the surface expression of dendritic GABABRs using an antibody directed against the extracellular domain of GABABR1 in unpermeabilized hippocampal neurons. The surface signal was normalized by the total dendritic GABABR1 levels after permeabilization6. As predicted, surface expression of GABABRs in ethanol-treated neurons was significantly higher (∼66% increase) relative to controls (Fig. 2h,i). This ethanol effect was consistent with what we previously observed following rapid antidepressant treatment of cultured hippocampal neurons6. Collectively, these results suggest that ethanol promotes the surface expression of GABABRs, and this is likely achieved by increasing GABABR2 protein levels.
FMRP regulates GABABR1 and GABABR2 expression
Next, we sought to identify the mechanism by which NMDAR antagonism increases GABABR2 expression. GABABR2 mRNA is present in the dendrites of hippocampal neurons34, suggesting that this mRNA may be locally regulated at the translational level. Thus, we examined RNA-binding factors that may regulate GABABR2 mRNA expression in dendrites. Notably, both GABABR1 and GABABR2 mRNAs are reported targets of FMRP, an RNA-binding protein that stalls translational elongation of its targets35,36.
To test the hypothesis that FMRP regulates GABABR mRNA translation, we first verified that (1) GABABR mRNAs bind to FMRP, and that (2) the absence of FMRP in knockout mice results in aberrant expression of GABABRs. Using a specific antibody against FMRP, bound mRNAs were isolated using RNA immunoprecipitation (RIP). GABABR1 and GABABR2 binding were assessed by reverse transcription (RT) and quantitative PCR. Indeed, GABABR1 and GABABR2 mRNAs were detected in the immunoprecipitate, along with CaMKIIα, a well-known FMRP mRNA target (Fig. 3a,b; uncropped representative qPCR gels, Supplementary Fig. 7d). The calcium channel accessory subunit Cacnα2δ2 mRNA is not a reported target for FMRP35 and was used as a negative control. Cacnα2δ2 mRNA was not detected in the FMRP RIP (Fig. 3b). In parallel, we used lysates isolated from brains of mice with a genetic deletion of the Fmr1 gene. We did not observe amplification of any of the mRNAs in Fmr1 KO brains, providing additional evidence for specific binding of FMRP to GABABR1 and GABABR2 mRNAs (Fig. 3a–c; uncropped blots, Supplementary Fig. 7b).
Next, we determined if FMRP regulates GABABR1 and GABABR2 protein levels. Genetic deletion of Fmr1 leads to the constitutive translation of FMRP target mRNAs and the loss of activity-dependent translation16. Protein levels of GABABR1 and GABABR2 were compared in hippocampal synaptoneurosomes from Fmr1 KO and wild-type (WT) mice (Fig. 3c–g). GABABR2 basal protein levels were elevated by ∼53% in Fmr1 KO hippocampi (Fig. 3g). GABABR1 protein levels also increased, albeit to a lesser extent than GABABR2 (Fig. 3f). Collectively, these data suggest that FMRP regulates the expression of GABABR1 and GABABR2.
Ethanol and rapid antidepressants reduce dendritic FMRP
As an initial test to determine if FMRP-regulated translation is linked to alcohol exposure, we compared FMRP target mRNAs35 with mRNAs that are differentially expressed in the hippocampi of alcohol-dependent humans37. Remarkably, 225 or ∼25% of verified FMRP target mRNAs overlap with mRNAs that are altered in alcohol-dependent individuals, suggesting a role for FMRP in aberrant protein levels observed in humans with AUD (Fig. 4a)35,37. We then determined whether exposure to acute ethanol (30 mM, 2 h) or Ro-25-6981 (10 μM, 2 h) alters FMRP expression in the dendrites of hippocampal neurons. Using immunofluorescence, we found that ethanol and Ro-25-6981 reduced FMRP levels by ∼38% and 45%, respectively (ethanol: Fig. 4b,c; Ro-25-6981: Supplementary Fig. 3). These data suggest that ethanol and Ro-25-6981 alter protein expression in an FMRP-dependent manner.
Ethanol-induced synthesis of GABABR2 requires FMRP
Due to the ethanol-induced decreases in FMRP, we hypothesized that FMRP is required for ethanol-induced expression of GABABRs. Specifically, if expression of GABABRs is constitutive and unregulated in Fmr1 KO mice, then ethanol-induced changes in GABABR expression should be absent in Fmr1 KO mice. Hippocampal synaptoneurosomes were isolated from WT and Fmr1 KO mice 30 min after i.p. injection of ethanol (2.5 g kg−1). Western blot analysis indicated that both GABABR1 and GABABR2 expression remained constant in vehicle- and ethanol-treated Fmr1 KO mice. As observed in Fig. 2, WT hippocampal synaptoneurosomes showed an ∼23% increase in GABABR2 but no change in GABABR1 expression (Fig. 5a–c; uncropped blots, Supplementary Fig. 7c). These data suggest that ethanol-induced changes in GABABR expression are dependent on FMRP translational regulation.
To determine whether protein synthesis is essential for the FMRP-dependent changes in GABABR expression, we measured ethanol-induced GABABRs in the presence of cycloheximide (CHX), a protein synthesis inhibitor. As demonstrated previously, ethanol did not influence the dendritic expression of GABABR1; however, co-treatment with cycloheximide increased GABABR1 expression by ∼22%. FMRP deletion did not affect the basal, ethanol-, or cycloheximide-induced dendritic protein expression of GABABR1 (Fig. 5d,e,h). For GABABR2, we again saw a significant ∼28% increase in dendritic expression with acute ethanol treatment; however, in the presence of cycloheximide the ethanol-induced increase was abolished. Notably in Fmr1 KO cultures, no change was observed with ethanol or ethanol+cycloheximide (Fig. 5f,g,i).
We next examined the requirement for protein synthesis and FMRP in ethanol-dependent surface expression of GABABRs. Using WT and Fmr1 KO hippocampal neurons, we measured ethanol-induced surface expression of GABABR1 with or without cycloheximide. Co-assembly of GABABR1 and R2 is required to express GABABR heterodimers in the membrane32. Thus, we predicted that the ethanol-induced increase in surface GABABRs would require FMRP-regulated synthesis of GABABR2. Again, acute ethanol increased dendritic surface GABABRs by ∼76%, and this was blocked by cycloheximide. In Fmr1 KO neurons there was no significant ethanol-induced change in surface GABABRs, but a decrease was observed with ethanol+cycloheximide (Fig. 6a–c). These data suggest that GABABR2 protein synthesis is required for the increased surface expression of the hetermultimeric receptor with ethanol exposure.
FMRP is reported to repress new protein synthesis. Considering the effects of the protein synthesis inhibitor, these data suggest that the ethanol-mediated reduction in FMRP results in the increase in dendritic GABABR2 by de novo protein synthesis. To provide more direct evidence, we performed bioorthogonal noncanonical amino-acid tagging (BONCAT) in conjunction with proximity ligation assay (PLA-Duolink)38. BONCAT+PLA can be used to detect new synthesis of proteins of interest, such as GABABR1 and GABABR2. Through click chemistry, noncanonical amino acids that are incorporated during mRNA translation are biotinylated. PLA, on the other hand, generates a fluorescent signal when two antibodies are within 30–40 nm of each other (that is, anti-GABABR1 or GABABR2 and anti-biotin). By combining these methods, we determined that ethanol treatment increases new protein synthesis of GABABR2 by ∼40%, but does not alter GABABR1 synthesis, similar to the GABABR changes induced by rapid antidepressants8. In Fmr1 KO dendrites, basal levels of GABABR2s increased by ∼48%, while a significant decrease was observed in GABABR1 levels. In addition, ethanol-induced translation of GABABR2 was lost in Fmr1 KO dendrites (Fig. 6d–i). These data provide additional evidence that the ethanol-induced increase in GABABR2 expression is due to new protein synthesis that requires the release of translational repression by FMRP.
Ethanol-induced GABABR plasticity requires FMRP
We previously demonstrated that rapid antidepressants shift GABABR signalling from opening potassium channels to increasing dendritic calcium6. To determine whether ethanol (30 mM, 2 h) causes the same plasticity in GABABR signalling, we performed fluorescence calcium imaging in cultured WT and Fmr1 KO hippocampal neurons. A transient rise or fall in calcium in dendritic compartments can be detected using a fluorescent indicator that exhibits changes in fluorescent properties depending on the amount of bound calcium39. We used baclofen, a GABABR agonist, to activate GABABRs in the presence or absence of ethanol. After establishing a baseline measurement, baclofen reduced dendritic calcium fluorescence in saline-treated WT neurons by ∼11%, a characteristic signature of GABABR signalling increasing outward potassium conductance8. However, in ethanol-treated WT neurons, baclofen induced distinct calcium waves and an overall averaged increase in calcium signal of ∼9% (Fig. 7a–c and Supplementary Fig. 4a,c). These results recapitulate our previous observations with NMDAR antagonists6. In addition, these findings in WT mouse neurons are consistent with what we observed in rat cultured hippocampal neurons treated with ethanol or the clinically relevant rapid antidepressant Ro-25-6981 (Supplementary Fig. 5). Unexpectedly, GABABR activation in saline-treated Fmr1 KO neurons failed to reduce the calcium signal. Moreover, in ethanol-treated Fmr1 KO neurons, GABABR activation failed to increase dendritic calcium signal (Fig. 7b,c and Supplementary Fig. 4b,c). These findings suggest that the loss of FMRP in Fmr1 KO dendrites decouples GABABRs from potassium channels. These results also suggest that the dynamic, ethanol-induced plasticity in GABABR signalling, which is observed with rapid antidepressants, requires FMRP6.
To further substantiate that FMRP regulates ethanol-dependent GABABR plasticity, we overexpressed FMRP in rat hippocampal neurons. Overexpression of FMRP did not alter the GABABR activation in saline-treated neurons because baclofen reduced the dendritic calcium signal. However, in ethanol-treated neurons, overexpressing FMRP blocked the ethanol-induced GABABR plasticity (Fig. 7d and Supplementary Fig. 4d,e). These results provide additional evidence that the dynamic reduction of FMRP with ethanol exposure is important for the expression of GABABR plasticity.
Antidepressant effect of ethanol on behaviour requires FMRP
Since FMRP is important for ethanol-induced GABABR plasticity, we examined antidepressant and anxiolytic-like effects of ethanol on behaviour in Fmr1 KO mice. Interestingly, ethanol administration did not affect the behaviours of Fmr1 KO mice in the splash and open field tests compared with saline-treated mice (Supplementary Fig. 6a–e). Surprisingly, the basal state of immobility in the FST in Fmr1 KO mice is equivalent to ethanol-injected WT mice (Fig. 8). To explore this paradox, we examined the requirement of GABABR activation in ethanol-induced decreases in immobility by using CGP-35348 to inhibit postsynaptic GABABRs. We previously showed that GABABR antagonism blocked the antidepressant-like behaviour produced by NMDAR antagonist in the FST6. GABABR inhibition alone did not affect the immobility of saline-injected WT mice in the FST, similar to what we observed previously (Fig. 8)6. CGP-35348, however, abolished the ethanol-induced antidepressant behaviour, demonstrating a requirement for GABABR activation in ethanol-triggered reduction of immobility. Neither ethanol, CGP-35348, nor ethanol+CGP-35348 treatment in Fmr1 KO mice produced immobility scores that were significantly different from saline-treated Fmr1 KO mice. These findings collectively demonstrate that GABABRs and FMRP are necessary to elicit the ethanol-mediated antidepressant response.
Emerging behavioural and molecular evidence demonstrate that NMDAR antagonists act as rapid antidepressants5,6,8,24,40. Because it has long been speculated that individuals with major depressive disorders self-medicate with alcohol, we examined whether ethanol, which blocks NMDARs9, acts through the same synaptic pathways as NMDAR antagonists. Until this study, the molecular mechanisms shared by alcohol and antidepressants were unexplored. Here we provide molecular and behavioural evidence that acute alcohol exposure elicits antidepressant-like behaviours that persist up to 24 h after administration (Fig. 1), supporting the hypothesis that ethanol initiates lasting antidepressant activity. We have previously demonstrated that NMDAR inhibition by rapid antidepressants induces two key molecular changes that are responsible for the rapid antidepressant response, namely (1) an increase in GABABR protein synthesis and (2) a shift in GABABR function that increases dendritic calcium signalling6,8. Our current work shows that these same signature changes are produced by acute ethanol exposure (Figs 5, 6, 7).
Surface expression of functional GABABRs requires the dimerization of GABABR1 and R2 subunits. Without GABABR2, GABABR1 is retained in the endoplasmic reticulum32. Our current studies show that the release of GABABR2 mRNA translational repression by FMRP is necessary for the ethanol-induced increase in surface GABABRs with NMDAR blockade (Figs 4, 6 and 8; Supplementary Fig. 3). Reduction of FMRP, as seen in animal models of FXS, is associated with elevated protein synthesis of target mRNAs16. While we have demonstrated that FMRP associates with GABABR1 and R2 mRNAs, the loss of FMRP has a profound effect on GABABR2 protein expression in dendrites. Constitutive loss of FMRP, as observed in many of its targets, abrogates stimulus-dependent mRNA transport and translation of target mRNAs41,42,43,44. We show that the ethanol-induced increase in GABABR2 protein is also absent in Fmr1 KO mice.
The question of why GABABR2 is uniquely affected by acute ethanol and its dependence on reduced FMRP levels is intriguing. Both GABABR1 and GABABR2 mRNAs were detected in the FMRP RIP (Fig. 3). Interestingly, in vivo FMRP influences GABABR1 expression to a lesser extent compared with GABABR2, suggesting that FMRP may act in concert with other repressors such as microRNAs to tightly regulate GABABR1 expression45. Moreover, our immunostaining and protein synthesis assays suggest that the increased protein levels of GABABR1 are not due to new protein synthesis in the dendrites, even in Fmr1 KO mice (Figs 5 and 6). These results indicate that the overall increase in synaptic GABABR1 expression in Fmr1 KO mice may be due to an increase in presynaptic GABABR1 expression, either through protein synthesis or increased protein stability. Notably, FMRP has been localized to axons and presynaptic terminals46,47. Further exploration into presynaptic and postsynaptic GABABR expression and function in AUD and FXS is warranted.
The ‘GABA hypoinhibition theory’ posits that loss of inhibition is a leading cause in many of the neurological symptoms observed in FXS48. While studies showing reduced inhibition in models of FXS have focused on decreased expression of GABAAR subunit mRNA and protein49, GABABR protein expression and dendritic signalling has not been explored. Interestingly, the GABABR agonist baclofen has shown promise in treating FXS. (R)-baclofen administration in vitro corrects the elevated basal protein synthesis normally seen in Fmr1 KO mice, and rescues synaptic abnormalities such as increased spine density50. In addition, baclofen administration reduced symptoms related to FXS in Fmr1 KO mice50,51. Recent studies have shown that the excitatory drive to fast spiking inhibitory neurons is reduced in the cortex of Fmr1 KO mice52. Our data expand on these findings by suggesting that postsynaptic GABABR coupling to inwardly rectifying G protein-coupled potassium channels (GIRK) is absent in Fmr1 KO neurons (Fig. 7). Collectively, these results may imply that the therapeutic effects of baclofen in the Fmr1 KO mouse may be due to the activation of presynaptic GABAB receptors that may in turn reduce glutamate release and reduce hyperactive metabatropic glutamate receptor mGluR signalling in the hippocampus53.
Changes in gene expression and protein synthesis are essential for normal neuroplasticity, but these crucial processes are dysregulated by drug addiction18,54. Several lines of evidence support parallel changes in GABABR mRNA translation/signalling as a result of NMDAR blockade that may be critical for alcohol actions. First, NMDAR antagonists mimic some effects of ethanol in humans55, suggesting common biochemical/electrophysiological signalling pathway(s). Second, changes in GABABR2 brain gene expression correlates with lifetime alcohol consumption, supporting a role for altered GABABR signalling in AUD56. Third, although controversial, the GABABR agonist baclofen may decrease alcohol consumption in some alcoholics57. In summary, our data define a common molecular paradigm for alcohol and rapid antidepressants, and identify a mechanism for the initial antidepressant effects of alcohol. A shift in GABABR signalling is observed with both rapid antidepressants and acute ethanol treatment, which may provide insight into the molecular basis for the high comorbidity between major depressive disorder and AUD.
Note added in proof: Spencer et al. found fmrp mediated changes ion channels with chronic ethanol exposure58.
Primary hippocampal neurons were prepared as previously described by Niere et al.43. Briefly, hippocampi were extracted from postnatal day 1–3 Sprague–Dawley rat pups, WT C57BL/6 mouse pups, or Fmr1-knockout (Fmr1 KO) mouse pups on a C57BL/6 background. The tissue was dissociated and plated in neurobasal A medium supplemented with B27, glutamine, and 1% fetal bovine serum. Cultures were plated at a density of ∼100,000 cells per 12 mm on glass coverslips that had been coated overnight with 50 μg ml−1 poly-D-lysine and 25 μg ml−1 laminin in borate buffer. Cultures were fed after 1 day in vitro (DIV), and media was replaced approximately once a week with either fresh rat culture media (neurobasal A supplemented with B27, glutamine and 3 μM AraC) or fresh mouse culture media (glial-conditioned media with 3 μM AraC) until cultures were used at DIV 14–21.
In vitro pharmacology
Primary hippocampal neurons were treated in ethanol vapour chambers according to a method adapted from Chandler et al.59. Ethanol vapour chambers were prepared by placing a reservoir of 31.5 mM ethanol (105% of the desired ethanol concentration, that is, 30 mM) in a plastic container with 24-well culture plates containing neuronal cultures in which 30 mM ethanol was added to the culture media. Chambers were filled with 95% O2/5% CO2 and cultures were incubated for 2 h at 37 °C. Cultures treated with vehicle (H2O) were incubated in the same manner but in the absence of ethanol. For calcium imaging, ethanol was added directly to HEPES-based artificial cerebral spinal fluid (ACSF in mM: 100 NaCl, 10 HEPES (pH 7.4), 3 KCl, 2 CaCl2, 1 MgCl2, 10 glucose) that was adjusted to match the osmolarity of cell culture media for live imaging. For GABABR activation neurons were treated with (R)-baclofen (50 μM, Tocris). For Figs 5 and 6, cultured hippocampal neurons were pre-treated with cycloheximide (50 μM, Tocris) for 10 min before ethanol treatment. For Supplementary Fig. 3, neurons were treated with Ro-25-6981 (10 μM, Tocris) or Veh (H2O) for 2 h. All cultures were treated at 14–21 DIV. Following treatment, cultures were immediately fixed or live imaged.
In vivo pharmacology
Male C57BL/6 mice (Charles Rivers) or Fmr1-knockout (Fmr1 KO) mice on a C57BL/6 background (at least 7 weeks old) were given i.p. injections of either 200 μl saline or 2.5 g kg−1 ethanol (in a volume of 200 μl saline)23. For Fig. 8, CGP-35348 (100 mg kg−1) was i.p. injected with or without ethanol (in a volume of 200 μl saline). All animals were housed four mice per cage according to genotype. All treatments were administered to one mouse per home cage. At the time of drug treatment, animals were coded by number. During the behavioural tasks, animal performance was video recorded, and then later blindly scored. In certain tasks (for example, open field), the animals were scored by a computer program and blinding was not necessary during that process. For western blots the hippocampi were isolated 30 min post injection and flash-frozen. All experiments were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and approved by the UT-Austin Institutional Animal Care and Use Committee (IACUC).
Primary neuronal cultures on glass coverslips were fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min, washed three times with phosphate-buffered saline (PBS) and permeabilized in 0.25% Triton X-100 in PBS for 5 min. For FMRP staining, neurons were fixed and permeabilized in 100% methanol at −20 °C for 10 min. Neurons were washed three times in PBS and then blocked (10% normal goat serum in PBS) for 30 min at room temperature. Primary antibodies were incubated in blocking buffer at 4 °C overnight. Neurons were washed three times for 10 min with PBS, and then incubated in secondary antibody in blocking buffer for 1 h at room temperature, and washed three times for 10 min with PBS before mounting in Fluoromount with DAPI (SouthernBiotech, 0100-20) as outlined in Sosanya et al.60. Surface staining was performed similarly to Workman et al.6. Neurons were first fixed in 4% PFA for 10 min on ice, washed three times in PBS, blocked with 3% normal goat serum, and then incubated in primary surface antibody in 3% blocking buffer overnight at 4 °C. Following primary surface antibody incubation, neurons were washed six times for 10 min each in PBS, then permeabilized with 0.25% Triton X-100 in PBS for 5 min, followed by three washes for 10 min each in PBS, and again incubated in primary total antibody in 3% blocking buffer overnight at 4 °C. Neurons were washed three times for 10 min each with PBS, and then incubated in secondary antibody in 3% blocking buffer for 1 h at room temperature, and finally washed four times for 10 min with PBS before mounting in Fluoromount with DAPI to slides. The primary antibodies used were: Total GABABR1 (1/50 dilution; Santa Cruz, sc-14006), Surface GABABR1 (1/200 dilution; Abcam, ab55051), GABABR2 (1/100; Neuromab 75-124), FMRP (1/500 dilution; Abcam ab17722), MAP2 (1/2000 dilution; Abcam ab5392), GFP (1/1,000 dilution; Aves, GFP-1020). Secondary antibodies included: Alexa488, 555 and 647 developed in goat (1/500 dilution; Life Technologies, A-11039, A-11017, A-31621, A-21430, A-21449, A-21240 and A-21237).
Adeno-associated viral vectors
The FMRP and tdTomato coding sequences were cloned into separate adeno-associated viral (AAV) vectors containing a mouse synapsin promoter, a woodchuck post-transcriptional regulatory element and SV40 poly-adenylation sequence between flanking AAV2 inverted terminal repeats. rAAVs were assembled using a modified helper-free system (Stratagene) as serotype 2/1 (rep/cap genes) viruses, and collected and purified over sequential caesium chloride gradients as previously described61. Viral titres were >1 × 109 infectious particles per microlitre. For FMRP and tdTomato co-infections, rAAV:mSYN-FMRP and rAAV:mSYN-tdTomato were mixed at a ratio of 4:1. One microlitre of the resulting rAAV mix was used per coverslip of primary cultured neurons. Imaging was performed ∼1 week after infection.
Live calcium imaging
Dissociated hippocampal cultures were prepared from WT and Fmr1 KO mice as described43. Neurons at 14–21 DIV were used for live calcium imaging. Neurons were treated as outlined in in vitro pharmacology above. Before imaging, cells were incubated in ACSF with Oregon Green 488 BAPTA-1 AM (OGB, 200 μM; 30 min; 37 °C; ThermoFisher) as described6. After OGB incubation, cells were transferred to fresh ACSF (37 °C) for imaging (1 frame per 20 s). Baseline calcium signal was imaged (1 min), after which (R)-baclofen (50 μM, Tocris) or vehicle (H2O) was added. For ethanol-treated cells, the neurons were incubated with OGB and imaged in ACSF containing ethanol (30 mM). Neurons were imaged for 800 s at room temperature. Quantification of the calcium signal was performed using Metamorph (Molecular Devices) as described6. Briefly, dendritic regions of interest (ROI) that were at least 5 μm from the soma were analysed. The mean intensity values for each ROI at each time were averaged as baseline (F0). The ROI intensity values obtained at each time point after the addition of baclofen or vehicle were averaged (F). The equation, ΔF/F=((F−F0)/F0), was used to measure the change in signal and data were plotted as a percentage of the baseline.
BONCAT-PLA was performed using Click-it Metabolic Labeling azidohomoalaine (AHA), Biotin-Alkyne and Click-iT Reaction buffer kit (Life Technologies). Proximity ligation assay (PLA) was performed using Duolink kit (Duolink, Sigma)38. Briefly, primary hippocampal neuronal cultures were incubated in a methionine-free ACSF media for 30 min. AHA was then added to the media just before neurons were treated with ethanol for 2 h as previously described. Neurons were fixed in 4% PFA for 15 min, washed two times for 5 min with 3% bovine serum albumin (BSA) in PBS, followed by permeabilization with 0.25% Triton X-100 in PBS for 15 min, and washed as before. Neurons were incubated for 30 min at room temperature in Cell Buffer Additive/Click-it Cocktail according to manufacturer directions. Neurons were washed as before and then blocked and incubated with primary antibody as previously described. Next, neurons were incubated in the appropriate PLA probes diluted in blocking buffer and secondary antibody at 37 °C for 1 h. Neurons were washed in RT Buffer A two times for 5 min, and incubated in ligation solution at 37 °C for 30 min, and washed again in Buffer A. Neurons were incubated in amplification solution at 37 °C for 2–3 h, followed by washing in RT Buffer B two times for 10 min and 1% buffer B for 1 min. Last, neurons were mounted to slides in Duolink mounting media for imaging. Primary and secondary antibodies included: GABABR1 (1/50 dilution; Santa Cruz, sc-14006), GABABR2 (1/100; Neuromab 75-124), MAP2 (1/2,000 dilution; Abcam, ab5392), biotin/α-rabbit (1/500; Sigma, SAB3700857), Alexa488 (1/500; Life Technologies, A-11039). PLA probes used: Rabbit Minus (1/5; Duolink, 82005), Mouse Plus (1/5; Duolink, 82001).
Microscopy and analysis
Images were acquired with a Leica SP5 confocal microscope under a × 63 oil immersion lens for fixed tissue or a × 63 water immersion lens for live imaging. Max projected images were used for immunostaining analysis from 10 μm Z-stacks of 1,024 × 1,024 pixels obtained using a 400-Hz scan rate60. For each experiment, all images were collected using the same settings. Fixed images were analysed using NIH imaging software ImageJ, and live imaging quantification was performed with Metamorph (Molecular Devices, Sunnyvale, CA). Background signal was determined by shifting the ROI adjacent to the dendrite being traced, but void of all processes. Dendritic signal was background subtracted and averaged every 10 μm using a customized R script.
Western blot analysis
Protein was isolated from hippocampal synaptoneurosomes prepared from male mice age 7–8 weeks treated with ethanol or vehicle as previously described. synaptoneurosomes were prepared by homogenizing hippocampal tissue in homogenization buffer (20 mM HEPES pH 7.4, 5 μM EDTA pH 8.0, and protease inhibitor cocktail). Homogenate was filtered through a 100-μm nylon filter followed by a 5-μm filter, and centrifuged at 14,000g for 20 min at 4 °C (ref. 62). The pellet was resuspended in RIPA buffer (150 mM NaCl; 10 mM Tris pH 7.4, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA and protease inhibitor cocktail) and centrifuged for 10 min. The supernatant was used for western blot analysis. Protein was separated on a 4–20% gradient SDS–polyacrylamide gel. The gel was then transferred to a nitrocellulose membrane, blocked in 5% non-fat dry milk in tris-buffered saline and 0.1% Tween20 (TBST) for 1 h, and incubated with primary antibody in blocking buffer overnight at 4 °C. The blot was washed in TBST three times for 10 min each, incubated in secondary antibody for 1 h, and washed as before. Blots were imaged using a LICOR Odyssey imaging system, and ImageJ was used for densitometry analysis. Representative images are pseudocoloured with black (lowest intensity at 0 pixels) to red (highest intensity at 255 pixels) using LICOR Image Studio software. Primary antibodies used consisted of: GABABR1 (1/400 dilution; Santa Cruz, sc-14006), GABABR2 (1/800; Neuromab 75-124), alpha-Tubulin (1/2,000 dilution; Sigma, T6074). Secondary antibodies included: anti-mouse-IR-Dye 800 (1/5,000 dilution excluding tubulin at 1/10,000 dilution; LICOR, 96-32210) and anti-rabbit Alexa680 (1/5,000 dilution; Invitrogen, A-21084).
Cortices from 6-week-old C57BL/6 and Fmr1 KO male mice were collected and flash-frozen on dry ice. RIP was performed by modified method of Jain et al. and Keene et al.63,64. Tissue was homogenized and lysed with a cordless pestle motor and disposable pellet mixers (VWR) in polysome lysis buffer (10 mM HEPES pH 7.0, 100 mM KCl, 25 mM EDTA, 5 mM MgCl2, 1 mM DTT, 0.5% NP-40) in a 1:1 tissue-buffer ratio. RNaseOUT (Thermo) and protease/phosphatase inhibitors (Halt Protease and Phosphatase Cocktail, Pierce Biotechnology) were freshly added to samples. Samples were rotated for 10 min at 4 °C to induce swelling and then flash-frozen on dry ice. Samples were thawed by holding between fingers at room temperature to lyse and nuclei were pelleted at 3,000g for 10 min. Lysates obtained above were pre-cleared by adding 50 μl of washed magnetic bead slurry (Protein A Dynabeads, Thermo) and rotating for 30 min at 4 °C. To bind the antibody to the beads, 50 μl of magnetic beads slurry was washed and then resuspended in eight volumes of NT-2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mM DTT, 0.05% NP-40 with RNaseOUT/protease and phosphatase inhibitors added fresh)+5% BSA. Ten micrograms of either FMRP (Abcam, ab17722) or IgG (Santa Cruz Biotechnologies, sc-2027) antibodies was added to the beads and rotated for 10 min at room temperature. Antibody-bound beads were washed four times with ice-cold NT-2 buffer. For the immunoprecipitation, 4.5 mg of protein from pre-cleared lysates was added to an RNase-free microcentrifuge tube containing the antibody-bound beads. Input collected at this step for downstream analysis was either 1% of the final pre-cleared lysate volume in the immunoprecipitation reaction (for immunoblotting) or 10% of the final pre-cleared lysate volume (to normalize in qPCR). The antibody–bead-lysate mixture was then diluted at a ratio of 1:5 with NET-2 buffer (20 mM EDTA pH 8.0, and 1 mM DTT in NT-2 buffer; RNaseOUT and protease/phosphatase inhibitors added fresh) and rotated for 1 h at room temperature. Beads were quickly washed six times in ice-cold NT-2 buffer and immediately resuspended in 350 μl TRI Reagent Solution (Ambion) for 10 min at room temperature. Beads were pelleted and the supernatant was removed and resuspended in 350 μl of absolute ethanol. RNA was extracted by applying ethanol-resuspended samples to spin column from the Direct-zol RNA MiniPrep Kit (Zymogen) according to the manufacturer’s instructions. Eluted RNA (25 μl) was DNase treated using the TURBO DNA-free kit (Thermo).
cDNA synthesis and quantitative real-time PCR
DNase-treated RNA samples were reverse-transcribed to complementary DNA using the iScript cDNA Synthesis Kit (Bio-Rad) in a 20 μl volume according to the manufacturer’s instructions. Quantitative real-time PCR (qRT–PCR) was performed in 20 μl reaction volume using the iQ SYBR Green Supermix (Bio-Rad) and primers for GABABR1 (GeneCopoeia, MQP031832), GABABR2 (GeneCopoeia, MQP026008), CaMKIIα (GeneCopoeia, MQP028785), and Cacna2δ2 (GeneCopoeia, MQP032309). qRT–PCR was run with the following protocol: 95 °C for 10 min, 40 cycles of 95 °C for 15 sec followed by 60 °C for 1 min, 95 °C for 1 min, and 55 °C for 1 min. Relative fold-enrichment was determined by the equation ΔΔCt=2−(Ct FMRP RIP−Ct IgG RIP)−(Ct FMRP input−Ct IgG input) (ref. 65).
Forced swim test
Male mice were tested in the forced swim test 24 h post injection as described previously6,8. Mice were individually placed into a cylinder containing 3 l of water (25 °C) for 6 min. Each session was video recorded and the last 4 min of the sessions were later scored blindly for immobility. Animals were scored for escape-directed behaviours. The water was replaced between animals. Experiments were repeated by three independent experimenters. Data were normalized by experimenter. Power analysis was performed in R Programming66 to predict sample size for all behavioural tests. This sample size was used as a guideline for the WT animals, however since transgenic animals were used, the exact sample size for each group may not have been possible. Transgenic animal sample size was as close as possible to that which was calculated, due to limitations of litter size.
Twenty-four hours after animals were injected i.p. with either saline or ethanol, they were studied in the open field test similar to Treit et al.31. Mice were individually placed in a 40 cm × 40 cm × 35 cm arena with opaque walls. Each test session lasted 30 min under 85 lux illumination. Sessions were video recorded and analysed via ANY-Maze (Stoelting, Wood Dale, IL). Mice were considered to be in the centre of the maze if they entered a 18.5 cm × 18.5 cm area in the centre of the apparatus. Mice were returned to their home cage at the end of the test session, and the arena was wiped down with 70% ethanol before the start of each run.
Two and a half hours after open field testing, animals underwent the splash test27,28. Cage mates were moved from their home cage to a holding cage, and each animal was individually tested in its home cage. Two-hundred microlitres of 10% sucrose was applied to the dorsal fur of the mouse. Mice were monitored and video recorded for 5 min and then moved to a different holding cage. Videos were later scored blindly for latency to initiate grooming and for total time spent grooming. Grooming behaviour included licking, grooming with forepaws, and scratching.
Power analysis was performed in R Programming66 to predict sample size. Prism software (GraphPad) was used for all statistical analyses. Statistical comparisons were made using one-way analysis of variance (ANOVA), two-way ANOVA, Student’s t-test or χ2-test with Yates. Outliers were determined using Grubbs’ test (α=0.05). All data are expressed as mean±s.e.m.
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This work was supported by an NIH-NIAAA pilot grant provided by the Integrated Neuroscience Initiative on Alcoholism (KRG), NSF grant IOS-1355158 (KRG), Department of Defense USAMRMC Award W81XWH-14-10061 (KRG), NIH-NIAAA grants AA012404 and AA013517 (RAH), NIH-NIAAA 5T32AA007471-29 (SAW), NSF Postdoctoral Research Fellowship in Biology DBI-1306528 (FN) and DBI-1103738 (LPC). We thank Dr Judson Chandler for advice regarding the treatment and maintenance of cultured neurons with ethanol. We thank Yesha Shah for help in scoring behavioural experiments. We thank Drs Jody Mayfield and R. Dayne Mayfield for helpful advice concerning this manuscript.
The authors declare no competing financial interests.
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Wolfe, S., Workman, E., Heaney, C. et al. FMRP regulates an ethanol-dependent shift in GABABR function and expression with rapid antidepressant properties. Nat Commun 7, 12867 (2016). https://doi.org/10.1038/ncomms12867
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