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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC 2012 September 25.
Published in final edited form as:
PMCID: PMC3457695
NIHMSID: NIHMS347954

Decrease in calcium concentration triggers neuronal retinoic acid synthesis during homeostatic synaptic plasticity

Abstract

Blockade of synaptic activity induces homeostatic plasticity, in part by stimulating synthesis of all-trans retinoic acid (RA), which in turn increases AMPA receptor synthesis. However, the synaptic signal that triggers RA synthesis remained unknown. Using multiple activity-blockade protocols that induce homeostatic synaptic plasticity, here we show that RA synthesis is activated whenever postsynaptic Ca2+-entry is significantly decreased, and that RA is required for up-regulation of synaptic strength under these homeostatic plasticity conditions, suggesting that Ca2+ plays an inhibitory role in RA synthesis. Consistent with this notion, we demonstrate that both transient Ca2+-depletion by membrane-permeable Ca2+-chelators and chronic blockage of L-type Ca2+-channels induces RA synthesis. Moreover, the source of dendritic Ca2+ entry that regulates RA synthesis is not specific as mild depolarization with KCl is sufficient to reverse synaptic scaling induced by L-type Ca2+-channel blocker. By expression of a dihydropyridine-insensitive L-type Ca2+-channel, we further show that RA acts cell-autonomously to modulate synaptic transmission. Our findings suggest that in synaptically active neurons, modest ‘basal’ levels of postsynaptic Ca2+ physiologically suppress RA synthesis, whereas in synaptically inactive neurons, decreases in the resting Ca2+-levels induce homeostatic plasticity, by stimulating synthesis of RA that then acts in a cell-autonomous fashion to increase AMPA receptor function.

Keywords: retinoic acid, homeostatic plasticity, AMPA receptor, synaptic scaling, dendritic calcium, L-type calcium channel

Introduction

Normal brain function requires that neurons operate within a dynamic range of overall activity, which is essential for optimal information coding. The processes that maintain network activity within this dynamic range are collectively called homeostatic plasticity (Turrigiano and Nelson, 2004; Davis, 2006). Synaptic scaling is a form of homeostatic plasticity in which synaptic strength is regulated in a multiplicative fashion (i.e., stronger synapses are changed proportionally more than weaker synapses), primarily through changes in the abundance of postsynaptic AMPA-type glutamate receptors (Turrigiano et al., 1998; Thiagarajan et al., 2005). Previously we reported a critical role for RA in the induction of synaptic scaling (Aoto et al., 2008). Inhibition of action potential firing by tetrodotoxin (TTX), along with blockade of NMDA-type glutamate receptors by D-2-amino-5-phosphonovalerate (APV), stimulated synthesis of RA in neurons, and RA was both necessary and sufficient to induce local translation of AMPA receptors in neuronal dendrites and to trigger synaptic scaling (Aoto et al., 2008). These findings placed RA into a key role in regulating synaptic strength. However, synaptic up-scaling can be induced by several different manipulations. For example, a form of synaptic scaling induced by prolonged blockade of action potential firing with TTX involves cell-wide, transcription-dependent changes (Turrigiano and Nelson, 2004). More recently, accumulating evidence supports the existence of a fast adaptive form of synaptic scaling that involves activation of protein synthesis locally in neuronal dendrites, allowing adjustment of synaptic strength at spatially discrete locations within a neuron (Ju et al., 2004; Sutton et al., 2006; Aoto et al., 2008). The latter form of synaptic scaling is induced by blockade of synaptic transmission mediated by a specific glutamate receptor type alone or concurrently with action potential blockade (Turrigiano et al., 1998; Ju et al., 2004; Thiagarajan et al., 2005; Sutton et al., 2006). Although RA has been shown to regulate dendritic protein synthesis in an activity-dependent manner, it is unknown how general the role of RA is in homeostatic plasticity, and what signal triggers RA synthesis.

In this study, we report that multiple manipulations that block glutamatergic synaptic transmission, alone or concurrently with action potential blockade, stimulate RA synthesis. Synaptic scaling induced by such manipulations is mediated by synaptic insertion of GluA2-lacking AMPA receptors, and can be blocked by RA synthesis inhibitors. Moreover, simply blocking L-type calcium channels or chelating intracellular calcium strongly activates RA synthesis. Together, our findings suggest that RA is a general component in multiple forms of synaptic scaling, whose expression is mediated by local synthesis and synaptic insertion of GluA2-lacking AMPA receptors, and that removing repression of RA synthesis by calcium–dependent signaling mechanisms underlies activity-regulated RA synthesis and synaptic up-scaling.

Materials and Methods

Drugs and Chemicals

The following drugs and chemicals were purchased from Sigma Aldrich: all-trans retinoic acid (RA), philanthotoxin-433 (PhTX), 4-diethylamino-benzaldehyde (DEAB), nifedipine, BAPTA-AM, and picrotoxin. Tetrodotoxin (TTX), D-(−)-2-Amino-5-phosphonopentanoic (D-APV) and 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) were purchased from Tocris Biosciences (Ellisville, MO). EGTA-AM was purchased from Invitrogen.

Cell Cultures and Drug Treatment

Primary hippocampal cultures were prepared from the brains of rats at embryonic day 22 and maintained in serum-free Neurobasal medium supplemented with B-27 and Glutamax (GIBCO-Brl, Grand Island, NY) for 2 to 3 weeks in vitro (Nam and Chen, 2005). Manipulations used to induce synaptic scaling in dissociated cultures include: TTX+APV (1 μM TTX+100 μM APV, 24 hours ); CNQX (10μM, 24 hours); TTX+CNQX (1 μM TTX+10 μM CNQX, 24 hours), TTX (1 μM, 48 hours), and nifedipine (10μM, 24 hours). Stock solution of 4-diethylamino-benzahldehyde (DEAB) in DMSO was freshly made right before treatment, and 10 μM DEAB was applied where indicated. Fifty-minute treatment of either 10μM BAPTA-AM or 10μM EGTA-AM was used to decrease the intracellular calcium level.

RARE assay

The 3xRARE-EGFP reporter construct is as described (Aoto et al., 2008). Three copies of the retinoic acid response element were placed upstream of a TK promoter driving EGFP. Dissociated cultures used for RARE imaging were transfected using Lipofectamine 2000 (Invitrogen) with a protocol described previously (Aoto et al., 2008), and were fixed with 4% paraformaldehyde (15 min, room temperature) and washed with PBS before mounting. Images were acquired and quantified as described previously (Nam and Chen, 2005) using an Olympus FV1000 BX61WI laser-scanning confocal microscope.

Electrophysiology

Whole-cell patch-clamp recordings were made at room temperature from 13–14 DIV cultured neurons, with 4–6 MΩ patch pipettes filled with an internal solution containing (in mM) 120 CsCl, 2 MgCl2, 5 EGTA, 10 HEPES, 0.3 Na3-GTP, 4 Na2-ATP (pH 7.35). Cultures were continuously superfused with external solution (in mM, 100 NaCl, 26 NaHCO3, 2.5 KCl, 11 glucose, 2.5 CaCl2, 1.3 MgSO4, 1.0 NaH2PO4). For mEPSC recording, tetrodotoxin (1 μM) and picrotoxin (100 μM) were included in the external saline. Cells were held at −60 mV. For PhTx recordings, 5 μM PhTx or vehicle control was bath perfused for 10 min before recording. Miniature responses were analyzed with Mini Analysis Program from Synaptosoft.

Statistical Analysis

Single-factor ANOVA was used for statistical analysis. Values are presented as mean ± SEM in the figures.

Results

RA synthesis is induced by multiple activity blockade protocols producing homeostatic plasticity

We first examined whether RA synthesis is stimulated by activity blockade with different protocols that induce synaptic scaling (Turrigiano et al., 1998; Ju et al., 2004; Thiagarajan et al., 2005; Sutton et al., 2006). We chose four manipulations: blocking action potentials alone with TTX for 24 hours and for 48 hours, blocking AMPA receptors with CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) for 24 hours, or blocking both action potentials and AMPA receptors with TTX and CNQX, respectively, for 24 hours. Additionally, we included a 24-hour TTX+APV treatment (blocking action potentials and NMDA receptors) as a positive control because of its well-characterized effects on RA synthesis (Aoto et al., 2008). All experiments were performed with cultured hippocampal neurons, which were transfected at 14 days in vitro (DIV) with a plasmid containing multiple copies of a RA response element (RARE) driving transcription of GFP to measure RA levels by monitoring the GFP intensity (Aoto et al., 2008).

Consistent with our previous report, 24 hr TTX+APV treatment significantly increased RA levels in neurons (Fig. 1A and B). Blocking AMPA receptor-mediated synaptic transmission alone (CNQX, 24 hr) or together with action potential blockade (TTX+CNQX, 24 hr) also significantly increased RA levels in neurons (Fig. 1A and B). Interestingly, TTX alone applied for either 24 or 48 hrs did not induce RA synthesis ((Aoto et al., 2008) and Fig. 1A and B), suggesting that blocking action potentials alone is insufficient for activating RA synthesis. All increases in RARE-GFP reporter expression were prevented by concurrent application of 4-diethylamino-benzaldehyde (DEAB), an inhibitor of RA synthesis (Russo et al., 1988)(Fig. 1A and B), confirming that the observed enhancement of GFP expression reflects an increase in RA synthesis. Additionally, expression of GFP from the same reporter plasmid lacking the RARE sequence failed to respond to activity blockade (Fig. 1C), suggesting that the increase in RARE-GFP expression is due to specific transcriptional activation by RA, and not due to a generally altered transcription or translation by activity blockade.

Figure 1
Prolonged blockade of synaptic transmission induces RA synthesis

RA is required for multiple forms of synaptic scaling

We next recorded miniature excitatory postsynaptic currents (mEPSCs) from neurons treated by activity blockade to evaluate changes in synaptic transmission. As expected, all four activity-blockade protocols tested (24 hour treatment of TTX+APV, CNQX, TTX+CNQX, or 48 hr TTX) significantly increased mEPSC amplitudes (Fig. 2A and B). The mEPSC frequency was not significantly changed in any of the groups (Fig. 2B, 14 DIV groups). Addition of DEAB to the activity blockade cocktail completely blocked the increases in mEPSC amplitudes induced by the TTX+APV, CNQX, or TTX+CNQX treatments (Fig. 2B). However, the mEPSC amplitude increases induced by 48 hr treatments with TTX alone were unaffected (Fig. 2B). This result, together with the RARE-GFP reporter expression result, demonstrates that RA is critically involved in synaptic scaling induced by blocking postsynaptic depolarization through glutamate receptor activation, but that a second type of synaptic scaling induced by TTX alone employs an RA-independent mechanism.

Figure 2
RA-dependence of synaptic scaling induced by different activity blockade protocols

RA is known as a transcriptional activator that functions during development. Recent evidence indicates that in mature neurons, RA regulates protein synthesis in a transcription-independent manner, and participates in synaptic scaling by activating local synthesis of the AMPA receptor GluA1 subunit, resulting in postsynaptic insertion of Ca2+-permeable homomeric GluA1-containing AMPA receptors (Aoto et al., 2008; Maghsoodi et al., 2008; Poon and Chen, 2008). We therefore examined the subunit composition of the newly inserted synaptic AMPA receptors induced by different synaptic scaling protocols. The increase in mEPSC amplitude in neurons treated with TTX+APV, CNQX, or TTX+CNQX was fully reversed by philanthotoxin-433 (PhTx), a blocker of GluA2-lacking AMPA receptors (Fig. 2C), indicating that the increase in synaptic strength is caused by synaptic insertion of AMPA receptors lacking GluA2. Synaptic scaling induced by a 48 hr treatment with TTX alone, however, was insensitive to PhTx treatment, consistent with previous reports showing that GluA1/GluA2 heteromeric AMPA receptors are incorporated into the synapses in response to this treatment (Wierenga et al., 2005).

Previous studies showed that synaptic scaling induced by prolonged blockade of AMPA receptors produces an increase in both mEPSC amplitude and frequency, with the latter effect mediated by postsynaptic synthesis and release of brain-derived neurotrophic factor (BDNF) (Thiagarajan et al., 2005; Jakawich et al., 2010; Lindskog et al., 2010). In our experiments, we employed cultured hippocampal neurons that are younger (14 DIV) than those in the published studies, and observed only a trend of an increase in mEPSC frequency that was not statistically significant (Fig. 2B and 2C). Given that BDNF expression in the brain increases steeply between postnatal day 15 and 20 (Schoups et al., 1995), we examined synaptic scaling induced by a 24 hr CNQX treatment in older cultured hippocampal neurons at 21 DIV. Indeed, blocking AMPA receptors for 24 hours in these older neurons induced a significant increase in both mEPSC amplitude and frequency (Fig. 2B and 2C). Consistent with a previous report (Jakawich et al., 2010), the increase in mEPSC amplitude was completely reversed by blockers specific for GluA2-lacking receptors such as PhTx, but the mEPSC frequency remained significantly higher than in control neurons even in the presence of PhTx (Fig. 2C). Importantly, this form of synaptic scaling, which involves changes in both pre- and post-synaptic compartments, is blocked by DEAB (Fig. 2B), indicating that RA synthesis surprisingly is also required for inducing these increases in mEPSC frequency, which is surprising since such increases are generally thought to represent a presynaptic effect.

Suppression of Ca2+-signaling is sufficient to induce RA synthesis

Why is RA synthesis selectively induced by glutamate receptor blockade but not by abrogating action potential generation? Although GluA2-containing AMPA receptors are virtually Ca2+-impermeable, activation of AMPA receptors is expected to induce sufficient depolarization to increase Ca2+-influx through Ca2+-permeable NMDA receptors and L-type Ca2+ channels. We therefore hypothesized that under resting conditions, RA-synthesis is suppressed by postsynaptic Ca2+-influx induced by spontaneous miniature excitatory synaptic events or by network activity.

To test this hypothesis, we applied the membrane permeable Ca2+-chelators BAPTA-AM and EGTA-AM to cultured neurons expressing the RARE-GFP reporter. Short (50 min) treatments of neurons with either BAPTA-AM or EGTA-AM dramatically stimulated RARE-regulated GFP expression (Fig. 3) but not GFP expression from a control plasmid lacking RAREs (normalized to control treatment: BAPTA-AM, 99% +/− 11.4%, n = 16; EGTA-AM, 119% +/− 6.7%, n = 12; p > 0.2). The RARE-dependent increase in GFP expression induced by the Ca2+-chelators was blocked by DEAB (Fig. 3), confirming that decreasing the postsynaptic Ca2+-concentration induces RA synthesis.

Figure 3
Calcium-dependent regulation of RA synthesis

To further test the regulation of RA synthesis by postsynaptic Ca2+ in the context of synaptic scaling, we examined whether RA synthesis can be activated by blockade of L-type Ca2+-channels. Primarily localized in the soma and dendrites (Hell et al., 1993), L-type Ca2+-channels significantly contribute to dendritic Ca2+-signaling. Prolonged blockade of L-type Ca2+-channels has been shown to mimic the effects of AMPA receptor blockade and induce synaptic scaling (Thiagarajan et al., 2005). Indeed, treating neurons for 24 hours with nifedipine, an L-type Ca2+-channel blocker, enhanced RARE-GFP reporter expression (Fig. 3), but not expression of the control GFP reporter (normalized to control treatment: 96.3% +/− 12.3%, n = 10, p > 0.8). DEAB again blocked the increase in the RARE-GFP signal (Fig. 3), suggesting that the increase is specifically due to RA synthesis but not a global increase in transcription or translation triggered by reduced Ca2+-levels in the neuron (Pang et al.). Consistent with previous reports (Thiagarajan et al., 2005), 24-hour nifedipine treatment in our hands also significantly increased excitatory synaptic transmission (Fig. 4). Importantly, DEAB treatment blocked nifedipine-induced synaptic scaling (Fig. 4A), further supporting the hypothesis that RA mediates the nifedipine-induced increase in synaptic strength. Consistent with RA’s role in activating dendritic GluA1 synthesis and promoting synaptic insertion of GluA1-containing homomeric AMPA receptors, PhTx reversed the increase in mEPSC amplitude seen in nifedipine-treated neurons (Fig. 4B). Somewhat surprisingly, blocking Ca2+-entry through NMDA receptors alone with APV failed to induce synaptic scaling even after 48 hours (Fig. 5), suggesting that under basal conditions when spontaneous action potential firing can occur, Ca2+ enters postsynaptic compartment mostly through non-NMDA receptor-mediated sources. This observation is consistent with the role of NMDA receptors as coincidence detectors and with the finding that most Ca2+-entry in response to release of presynaptic glutamate alone (without pairing with postsynaptic action potential) is mediated by voltage-gated Ca2+-channels, with NMDA receptor activation accounting for less than 20% of the total Ca2+-entry (Schiller et al., 1998).

Figure 4
RA mediates nifedipine-induced synaptic scaling
Figure 5
Blocking NMDA receptor alone does not induce synaptic scaling

The fact that blocking Ca2+-entry through either synaptic glutamate receptors or L-type Ca2+-channels induces RA synthesis suggests that the source of Ca2+-entry is not important for suppression of RA synthesis, and that derepression of RA synthesis requires only a partial decrease in Ca2+-entry since blocking AMPA receptor or L-type Ca2+-channels is sufficient for stimulating RA synthesis. To further test this hypothesis, we asked whether we could block nifedipine-induced synaptic scaling by depolarizing neurons using a moderate increase in the external K+ concentration, which would be expected to activate Ca2+-influx via other channels. Indeed, depolarizing neurons with 15 mM external KCl completely prevented synaptic scaling induced by nifedipine (Fig. 6), although KCl treatment alone did not alter the mEPSC amplitude or frequency (Fig. 6).

Figure 6
Moderate depolarization of neurons by KCl inhibits nifedipine-induced synaptic scaling

RA-mediated synaptic scaling acts cell-autonomously

The question arises whether RA, which enhances AMPA receptor synthesis and insertion in neurons, is produced in the silenced neurons and acts cell-autonomously in the same neuron. For example, it is possible that RA is synthesized in neurons with reduced synaptic transmission and secreted in a paracrine fashion to neighboring neurons to effect changes in synaptic strength. It is also possible that RA is synthesized in astrocytes and diffuses into neurons to stimulate AMPA receptor function. This possibility is raised by the fact that astrocytes express AMPA and NMDA receptors (Lin and Bergles, 2004; Ge et al., 2006; Cahoy et al., 2008a), L-type Ca2+ channels (Cahoy et al., 2008b), and RA biosynthetic enzymes (Wang et al., 2011). Since all of our pharmacological manipulations affect neurons and glia cells alike, these manipulations could have induced RA synthesis in astrocytes instead of neurons.

To address this important question, we made use of a mutant, dihydropyridine (DHP)-insensitive L-type Ca2+-channel, α1C/DHPi, that contains two amino acid substitutions (T1039Y and Q1043M) which render it insensitive to nifedipine blockade (Hockerman et al., 2000). We sparsely transfected cultured neurons with a plasmid encoding α1C/DHPi and co-expressing GFP (for identification of transfected neurons), and treated the neurons with nifedipine for 24 hours. Given our low transfection efficiency (less than 5%), only a few neurons expressed the nifedipine- resistant Ca2+-channel, while most of surrounding neurons and glial cells were untransfected and therefore susceptible to nifedipine blockade. If RA synthesized in these untransfected neurons and glial cells diffused into α1C/DHPi-transfected neurons, synaptic scaling should still be induced in the α1C/DHPi-expressing neurons by chronic nifedipine treatments.

Recordings of mEPSCs revealed that whereas untransfected neurons showed significant increases in both the amplitude and frequency of mEPSCs, transfected neurons expressing α1C/DHPi exhibited no synaptic scaling (Fig. 7). Thus, RA synthesized in neighboring neurons and glial cells does not seem to be sufficient to support synaptic scaling via diffusion, at least in dissociated cultures. This result strongly suggests that during synaptic scaling, RA is most likely induced locally in silenced neurons and primarily acts cell-autonomously to stimulate AMPA receptor synthesis and insertion.

Figure 7
RA acts cell-autonomously to scale up excitatory synaptic strength

Discussion

Our previous work demonstrated that RA directly potentiates excitatory synaptic transmission by increasing the postsynaptic AMPA receptor response in a synapse, that RA synthesis is induced during synaptic silencing to mediate homeostatic plasticity, and that blocking RA synthesis prevents the increase in AMPA receptor responses induced by synaptic silencing (Aoto et al., 2008; Soden and Chen, 2010). These results raised three major questions: What postsynaptic signaling pathway mediates the induction of RA synthesis upon synaptic silencing? Is RA synthesized in the ‘silenced’ neurons or in glia? If synthesized in neurons, does RA act cell-autonomously in the neuron in which it is synthesized, or does it act diffusely in a paracrine fashion?

To address these questions, we here examined which of the multiple forms of synaptic scaling previously described (Turrigiano et al., 1998; Ju et al., 2004; Thiagarajan et al., 2005; Sutton et al., 2006) involves stimulation of RA synthesis. Strikingly but consistent with previous data, we find that the various protocols that lead to synaptic scaling can be classed into two groups: The first group (RA-independent) involves chronic treatment of neurons with TTX alone, which results in an increase in AMPA response sizes as measured by the amplitude of mEPSCs without inducing RA synthesis. This increase is insensitive to either the RA synthesis inhibitor DEAB or the AMPA receptor blocker PhTx-433 that only acts on GluA2-lacking receptors (Fig. 1 and and2).2). The second RA-dependent group involves chronic treatment of neurons with either a combination of TTX and APV, with CNQX alone, or with CNQX in combination with TTX, which induced RA synthesis and produced an increase in AMPA receptor response that was blocked by either DEAB or PhTx (Fig. 1 and and22).

In considering what differentiates the two classes of synaptic scaling mechanisms, we noted that TTX treatment alone does not decrease the Ca2+-influx into postsynaptic neurons to below the level induced by miniature synaptic transmission, whereas all other treatments are expected to reduce Ca2+-influx below that level. Thus, we examined whether simply lowering the neuronal Ca2+-levels using membrane-permeable Ca2+-chelators would induce RA synthesis, and observed a strong stimulation of RA synthesis that was blocked by DEAB (Fig. 3). Next, we asked whether blocking L-type Ca2+-channels with nifedipine could also achieve this effect, and found that chronic nifedipine treatments indeed induced RA synthesis and increased the mEPSC amplitude (Fig. 3 and and44).

The results described in Fig. 14 suggest a simple and straightforward mechanism mediating RA-dependent synaptic scaling induced by synaptic silencing (Fig. 8). Under normal conditions (no activity blockade), there are two main processes leading to a rise in dendritic Ca2+ levels. The first process involves activation of synaptic AMPA receptors, which causes depolarization of the postsynaptic membrane. Subsequent opening of voltage-dependent Ca2+ channels (VDCCs) and to a lesser extent synaptic NMDA receptors induces large amounts of Ca2+ entry into dendrites (the AMPAR-NMDAR and the AMPAR-VDCC pathways). The second process is activation of dendritic VDCCs by direct postsynaptic neuronal spiking (the AP-VDCC pathway). When one and/or the other process operate, RA synthesis is completely shut off. When AP firing is blocked by TTX, the AP-VDCC pathway is blocked, but significant amount of Ca2+ can still enter through the AMPAR-NMDAR and the AMPAR-VDCC pathways due to preserved miniature synaptic transmission, and RA synthesis remains suppressed. Further reduction of dendritic Ca2+ levels by addition of APV or CNQX on top of TTX triggers RA synthesis (Fig. 8). Alternatively, nifedipine alone, which blocks most if not all Ca2+ influx through dendritic VDCCs, effectively triggers RA synthesis by inhibiting both AMPAR-VDCC and AP-VDCC pathways. Although synaptic NMDAR activation does contribute to dendritic Ca2+ levels, its contribution is limited unless both pre- and post-synaptic neurons fire together, a coincidence that is rare in normal neural networks in culture. This explains the lack of effects of APV alone treatment on RA synthesis. In summary, the amount of postsynaptic Ca2+-influx mediated by miniature synaptic events suppresses RA synthesis, whereas under conditions of reduced Ca2+-influx RA synthesis is stimulated. Since partial lowering of Ca2+-influx by blocking either glutamate receptors or L-type Ca2+-channels is sufficient to de-repress RA synthesis, RA synthesis appears to be tightly regulated by Ca2+. The overall mechanism proposed here (Fig. 8) was further confirmed by the finding that the increase in AMPA receptor responses induced by nifedipine could be prevented by chronic moderate depolarization of neurons using 15 mM KCl, which enhances Ca2+-influx via alternative routes (e.g., NMDA receptors and other types of Ca2+-channels; Fig. 6). The parsimonious nature of the mechanism of regulating synaptic scaling via RA proposed suggests a novel Ca2+-dependent signaling pathway in neurons in which RA plays a central role (Fig. 8).

Figure 8
A model for activity-dependent RA synthesis in neurons

How does Ca2+ signaling regulate RA synthesis? RA has long been known to play a critical role in early brain development, but the embryonic brain is devoid of RA synthesis. Instead, during early development the brain is flanked by two regions of extremely high expression of retinal dehydrogenase (RALDH, a major enzyme in RA synthesis) along the rostral-caudal axis (Smith et al., 2001b; Niederreither et al., 2002b), which provide the RA gradient required for the morphogenesis of the early hindbrain (Gavalas and Krumlauf, 2000b). This situation is strikingly different from the adult brain where RA is synthesized almost exclusively within the brain (Werner and Deluca, 2002), and the rate of RA synthesis is very high, twice the rate as in the retinoid-rich liver (Dev et al., 1993b). It is generally believed that RALDHs are critical determinants for the sites of RA action (Wagner et al., 2002), making them attractive candidates subjected to modulation by activity. However, very little information is available regarding how neuronal activity may affect enzymatic activities of RALDHs. Further investigation is required to uncover the components of this novel signaling pathway.

Interestingly, the increase in postsynaptic AMPA receptors produced by RA specifically stimulates the local synthesis and synaptic insertion of Ca2+-permeable GluA2-lacking AMPA receptors. Given the negative regulation of RA synthesis by dendritic Ca2+, it is conceivable that activation of Ca2+-permeable AMPA receptors serves as a negative feedback regulation for RA synthesis, and therefore allows rapid stabilization of synaptic strength after homeostatic adjustment. The connection between decreased postsynaptic Ca2+ and RA synthesis suggests that RA signaling has wide implications for regulating synaptic strength, and may have a broad impact on synaptic function, as synaptic Ca2+ signaling and dendritic protein synthesis are also important for use-dependent forms of synaptic plasticity (e.g. Hebbian-type plasticity).

The dependence of RA synthesis on nifedipine-sensitive L-type Ca2+-channels enabled us to test whether RA is produced locally in silenced neurons, and whether the RA thus produced acts cell-autonomously in the same neuron, or provides a diffusible signal to surrounding neurons. When we expressed an L-type Ca2+-channel mutant that is nifedipine-resistant in a small subset of neurons in our culture, and analyzed nifedipine-induced synaptic scaling in these neurons, we found that synaptic scaling was blocked, despite the fact that all surrounding neurons scaled. This result indicates that RA is synthesized locally in a neuron and acts primarily in the same neuron to induce synaptic AMPA receptor synthesis. The result was surprising given the diffusible nature of RA and its ability to regulate synaptic strength when added to the culture medium at high concentrations (Aoto et al., 2008). Although the present data indicate that the RA levels produced physiologically in a neuron do not suffice for a long-range diffusible signal, because neuronal density is lower in dissociate culture systems than in vivo, our result does not exclude the possibility that in densely packed tissues, RA may diffuse between neuronal cell bodies and dendrites.

In suggesting a general cell-autonomous role for RA in regulating synaptic strength in neurons, our data also raise a series of new questions. At present, little is known about the physiological importance of homeostatic plasticity, and we do not know under what conditions in vivo RA signaling modulates neural circuits, a question that will require sophisticated mouse genetics and pharmacology experiments in vivo to address. Another important question concerns the changes in mEPSC frequency that we observed during synaptic scaling consistent with previous papers (Thiagarajan et al., 2005; Jakawich et al., 2010). Since these changes may depend on BDNF as a secreted signal (Jakawich et al., 2010) but as we show here are also RA-dependent, it may be possible that RA not only stimulates AMPA receptor synthesis and insertion, but also BDNF secretion, which then takes the initially cell-autonomous action of RA to the presynaptic partners that send their inputs to the RA-synthesising neuron. This exciting possibility broadens the possible scope of RA functions and again will involve sophisticated approaches to test.

Acknowledgments

The research was supported by the David and Lucile Packard Foundation, the W. M. Keck Foundation, and NIMH grants 1P50MH86403 and 1R01MH091193 (L.C.).

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