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 ( and ). 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 ( and ).
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 (). 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 ( and ).
The results described in – suggest a simple and straightforward mechanism mediating RA-dependent synaptic scaling induced by synaptic silencing (). 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 (). 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 () 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; ). 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 ().
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.