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Mossy Fiber LTP: A Presynaptic cAMP-Dependent Form of Plasticity☆
Mossy Fiber LTP: A Presynaptic cAMP-Dependent Form of Plasticityq Herman B Fernandes and Anis Contractor, Northwestern University Feinberg School of Medicine, Chicago, IL, USA © 2017 Elsevier Inc. All rights reserved.
Introduction The Mossy Fiber Synapse Expression Mechanisms of Mossy Fiber LTP Involvement of cAMP Signaling in Mossy Fiber LTP Downstream Targets of cAMP Potential Substrates of PKA Involved in Elevation of Transmitter Release State Dependant cAMP Signaling at Mossy Fiber to Interneuron Synapses Mossy Fiber LTD cAMP Dependency Comparison With Other cAMP Dependent Forms of LTP Outstanding Questions Further Reading
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Introduction Mossy fiber synapses in the hippocampus are formed by the axons of the granule cells and their postsynaptic targets in the hilar and CA3 regions of the hippocampus. The excitatory synapses formed with the principal pyramidal neurons exhibit a number of unique structural and functional properties, including a distinctive form of long-term potentiation (LTP) that is expressed in the presynaptic terminal and is dependent on cyclic AMP (cAMP) signaling. Here we review the current state of our understanding of this form of synaptic plasticity.
The Mossy Fiber Synapse The hippocampus is a widely studied structure because of the well-described involvement in memory processing and formation. The commonly referenced circuitry of excitatory synaptic connections in this structure is relatively simple with an almost linear progression in connectivity, and therefore information flow, between the granule neurons in the dentate gyrus, which receive the largest proportion of extrinsic connections from the cortex, through the CA3 and CA1 regions and outputting to the subiculum and cortex. However, in reality this simplified scheme is somewhat more complex; in the CA3 region, in particular, extensive axon collaterals of the pyramidal neurons make synapses onto other pyramidal neurons and local circuit interneurons. Neurons in the CA1 and CA3 also receive a direct perforant path connection from the cortex. Whilst the absolute number of CA3 collateral synapses per pyramidal neuron is much greater, the connection between the granule cell axons, termed the mossy fibers, and the CA3 pyramids have the largest impact on CA3 pyramidal excitability. The mossy fiber axons extend from the dentate gyrus, traversing through the hilus, and projecting through the stratum lucidum. This long axonal projection (upwards of 3 mm in the rat) forms en passant connections in the CA3 with the proximal dendrites of the pyramidal neurons and the local circuit interneurons. In a stunning example of target cell specific synaptic segregation, the connections to the pyramidal neuron and interneuron have completely divergent functional properties. Connections onto the pyramidal neurons are formed by large mossy fiber boutons that are typically 2–8 mm in diameter. From these large synaptic terminals, filopodial extensions reach out tens of micrometers to form synaptic contacts exclusively with interneurons. The main mossy fiber bouton synapses onto complex, multi-headed spines, termed thorny excrescences, which extend from the proximal dendrites of the CA3 pyramidal neurons; each terminal contains multiple release sites that are apposed to multiple postsynaptic densities on the same spine. This review focuses almost exclusively on the mossy fiber connection to the principal CA3 pyramidal neurons.
Expression Mechanisms of Mossy Fiber LTP Long-term potentiation (LTP) was first described in the hippocampus by pioneering work from Tim Bliss and Terje Lømo in the early part of the 1970s. These seminal studies quickly led to a large and sustained effort to describe a synaptic phenomenon that is now widely believed to be a cellular correlate of memory formation. LTP at the mossy fiber to CA3 pyramidal neuron synapse was described a decade after these initial findings, and soon proved to be mechanistically distinct from LTP in the CA1 region of the hippocampus and the dentate gyrus. Several important findings about the induction of mossy fiber LTP were described in these early studies. For instance, several groups found that NMDA receptor activation, a defining characteristic of LTP at most other synapses, was not required for the induction of mossy fiber LTP. Whereas some aspects of mossy fiber LTP remain controversial, in particular
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Mossy Fiber LTP: A Presynaptic cAMP-Dependent Form of Plasticity
the requirement of postsynaptic Ca2þ signaling for induction, other aspects, such as the expression mechanisms, are more firmly established. Several coincident lines of evidence have confirmed that mossy fiber to CA3 pyramidal neuron LTP is expressed in the presynaptic terminal as a long lasting increase in glutamate release probability. The evidence for this includes clear reductions in paired pulse ratio, increased rate of open channel block of NMDA receptors, and quantal variance analysis, which strongly supports an increase in transmitter release probability following induction of LTP.
Involvement of cAMP Signaling in Mossy Fiber LTP A complete description that incorporates all the published findings for the expression mechanisms of mossy fiber LTP has not been established. It seems likely that a consensus on a simple unitary model is likely to be elusive, because several parallel pathways may underlie mossy fiber LTP. Despite this, the involvement of several molecules has been firmly established using molecular/genetic techniques, and more acute pharmacological manipulation. A major signaling pathway, that has overwhelming evidence supporting its involvement in the expression of mossy fiber LTP, is the cyclic AMP (cAMP) cascade resulting in the activation of the cAMP dependent protein kinase PKA. The earliest evidence that cAMP might be involved in mossy fiber LTP came from pharmacological manipulations. Application of the diterpene forskolin, which directly activates adenylyl cyclase to elevate cAMP, produces a longlasting enhancement of mossy fiber transmission. Similarly, analogs of cAMP also potentiate mossy fiber transmission. Forskolin potentiation and activity-induced LTP mutually occlude one another, suggesting, but not explicitly confirming, that elevation of cAMP is involved in mossy fiber LTP. Further support for a role of cAMP elevation in mossy fiber LTP comes from studies in knockout mice. Evidence suggests that elevations in presynaptic Ca2þ are required for the LTP cascade, specifically through the activation of presynaptic R-type voltage-dependent Ca2þ channels. Therefore, the focus has been on Ca2þ-activated forms of adenylyl cyclase as the effectors of this rise in cAMP. There are two Ca2þ-sensitive adenylyl cyclases expressed in the CNS: adenylyl cyclase 1 (AC1) and adenylyl cyclase 8 (AC8). Knockout mice for either one of these proteins show impaired mossy fiber LTP, further supporting the role of cAMP signaling at this synapse.
Downstream Targets of cAMP Elevations in cAMP may have several potential downstream targets that could mediate changes in release probability. At the mossy fiber synapse, the emphasis has been on the cAMP dependent protein kinase PKA; however, it is also worth mentioning the other potential effectors that might be involved. At the neuromuscular junction of both crayfish and Drosophila, cAMP elevation potentiates transmitter release by acting directly on presynaptic hyperpolarization-activated cyclic nucleotide-gated channels that underlie Ih. A similar role for presynaptic Ih in mossy fiber LTP is controversial. Incubation of ZD7288, an Ih channel blocker, reduces LTP and forskolin-mediated potentiation of mossy fiber transmission. However, the interpretation of this finding is complicated as ZD7288 was found to have off-target actions on synapses, and by itself causes a robust depression of mossy fiber basal synaptic transmission. Presynaptic Ih therefore is unlikely to play a substantive role in mossy fiber LTP. The consensus target of cAMP in many biological processes is PKA and, as alluded to above, there is substantial evidence that this kinase plays a central role in mossy fiber LTP. Inhibitors of PKA block mossy fiber LTP and targeted mutation of either regulatory or catalytic subunits of PKA has specific effects on mossy fiber LTP. While it is commonly assumed that PKA in the presynaptic terminal plays a critical role, it is worth noting here that inclusion of PKA inhibitory peptide in the postsynaptic cell can also block the establishment of mossy fiber LTP, possibly suggesting a more complex scheme where both pre and postsynaptic PKA activity is required for the full expression of mossy fiber LTP.
Potential Substrates of PKA Involved in Elevation of Transmitter Release All available evidence seems to suggest that PKA is central to mossy fiber LTP; therefore, there has been a concerted effort to uncover the molecular substrates of this enzyme that could underlie the sustained increase in transmitter release. Two molecules that have received particular scrutiny for their role in mossy fiber LTP are Rab3a and RIM1a. Rab3a is a vesicle-associated GTPase, which has been implicated in vesicle fusion. Rab3a is not itself a substrate of PKA, at least in in vitro assays, but it interacts with other synapseassociated proteins that are. RIM1a is an active zone protein that is thought to form a presynaptic scaffold. The involvement of both these molecules in mossy fiber LTP has been determined using examination of knockout mice. The model that has emerged from these studies suggests that phosphorylation of RIM1a, and its interaction with Rab3a, is required for the long lasting increase in neurotransmitter release. While the exact roles of these proteins are still unknown, the clear impairment in mossy fiber LTP in knockout mice put these two molecules at the center of efforts to uncover the molecular basis for plasticity at this synapse. Rim1a also interacts with the active zone protein Munc13-1, which is involved in vesicle priming. In experiments that disrupted the interaction between Munc13-1 and RIM1a mossy fiber LTP was impaired, suggesting that this interaction is key to the function of RIM1a in MF-LTP, and hence a critical component of the cAMP-PKA-Rim1a effector pathway at mossy fiber synapses. If the cAMP-PKA-RIM1a effector pathway were critical for plasticity at mossy fiber synapses, one might assume that forskolininduced synaptic potentiation would be abrogated in RIM1a knockout mice, and potentially disrupted in Rab3a knockout mice.
Mossy Fiber LTP: A Presynaptic cAMP-Dependent Form of Plasticity
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However, normal forskolin-induced potentiation is observed in both of these knockout mice, suggesting that other parallel mechanisms may in fact mediate cAMP signaling. Synaptotagmin-12 (Syt12) is an isoform of Syt that does not function as a Ca2þ sensor, but is notable as a target of PKA, which phosphorylates Syt12 at residue S97. To investigate the potential involvement of Syt12 in cAMP-PKA-dependent mossy fiber LTP, two lines of mutant mice were generated, one in which a knock-in S97A point mutation rendered Syt12 phosphorylationincompetent, and another in which Syt12 was knocked out. Unlike what is observed in Rab3a and Rim1a KO mice, ablation of Syt12 function impairs both mossy fiber LTP and forskolin-mediated potentiation of synaptic release from mossy fiber boutons, suggesting the function of Syt12 is situated at a mechanistic convergence point common to both of these induction pathways. Recently, there has been considerable interest in another downstream target of cAMP, the cAMP-sensitive guanine nucleotide exchanging factor (GEF), Epac. This GEF has been implicated in synaptic potentiation at MNTB neurons in the brainstem, and also in potentiation of transmission at the Drosophila neuromuscular junction. Epac is expressed at high levels in the hippocampus, and recently the involvement of Epac proteins in hippocampal synaptic transmission has been interrogated with surprising results. Using knockout animals for Epac1 and/or Epac2, a role for Epac proteins in CA1 neurotransmission was demonstrated, acting through control of the transcription of miR-124, which in turn suppresses translation of the transcription factor Zif268 and impairs LTP at Schaffer collateral-CA1 synapses. A significant impact on neurotransmission was only observed in the doubleKO, suggesting that either Epac isoform could mitigate the absence of the other at this particular synapse. More recently, a critical role for the Epac2 isoform was uncovered at the mossy fiber - CA3 synapse. In Epac2/ mice mossy fiber LTP was reduced, but not absent, while short-term plasticity remained unaffected. Using selective inhibitors of PKA in the Epac2/ animals, it was found that both Epac2 and PKA are involved in mediating forskolin-induced potentiation of MF responses, suggesting that both cAMP effector pathways are available to be actively recruited in the MF terminal. The size of the readily releasable pool (RRP) of synaptic vesicles was also reduced in Epac2/ mice, suggesting a possible mechanism for the involvement of Epac2 in promoting neurotransmitter release at synaptic terminals. Further evidence for Epac2 in influencing the size of the RRP has come from studies of neurotransmitter release from cortical synaptosomes. Epac2 promotes the interaction between Munc13-1, Rim1a and Rab3a to enhance release probability. The activation of Epac by the selective agonist 8-pCPT promoted the translocation of Munc13-1 from the cytosol to the plasma membrane and the association of Rab3a with Rim1a, and concurrently increased the number of synaptic vesicles available for release. These recent data strongly suggest that Epac2 serves as an effector for the actions of cAMP for the enhancement of vesicular release probability in mossy fiber LTP, in a pathway operating in parallel to PKA activity. Several potential PKA substrates have been excluded as mediators of mossy fiber plasticity. Knockout mice in which synapsin I and II or rabphilin are ablated express normal mossy fiber LTP, suggesting that these three PKA substrates play no role. Therefore, while there is evidence that activation of PKA phosphorylates Syt12 and RIM1a, and possibly facilitates interactions with the active zone proteins Rab3a and Munc13-1 resulting in a sustained increase in transmitter release, this model remains incomplete and is likely to undergo further revision as additional studies uncover more about how PKA and Epac2 exert both their individual and cooperative actions in mossy fiber LTP.
State Dependant cAMP Signaling at Mossy Fiber to Interneuron Synapses As noted above, the mossy fiber axons make contact onto both pyramidal neurons and stratum lucidum interneurons in the CA3 region. These two synaptic connections demonstrate a remarkable segregation in their functional properties. High frequency stimulation of mossy fibers, which causes LTP at pyramidal cell synapses, actually depresses mossy fiber interneuron synapses through activation of presynaptic mGluR7 and PKC signaling. Naïve interneuron synapses are not potentiated by elevating cAMP by application of forskolin, suggesting that these synapses are under regulation by fundamentally different mechanisms, despite being formed by the same axons and being closely spatially arranged (filopodial synapses onto interneurons emanate from the main mossy fiber boutons that form the synapses onto the pyramidal neuron). However, one interesting report demonstrated that activation of mGluR7, and depression of the mossy fiber interneuron synapse, uncovers a subsequent sensitivity to cAMP-dependent potentiation. This state-dependent responsiveness to cAMP suggests that this signaling pathway is more ubiquitous in mossy fibers than was originally thought to be the case.
Mossy Fiber LTD cAMP Dependency The functional inverse of LTP is long-term depression (LTD) of synaptic transmission. A simplistic view about LTD is that it should also engage the same molecular machinery as LTP, but in reverse. Whilst it turns out that things are usually more complicated, there are many examples of molecules that have central roles in both LTD mechanisms and in LTP. At the mossy fiber to pyramidal cell synapse LTD involves cAMP signaling, and therefore may engage at least some of the same machinery as LTP. Mossy fiber LTD can be induced by long trains of low frequency stimulation or by the application of group II mGluR agonist combined with activity. LTD at this synapse is blocked by inhibitors of PKA. In initial studies, it was also reported that LTD was absent in Rab3a knockout mice. These results had established a model that seemed to involve the same molecular
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Mossy Fiber LTP: A Presynaptic cAMP-Dependent Form of Plasticity
players as mossy fiber LTP. However, later studies by the same group could not replicate this finding in Rab3a knockout mice, bringing this model into some doubt. Examination of knockout mice for the other prominent player in mossy fiber LTP, RIM1a, is also consistent with a separate molecular pathway for LTD. In fact, the magnitude of mossy fiber LTD in RIM1a knockout mice is enhanced, possibly due to a masked potentiation occurring in parallel with LTD with low frequency stimulation at some synapses. Therefore, it seems that LTD of mossy fiber to pyramidal cell synapses, while involving cAMP signaling, is unlikely to be an inverse process of LTP, but instead involves an alternate pathway whose components are still unknown.
Comparison With Other cAMP Dependent Forms of LTP Three other synapses have been demonstrated to have forms of LTP similar to that at the mossy fiber pyramidal cell synapse. Corticothalamic fibers originating in layer VI of the neocortex form synapses onto neurons in the ventrobasal thalamus. LTP at corticothalamic synapses is NMDA receptor independent and expressed presynaptically. These synapses are also subject to forskolin potentiation and LTP can be blocked by PKA inhibitors. Beyond these initial findings, it is not clear whether corticothalamic synapses utilize the same molecular machinery as mossy fibers for the expression of LTP. In the cerebellum, parallel fiber axons of the granule cells form synapses onto Purkinje cell dendrites in the molecular layer. Parallel fiber transmission is potentiated by forskolin, which occludes subsequent LTP. Furthermore, parallel fiber LTP induction is blocked by inhibitors of PKA, and is almost absent in RIM1a knockout mice, suggesting that parallel fibers and mossy fibers utilize similar LTP mechanisms. In a reduced co-culture system, reintroduction of RIM1a rescued parallel fiber LTP, but expression of a mutant of a putative phosphorylation site of RIM1a (serine 413) did not, and itself acted as a dominant negative to block LTP in wild-type cultures. These experiments have further reinforced the central role of phosphorylation of RIM1a in the expression of presynaptic cAMP dependent forms of LTP. A final example of presynaptic LTP that is dependent on cAMP signaling is at the corticoamygdala synapse. Although the mechanisms underlying this form of LTP are likely to be different than those at mossy fiber synapses (for instance, NMDA receptor activation is required for induction), some similarities exist. Synaptic transmission is potentiated by forskolin and this occludes subsequent LTP induction, and PKA inhibitors also block LTP. Interestingly, corticoamygdala LTP is impaired in Rab3a knockout mice, but is not significantly impaired in RIM1a knockouts, perhaps suggesting a different signaling pathway through a different, as yet unidentified, PKA substrate.
Outstanding Questions Whilst evidence from numerous studies has begun to dissect the molecular pathways that underlie LTP at mossy fiber synapses, it seems we are some way from consolidating all the available evidence into a single model. There is strong evidence that cAMP signaling plays a central role in the expression mechanisms; however, it is clear that it exerts its effects through multiple pathways. Evidence has demonstrated the importance of Rab3a, Munc13-1, as well as Syt12 and RIM1a, the putative downstream substrates of PKA in mossy fiber LTP. However additional studies have demonstrated that parallel cAMP dependent pathways are involved, with the recent emergence of Epac2 as a co-mediator of mossy fiber LTP. The role of presynaptic Ih has been disputed at mossy fiber synapses, but, at least at other synapses, it does play a role in regulating transmitter release. Similarly, many of the early studies have relied upon inhibitors whose specificity is somewhat unreliable, and there remain some inconsistencies in the literature regarding the induction and expression of mossy fiber LTP. Amongst these is the finding that postsynaptic mechanisms may play a role in the induction process. Recently, there has been evidence that B-ephrin signaling is also required for mossy fiber LTP. It remains to be determined whether a consensus can be established, and whether in fact mossy fiber LTP might turn out to be more complex than first thought.
Further Reading Castillo, P.E., Schoch, S., Schmitz, F., Sudhof, T.C., Malenka, R.C., 2002. RIM1alpha is required for presynaptic long-term potentiation. Nature 415, 327–330. Castillo, P.E., Janz, R., Sudhof, T.C., Tzounopoulos, T., Malenka, R.C., Nicoll, R.A., 1997. Rab3A is essential for mossy fibre long-term potentiation in the hippocampus. Nature 388, 590–593. Fernandes, H.B., Riordan, S., Nomura, T., Remmers, C.L., Kraniotis, S., Marshall, J.J., Kukreja, L., Vassar, R., Contractor, A., 2015. Epac2 mediates cAMP-dependent potentiation of neurotransmission in the Hippocampus. J. Neurosci. 35, 6544–6553. Henze, D.A., Urban, N.N., Barrionuevo, G., 2000. The multifarious hippocampal mossy fiber pathway: a review. Neuroscience 98, 407–427. Kaeser, P.S., Sudhof, T.C., 2005. RIM function in short- and long-term synaptic plasticity. Biochem. Soc. Trans. 33, 1345–1349. q
Change History: January 2016. H. Fernandes and A. Contractor updated the text and ‘Further Reading’ with updated citations.
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Kaeser-Woo, Y.J., Younts, T.J., Yang, X., Zhou, P., Wu, D., Castillo, P.E., Südhof, T.C., 2013. Synaptotagmin-12 phosphorylation by cAMP-dependent protein kinase is essential for hippocampal mossy fiber LTP. J. Neurosci. 33, 9769–9780. Malenka, R.C., Bear, M.F., 2004. LTP and LTD: an embarrassment of riches. Neuron 44, 5–21. Nicoll, R.A., Schmitz, D., 2005. Synaptic plasticity at hippocampal mossy fibre synapses. Nat. Rev. Neurosci. 6, 863–876. Weisskopf, M.G., Castillo, P.E., Zalutsky, R.A., Nicoll, R.A., 1994. Mediation of hippocampal mossy fiber long-term potentiation by cyclic AMP. Science 265, 1878–1882. Yang, Y., Calakos, N., 2011. Munc13-1 is required for presynaptic long-term potentiation. J. Neurosci. 31, 12053–12057.
Introduction The Mossy Fiber Synapse Expression Mechanisms of Mossy Fiber LTP Involvement of cAMP Signaling in Mossy Fiber LTP Downstream Targets of cAMP Potential Substrates of PKA Involved in Elevation of Transmitter Release State Dependant cAMP Signaling at Mossy Fiber to Interneuron Synapses Mossy Fiber LTD cAMP Dependency Comparison With Other cAMP Dependent Forms of LTP Outstanding Questions Further Reading
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Introduction Mossy fiber synapses in the hippocampus are formed by the axons of the granule cells and their postsynaptic targets in the hilar and CA3 regions of the hippocampus. The excitatory synapses formed with the principal pyramidal neurons exhibit a number of unique structural and functional properties, including a distinctive form of long-term potentiation (LTP) that is expressed in the presynaptic terminal and is dependent on cyclic AMP (cAMP) signaling. Here we review the current state of our understanding of this form of synaptic plasticity.
The Mossy Fiber Synapse The hippocampus is a widely studied structure because of the well-described involvement in memory processing and formation. The commonly referenced circuitry of excitatory synaptic connections in this structure is relatively simple with an almost linear progression in connectivity, and therefore information flow, between the granule neurons in the dentate gyrus, which receive the largest proportion of extrinsic connections from the cortex, through the CA3 and CA1 regions and outputting to the subiculum and cortex. However, in reality this simplified scheme is somewhat more complex; in the CA3 region, in particular, extensive axon collaterals of the pyramidal neurons make synapses onto other pyramidal neurons and local circuit interneurons. Neurons in the CA1 and CA3 also receive a direct perforant path connection from the cortex. Whilst the absolute number of CA3 collateral synapses per pyramidal neuron is much greater, the connection between the granule cell axons, termed the mossy fibers, and the CA3 pyramids have the largest impact on CA3 pyramidal excitability. The mossy fiber axons extend from the dentate gyrus, traversing through the hilus, and projecting through the stratum lucidum. This long axonal projection (upwards of 3 mm in the rat) forms en passant connections in the CA3 with the proximal dendrites of the pyramidal neurons and the local circuit interneurons. In a stunning example of target cell specific synaptic segregation, the connections to the pyramidal neuron and interneuron have completely divergent functional properties. Connections onto the pyramidal neurons are formed by large mossy fiber boutons that are typically 2–8 mm in diameter. From these large synaptic terminals, filopodial extensions reach out tens of micrometers to form synaptic contacts exclusively with interneurons. The main mossy fiber bouton synapses onto complex, multi-headed spines, termed thorny excrescences, which extend from the proximal dendrites of the CA3 pyramidal neurons; each terminal contains multiple release sites that are apposed to multiple postsynaptic densities on the same spine. This review focuses almost exclusively on the mossy fiber connection to the principal CA3 pyramidal neurons.
Expression Mechanisms of Mossy Fiber LTP Long-term potentiation (LTP) was first described in the hippocampus by pioneering work from Tim Bliss and Terje Lømo in the early part of the 1970s. These seminal studies quickly led to a large and sustained effort to describe a synaptic phenomenon that is now widely believed to be a cellular correlate of memory formation. LTP at the mossy fiber to CA3 pyramidal neuron synapse was described a decade after these initial findings, and soon proved to be mechanistically distinct from LTP in the CA1 region of the hippocampus and the dentate gyrus. Several important findings about the induction of mossy fiber LTP were described in these early studies. For instance, several groups found that NMDA receptor activation, a defining characteristic of LTP at most other synapses, was not required for the induction of mossy fiber LTP. Whereas some aspects of mossy fiber LTP remain controversial, in particular
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Mossy Fiber LTP: A Presynaptic cAMP-Dependent Form of Plasticity
the requirement of postsynaptic Ca2þ signaling for induction, other aspects, such as the expression mechanisms, are more firmly established. Several coincident lines of evidence have confirmed that mossy fiber to CA3 pyramidal neuron LTP is expressed in the presynaptic terminal as a long lasting increase in glutamate release probability. The evidence for this includes clear reductions in paired pulse ratio, increased rate of open channel block of NMDA receptors, and quantal variance analysis, which strongly supports an increase in transmitter release probability following induction of LTP.
Involvement of cAMP Signaling in Mossy Fiber LTP A complete description that incorporates all the published findings for the expression mechanisms of mossy fiber LTP has not been established. It seems likely that a consensus on a simple unitary model is likely to be elusive, because several parallel pathways may underlie mossy fiber LTP. Despite this, the involvement of several molecules has been firmly established using molecular/genetic techniques, and more acute pharmacological manipulation. A major signaling pathway, that has overwhelming evidence supporting its involvement in the expression of mossy fiber LTP, is the cyclic AMP (cAMP) cascade resulting in the activation of the cAMP dependent protein kinase PKA. The earliest evidence that cAMP might be involved in mossy fiber LTP came from pharmacological manipulations. Application of the diterpene forskolin, which directly activates adenylyl cyclase to elevate cAMP, produces a longlasting enhancement of mossy fiber transmission. Similarly, analogs of cAMP also potentiate mossy fiber transmission. Forskolin potentiation and activity-induced LTP mutually occlude one another, suggesting, but not explicitly confirming, that elevation of cAMP is involved in mossy fiber LTP. Further support for a role of cAMP elevation in mossy fiber LTP comes from studies in knockout mice. Evidence suggests that elevations in presynaptic Ca2þ are required for the LTP cascade, specifically through the activation of presynaptic R-type voltage-dependent Ca2þ channels. Therefore, the focus has been on Ca2þ-activated forms of adenylyl cyclase as the effectors of this rise in cAMP. There are two Ca2þ-sensitive adenylyl cyclases expressed in the CNS: adenylyl cyclase 1 (AC1) and adenylyl cyclase 8 (AC8). Knockout mice for either one of these proteins show impaired mossy fiber LTP, further supporting the role of cAMP signaling at this synapse.
Downstream Targets of cAMP Elevations in cAMP may have several potential downstream targets that could mediate changes in release probability. At the mossy fiber synapse, the emphasis has been on the cAMP dependent protein kinase PKA; however, it is also worth mentioning the other potential effectors that might be involved. At the neuromuscular junction of both crayfish and Drosophila, cAMP elevation potentiates transmitter release by acting directly on presynaptic hyperpolarization-activated cyclic nucleotide-gated channels that underlie Ih. A similar role for presynaptic Ih in mossy fiber LTP is controversial. Incubation of ZD7288, an Ih channel blocker, reduces LTP and forskolin-mediated potentiation of mossy fiber transmission. However, the interpretation of this finding is complicated as ZD7288 was found to have off-target actions on synapses, and by itself causes a robust depression of mossy fiber basal synaptic transmission. Presynaptic Ih therefore is unlikely to play a substantive role in mossy fiber LTP. The consensus target of cAMP in many biological processes is PKA and, as alluded to above, there is substantial evidence that this kinase plays a central role in mossy fiber LTP. Inhibitors of PKA block mossy fiber LTP and targeted mutation of either regulatory or catalytic subunits of PKA has specific effects on mossy fiber LTP. While it is commonly assumed that PKA in the presynaptic terminal plays a critical role, it is worth noting here that inclusion of PKA inhibitory peptide in the postsynaptic cell can also block the establishment of mossy fiber LTP, possibly suggesting a more complex scheme where both pre and postsynaptic PKA activity is required for the full expression of mossy fiber LTP.
Potential Substrates of PKA Involved in Elevation of Transmitter Release All available evidence seems to suggest that PKA is central to mossy fiber LTP; therefore, there has been a concerted effort to uncover the molecular substrates of this enzyme that could underlie the sustained increase in transmitter release. Two molecules that have received particular scrutiny for their role in mossy fiber LTP are Rab3a and RIM1a. Rab3a is a vesicle-associated GTPase, which has been implicated in vesicle fusion. Rab3a is not itself a substrate of PKA, at least in in vitro assays, but it interacts with other synapseassociated proteins that are. RIM1a is an active zone protein that is thought to form a presynaptic scaffold. The involvement of both these molecules in mossy fiber LTP has been determined using examination of knockout mice. The model that has emerged from these studies suggests that phosphorylation of RIM1a, and its interaction with Rab3a, is required for the long lasting increase in neurotransmitter release. While the exact roles of these proteins are still unknown, the clear impairment in mossy fiber LTP in knockout mice put these two molecules at the center of efforts to uncover the molecular basis for plasticity at this synapse. Rim1a also interacts with the active zone protein Munc13-1, which is involved in vesicle priming. In experiments that disrupted the interaction between Munc13-1 and RIM1a mossy fiber LTP was impaired, suggesting that this interaction is key to the function of RIM1a in MF-LTP, and hence a critical component of the cAMP-PKA-Rim1a effector pathway at mossy fiber synapses. If the cAMP-PKA-RIM1a effector pathway were critical for plasticity at mossy fiber synapses, one might assume that forskolininduced synaptic potentiation would be abrogated in RIM1a knockout mice, and potentially disrupted in Rab3a knockout mice.
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However, normal forskolin-induced potentiation is observed in both of these knockout mice, suggesting that other parallel mechanisms may in fact mediate cAMP signaling. Synaptotagmin-12 (Syt12) is an isoform of Syt that does not function as a Ca2þ sensor, but is notable as a target of PKA, which phosphorylates Syt12 at residue S97. To investigate the potential involvement of Syt12 in cAMP-PKA-dependent mossy fiber LTP, two lines of mutant mice were generated, one in which a knock-in S97A point mutation rendered Syt12 phosphorylationincompetent, and another in which Syt12 was knocked out. Unlike what is observed in Rab3a and Rim1a KO mice, ablation of Syt12 function impairs both mossy fiber LTP and forskolin-mediated potentiation of synaptic release from mossy fiber boutons, suggesting the function of Syt12 is situated at a mechanistic convergence point common to both of these induction pathways. Recently, there has been considerable interest in another downstream target of cAMP, the cAMP-sensitive guanine nucleotide exchanging factor (GEF), Epac. This GEF has been implicated in synaptic potentiation at MNTB neurons in the brainstem, and also in potentiation of transmission at the Drosophila neuromuscular junction. Epac is expressed at high levels in the hippocampus, and recently the involvement of Epac proteins in hippocampal synaptic transmission has been interrogated with surprising results. Using knockout animals for Epac1 and/or Epac2, a role for Epac proteins in CA1 neurotransmission was demonstrated, acting through control of the transcription of miR-124, which in turn suppresses translation of the transcription factor Zif268 and impairs LTP at Schaffer collateral-CA1 synapses. A significant impact on neurotransmission was only observed in the doubleKO, suggesting that either Epac isoform could mitigate the absence of the other at this particular synapse. More recently, a critical role for the Epac2 isoform was uncovered at the mossy fiber - CA3 synapse. In Epac2/ mice mossy fiber LTP was reduced, but not absent, while short-term plasticity remained unaffected. Using selective inhibitors of PKA in the Epac2/ animals, it was found that both Epac2 and PKA are involved in mediating forskolin-induced potentiation of MF responses, suggesting that both cAMP effector pathways are available to be actively recruited in the MF terminal. The size of the readily releasable pool (RRP) of synaptic vesicles was also reduced in Epac2/ mice, suggesting a possible mechanism for the involvement of Epac2 in promoting neurotransmitter release at synaptic terminals. Further evidence for Epac2 in influencing the size of the RRP has come from studies of neurotransmitter release from cortical synaptosomes. Epac2 promotes the interaction between Munc13-1, Rim1a and Rab3a to enhance release probability. The activation of Epac by the selective agonist 8-pCPT promoted the translocation of Munc13-1 from the cytosol to the plasma membrane and the association of Rab3a with Rim1a, and concurrently increased the number of synaptic vesicles available for release. These recent data strongly suggest that Epac2 serves as an effector for the actions of cAMP for the enhancement of vesicular release probability in mossy fiber LTP, in a pathway operating in parallel to PKA activity. Several potential PKA substrates have been excluded as mediators of mossy fiber plasticity. Knockout mice in which synapsin I and II or rabphilin are ablated express normal mossy fiber LTP, suggesting that these three PKA substrates play no role. Therefore, while there is evidence that activation of PKA phosphorylates Syt12 and RIM1a, and possibly facilitates interactions with the active zone proteins Rab3a and Munc13-1 resulting in a sustained increase in transmitter release, this model remains incomplete and is likely to undergo further revision as additional studies uncover more about how PKA and Epac2 exert both their individual and cooperative actions in mossy fiber LTP.
State Dependant cAMP Signaling at Mossy Fiber to Interneuron Synapses As noted above, the mossy fiber axons make contact onto both pyramidal neurons and stratum lucidum interneurons in the CA3 region. These two synaptic connections demonstrate a remarkable segregation in their functional properties. High frequency stimulation of mossy fibers, which causes LTP at pyramidal cell synapses, actually depresses mossy fiber interneuron synapses through activation of presynaptic mGluR7 and PKC signaling. Naïve interneuron synapses are not potentiated by elevating cAMP by application of forskolin, suggesting that these synapses are under regulation by fundamentally different mechanisms, despite being formed by the same axons and being closely spatially arranged (filopodial synapses onto interneurons emanate from the main mossy fiber boutons that form the synapses onto the pyramidal neuron). However, one interesting report demonstrated that activation of mGluR7, and depression of the mossy fiber interneuron synapse, uncovers a subsequent sensitivity to cAMP-dependent potentiation. This state-dependent responsiveness to cAMP suggests that this signaling pathway is more ubiquitous in mossy fibers than was originally thought to be the case.
Mossy Fiber LTD cAMP Dependency The functional inverse of LTP is long-term depression (LTD) of synaptic transmission. A simplistic view about LTD is that it should also engage the same molecular machinery as LTP, but in reverse. Whilst it turns out that things are usually more complicated, there are many examples of molecules that have central roles in both LTD mechanisms and in LTP. At the mossy fiber to pyramidal cell synapse LTD involves cAMP signaling, and therefore may engage at least some of the same machinery as LTP. Mossy fiber LTD can be induced by long trains of low frequency stimulation or by the application of group II mGluR agonist combined with activity. LTD at this synapse is blocked by inhibitors of PKA. In initial studies, it was also reported that LTD was absent in Rab3a knockout mice. These results had established a model that seemed to involve the same molecular
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Mossy Fiber LTP: A Presynaptic cAMP-Dependent Form of Plasticity
players as mossy fiber LTP. However, later studies by the same group could not replicate this finding in Rab3a knockout mice, bringing this model into some doubt. Examination of knockout mice for the other prominent player in mossy fiber LTP, RIM1a, is also consistent with a separate molecular pathway for LTD. In fact, the magnitude of mossy fiber LTD in RIM1a knockout mice is enhanced, possibly due to a masked potentiation occurring in parallel with LTD with low frequency stimulation at some synapses. Therefore, it seems that LTD of mossy fiber to pyramidal cell synapses, while involving cAMP signaling, is unlikely to be an inverse process of LTP, but instead involves an alternate pathway whose components are still unknown.
Comparison With Other cAMP Dependent Forms of LTP Three other synapses have been demonstrated to have forms of LTP similar to that at the mossy fiber pyramidal cell synapse. Corticothalamic fibers originating in layer VI of the neocortex form synapses onto neurons in the ventrobasal thalamus. LTP at corticothalamic synapses is NMDA receptor independent and expressed presynaptically. These synapses are also subject to forskolin potentiation and LTP can be blocked by PKA inhibitors. Beyond these initial findings, it is not clear whether corticothalamic synapses utilize the same molecular machinery as mossy fibers for the expression of LTP. In the cerebellum, parallel fiber axons of the granule cells form synapses onto Purkinje cell dendrites in the molecular layer. Parallel fiber transmission is potentiated by forskolin, which occludes subsequent LTP. Furthermore, parallel fiber LTP induction is blocked by inhibitors of PKA, and is almost absent in RIM1a knockout mice, suggesting that parallel fibers and mossy fibers utilize similar LTP mechanisms. In a reduced co-culture system, reintroduction of RIM1a rescued parallel fiber LTP, but expression of a mutant of a putative phosphorylation site of RIM1a (serine 413) did not, and itself acted as a dominant negative to block LTP in wild-type cultures. These experiments have further reinforced the central role of phosphorylation of RIM1a in the expression of presynaptic cAMP dependent forms of LTP. A final example of presynaptic LTP that is dependent on cAMP signaling is at the corticoamygdala synapse. Although the mechanisms underlying this form of LTP are likely to be different than those at mossy fiber synapses (for instance, NMDA receptor activation is required for induction), some similarities exist. Synaptic transmission is potentiated by forskolin and this occludes subsequent LTP induction, and PKA inhibitors also block LTP. Interestingly, corticoamygdala LTP is impaired in Rab3a knockout mice, but is not significantly impaired in RIM1a knockouts, perhaps suggesting a different signaling pathway through a different, as yet unidentified, PKA substrate.
Outstanding Questions Whilst evidence from numerous studies has begun to dissect the molecular pathways that underlie LTP at mossy fiber synapses, it seems we are some way from consolidating all the available evidence into a single model. There is strong evidence that cAMP signaling plays a central role in the expression mechanisms; however, it is clear that it exerts its effects through multiple pathways. Evidence has demonstrated the importance of Rab3a, Munc13-1, as well as Syt12 and RIM1a, the putative downstream substrates of PKA in mossy fiber LTP. However additional studies have demonstrated that parallel cAMP dependent pathways are involved, with the recent emergence of Epac2 as a co-mediator of mossy fiber LTP. The role of presynaptic Ih has been disputed at mossy fiber synapses, but, at least at other synapses, it does play a role in regulating transmitter release. Similarly, many of the early studies have relied upon inhibitors whose specificity is somewhat unreliable, and there remain some inconsistencies in the literature regarding the induction and expression of mossy fiber LTP. Amongst these is the finding that postsynaptic mechanisms may play a role in the induction process. Recently, there has been evidence that B-ephrin signaling is also required for mossy fiber LTP. It remains to be determined whether a consensus can be established, and whether in fact mossy fiber LTP might turn out to be more complex than first thought.
Further Reading Castillo, P.E., Schoch, S., Schmitz, F., Sudhof, T.C., Malenka, R.C., 2002. RIM1alpha is required for presynaptic long-term potentiation. Nature 415, 327–330. Castillo, P.E., Janz, R., Sudhof, T.C., Tzounopoulos, T., Malenka, R.C., Nicoll, R.A., 1997. Rab3A is essential for mossy fibre long-term potentiation in the hippocampus. Nature 388, 590–593. Fernandes, H.B., Riordan, S., Nomura, T., Remmers, C.L., Kraniotis, S., Marshall, J.J., Kukreja, L., Vassar, R., Contractor, A., 2015. Epac2 mediates cAMP-dependent potentiation of neurotransmission in the Hippocampus. J. Neurosci. 35, 6544–6553. Henze, D.A., Urban, N.N., Barrionuevo, G., 2000. The multifarious hippocampal mossy fiber pathway: a review. Neuroscience 98, 407–427. Kaeser, P.S., Sudhof, T.C., 2005. RIM function in short- and long-term synaptic plasticity. Biochem. Soc. Trans. 33, 1345–1349. q
Change History: January 2016. H. Fernandes and A. Contractor updated the text and ‘Further Reading’ with updated citations.
Mossy Fiber LTP: A Presynaptic cAMP-Dependent Form of Plasticity
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Kaeser-Woo, Y.J., Younts, T.J., Yang, X., Zhou, P., Wu, D., Castillo, P.E., Südhof, T.C., 2013. Synaptotagmin-12 phosphorylation by cAMP-dependent protein kinase is essential for hippocampal mossy fiber LTP. J. Neurosci. 33, 9769–9780. Malenka, R.C., Bear, M.F., 2004. LTP and LTD: an embarrassment of riches. Neuron 44, 5–21. Nicoll, R.A., Schmitz, D., 2005. Synaptic plasticity at hippocampal mossy fibre synapses. Nat. Rev. Neurosci. 6, 863–876. Weisskopf, M.G., Castillo, P.E., Zalutsky, R.A., Nicoll, R.A., 1994. Mediation of hippocampal mossy fiber long-term potentiation by cyclic AMP. Science 265, 1878–1882. Yang, Y., Calakos, N., 2011. Munc13-1 is required for presynaptic long-term potentiation. J. Neurosci. 31, 12053–12057.