Activation of protein kinase C by presynaptic FLRFamide receptors facilitates transmitter release at an aplysia cholinergic synapse

Modulation of evoked quantal transmitter release by protein kinase C (PKC) was investigated at an identified cholinergic neuro-neuronal synapse of the Aplysia buccal ganglion. Evoked acetylcholine release was increased by a diacylglycerol analog that activates PKC and was decreased by H-7, a blocker of PKC. FLRFamide facilitated evoked quantal release by increasing presynaptic Ca2+ influx. The inhibition of PKC by H-7 prevented both the increase of presynaptic Ca2+ influx and the facilitation of evoked acetylcholine release induced by the activation of presynaptic FLRFamide receptors. These results provide evidence that the activation of PKC could be a step in the intracellular pathway by which FLRFamide receptors increase evoked quantal acetylcholine release.     

Nitric oxide and carbon monoxide as possible retrograde messengers in hgippocampal long-term potentiation

We have been investigating the hypothesis that the membrane-permeant molecules nitric oxide (NO) and carbon monoxide(CO) may act as retrograde messengers during long-term potentiation (LTP). Inhibitors of either NO synthase or heme oxygenase, the enzyme that produces CO, blocked induction of LTP in the CA1 region of hippocampal slices. Brief application of either NO or CO to slices produced a rapid and long-lasting increase in the size of synaptic potentials if, and only if, the application occurred at the same time as weak tetanic stimulation of the presynaptic fibers. The long-term enhancement by NO or CO was spatially restricted to synapses from active presynaptic fibers and appeared to involve mechanisms utilized by LTP, occluding the subsequent induction of LTP by strong tetanic stimulation. The enhancement by No or CO was not blocked by the NMDA receptor blocker APV, suggesting that NO and CO act downstream for the NMDA receptor. In other systems, both NO and CO produce many of their effects by activation of soluble guanylyl cyclase nd cGMP-dependent protein kinase. An inhibitor of soluble guabylyl cyclase blocked the induction of normal LTP. Conversely, membrane-permeabel analog 8-Br-cGMP produced a rapid onset and long-lasting synaptic enhancement if, and only if, it was applied at the same time as weak presynaptic stimulation. Similarly, two inhibitors of cGMP-dependent protein kinase blocked the induction of normal LTP, and a selective activator of cGMP-dependent protein kinase produced activity-dependent long-lasting synaptic enhancement. 8-Br-cGMP also produced and activity-dependent, long-lasting increase in the amplitude of evoked synaptic current between pairs of hippocampal neurons in dissociated cell culture. In addition, 8-Br-cGMP, like NO, produced a long-lasting increase in the frequency of spontaneous miniature synaptic currents. These results are consistent with the hypothesis that NO and CO, either alone or in combination, serve as retrograde messengers that produce activity-dependent presynaptic enhancement, perhaps by stimulating soluble guanbylyl cyclase and cGMP-dependent protein kinase, during LTP in hippocampus. 1994 John Wiley & Sons, Inc.     

Post-tetanic increase in the fast-releasing synaptic vesicle pool at the expense of the slowly releasing pool

Post-tetanic potentiation (PTP) at the calyx of Held synapse is caused by increases not only in release probability (P_r) but also in the readily releasable pool size estimated from a cumulative plot of excitatory post-synaptic current amplitudes (RRP_cum), which contribute to the augmentation phase and the late phase of PTP, respectively. The vesicle pool dynamics underlying the latter has not been investigated, because PTP is abolished by presynaptic whole-cell patch clamp. We found that supplement of recombinant calmodulin to the presynaptic pipette solution rescued the increase in the RRP_cum after high-frequency stimulation (100 Hz for 4-s duration, HFS), but not the increase in P_r. Release-competent synaptic vesicles (SVs) are heterogeneous in their releasing kinetics. To investigate post-tetanic changes of fast and slowly releasing SV pool (FRP and SRP) sizes, we estimated quantal release rates before and 40 s after HFS using the deconvolution method. After HFS, the FRP size increased by 19.1% and the SRP size decreased by 25.4%, whereas the sum of FRP and SRP sizes did not increase. Similar changes in the RRP were induced by a single long depolarizing pulse (100 ms). The post-tetanic complementary changes of FRP and SRP sizes were abolished by inhibitors of myosin II or myosin light chain kinase. The post-tetanic increase in the FRP size coupled to a decrease in the SRP size provides the first line of evidence for the idea that a slowly releasing SV can be converted to a fast releasing one.     

Mechanism of P2X7 receptor-dependent enhancement of neuromuscular transmission in pannexin 1 knockout mice

P2X7 receptors are present in presynaptic membranes of motor synapses, but their regulatory role in modulation of neurotransmitter release remains poorly understood. P2X7 receptors may interact with pannexin 1 channels to form a purinergic signaling unit. The potential mechanism of P2X7 receptor-dependent modulation of acetylcholine (ACh) release was investigated by recording miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in neuromuscular junctions of wild-type (WT) and pannexin 1 knockout (Panx1^?/?) mice. Modulation of P2X7 receptors with the selective inhibitor A740003 or the selective agonist BzATP did not alter the parameters of either spontaneous or evoked ACh release in WT mice. In Panx1^?/? mice, BzATP-induced activation of P2X7 receptors resulted in a uniformly increased quantal content of EPPs during a short stimulation train. This effect was accompanied by an increase in the size of the readily releasable pool, while the release probability did not change. Inhibition of calmodulin by W-7 or of calcium/calmodulin-dependent kinase II (CaMKII) by KN-93 completely prevented the potentiating effect of BzATP on the EPP quantal content. The blockade of L-type calcium channels also prevented BzATP action on evoked synaptic activity. Thus, the activation of presynaptic P2X7 receptors in mice lacking pannexin 1 resulted in enhanced evoked ACh release. Such enhanced release was provoked by triggering the calmodulin- and CaMKII-dependent signaling pathway, followed by activation of presynaptic L-type calcium channels. We suggest that in WT mice, this pathway is downregulated due to pannexin 1-dependent tonic activation of inhibitory presynaptic purinergic receptors, which overcomes P2X7-mediated effects.     

Postsynaptic TrkC and presynaptic PTPσ function as a bidirectional excitatory synaptic organizing complex

Neurotrophin receptor tyrosine kinases (Trks) have well-defined trophic roles in nervous system development through kinase activation by neurotrophins. Yet Trks have typical cell-adhesion domains and express noncatalytic isoforms, suggesting additional functions. Here we discovered noncatalytic TrkC in an unbiased hippocampal neuron-fibroblast coculture screen for proteins that trigger differentiation of neurotransmitter release sites in axons. All TrkC isoforms, but not TrkA or TrkB, function directly in excitatory glutamatergic synaptic adhesion by neurotrophin-independent high-affinity trans binding to axonal protein tyrosine phosphatase receptor PTPσ. PTPσ triggers and TrkC mediates clustering of postsynaptic molecules in dendrites, indicating bidirectional synaptic organizing functions. Effects of a TrkC-neutralizing antibody that blocks TrkC-PTPσ interaction and TrkC knockdown in culture and in?vivo reveal essential roles of TrkC-PTPσ in glutamatergic synapse formation. Thus, postsynaptic TrkC trans interaction with presynaptic PTPσ generates bidirectional adhesion and recruitment essential for excitatory synapse development and positions these signaling molecules at the center of synaptic pathways.     

Synaptic transmission in the sixth ganglion of the cockroach: action of 4-aminopyridine.

1. Study was made of the action of 4-aminopyridine (5 X 10(-5) M) on synaptic transmission in the last abdominal ganglion of Periplaneta americana. The 'oil-gap' technique was used to record postsynaptic events in a single giant axon. 2. 4-AP quickly increased the 'background' of postsynaptic activity, which consisted of 'spontaneous' unitary EPSPs and IPSPs. Postsynaptic spikes were also propagated. 3. Both evoked EPSPs (stimulation of cercal nerve XI) and evoked IPSPs (stimulation of cercal nerve X) were greatly increased in amplitude although their duration (half-time) was unaltered. 4. 4-AP triggered presynaptic action potentials in the cercal nerves (recorded with external electrodes). These 'antidromic' potentials appeared singly or sometimes repetitively, especially after electrical stimulation of the cercal nerves. They were often in monosynaptic correlation with unitary EPSPs. 5. Neither the resting potential nor the postsynaptic membrane resistance was modified. 6. There were no changes in the equilibrium potentials of the ions involved in postsynaptic events. 7. The results may be essentially explained by an increase in transmitter release after 4-AP treatment, which may be partly the result of a rise in presynaptic terminal excitability, and partly the result of a lengthening of the presynaptic action potentials.     

Different regulation of Purkinje cell dendritic development in cerebellar slice cultures by protein kinase Cα and ‐β

Activity of protein kinase C (PKC), and in particular the PKCγ‐isoform, has been shown to strongly affect and regulate Purkinje cell dendritic development, suggesting an important role for PKC in activity‐dependent Purkinje cell maturation. In this study we have analyzed the role of two additional Ca²⁺‐dependent PKC isoforms, PKCα and ‐β, in Purkinje cell survival and dendritic morphology in slice cultures using mice deficient in the respective enzymes. Pharmacological PKC activation strongly reduced basal Purkinje cell dendritic growth in wild‐type mice whereas PKC inhibition promoted branching. Purkinje cells from mice deficient in PKCβ, which is expressed in two splice forms by granule but not Purkinje cells, did not yield measurable morphological differences compared to respective wild‐type cells under either experimental condition. In contrast, Purkinje cell dendrites in cultures from PKCα‐deficient mice were clearly protected from the negative effects on dendritic growth of pharmacological PKC activation and showed an increased branching response to PKC inhibition as compared to wild‐type cells. Together with our previous work on the role of PKCγ, these data support a model predicting that normal Purkinje cell dendritic growth is mainly regulated by the PKCγ‐isoform, which is highly activated by developmental processes. The PKCα isoform in this model forms a reserve pool, which only becomes activated upon strong stimulation and then contributes to the limitation of dendritic growth. The PKCβ isoform appears to not be involved in the signaling cascades regulating Purkinje cell dendritic maturation in cerebellar slice cultures. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 95–109, 2003     

The actin cytoskeleton and neurotransmitter release: an overview

Here we review evidence that actin and its binding partners are involved in the release of neurotransmitters at synapses. The spatial and temporal characteristics of neurotransmitter release are determined by the distribution of synaptic vesicles at the active zones, presynaptic sites of secretion. Synaptic vesicles accumulate near active zones in a readily releasable pool that is docked at the plasma membrane and ready to fuse in response to calcium entry and a secondary, reserve pool that is in the interior of the presynaptic terminal. A network of actin filaments associated with synaptic vesicles might play an important role in maintaining synaptic vesicles within the reserve pool. Actin and myosin also have been implicated in the translocation of vesicles from the reserve pool to the presynaptic plasma membrane. Refilling of the readily releasable vesicle pool during intense stimulation of neurotransmitter release also implicates synapsins as reversible links between synaptic vesicles and actin filaments. The diversity of actin binding partners in nerve terminals suggests that actin might have presynaptic functions beyond synaptic vesicle tethering or movement. Because most of these actin-binding proteins are regulated by calcium, actin might be a pivotal participant in calcium signaling inside presynaptic nerve terminals. However, there is no evidence that actin participates in fusion of synaptic vesicles.     

Protein kinase C delta induces Src kinase activity via activation of the protein tyrosine phosphatase PTP alpha

Previously we have shown that protein kinase C (PKC)-mediated reorganization of the actin cytoskeleton in smooth muscle cells is transmitted by the non-receptor tyrosine kinase, Src. Several authors have described how 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of cells results in an increase of Src activity, but the mechanism of the PKC-mediated Src activation is unknown. Using PKC isozymes purified from Spodoptera frugiperda insect cells, we show here that PKC is not able to activate Src directly. Our data reveal that the PKC-dependent Src activation occurs via the activation of the protein tyrosine phosphatase (PTP) PTP alpha. PTP alpha becomes activated in vivo after TPA stimulation. Further, we show that PKC delta phosphorylates and activates only PTP alpha in vitro but not any other of the TPA-responsive PKC isozymes that are expressed in A7r5 rat aortic smooth muscle cells. To further substantiate our data, we show that cells lacking PKC delta have a markedly reduced PTP alpha and Src activity after 12-O-tetradecanoylphorbol-13-acetate stimulation. These data support a model in which the main mechanism of 12-O-tetradecanoylphorbol-13-acetate-induced Src activation is the direct phosphorylation and activation of PTP alpha by PKC delta, which in turn dephosphorylates and activates Src.     

Two synaptic vesicle pools,vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction

Drosophila neuromuscular junctions (DNMJs) are malleable and its synaptic strength changes with activities. Mobilization and recruitment of synaptic vesicles (SVs), and replenishment of SV pools in the presynaptic terminal are involved in control of synaptic efficacy. We have studied dynamics of SVs using a fluorescent styryl dye, FM1-43, which is loaded into SVs during endocytosis and released during exocytosis, and identified two SV pools. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low frequency nerve stimulation and releases FM1-43 during exocytosis induced by high K⁺. The ECP locates close to release sites in the periphery of presynaptic boutons. The reserve pool (RP) is loaded and unloaded only during high frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the efficacy of synaptic transmission during low frequency neuronal firing. An increase of cAMP facilitates SV movement from RP to ECP. Post-tetanic potentiation (PTP) correlates well with recruitment of SVs from RP. Neither PTP nor post-tetanic recruitment of SVs from RP occurs in memory mutants that have defects in the cAMP/PKA cascade. Cyotochalasin D slows mobilization of SVs from RP, suggesting involvement of actin filaments in SV movement. During repetitive nerve stimulation the ECP is replenished, while RP replenishment occurs after tetanic stimulation in the absence of external Ca²⁺. Mobilization of internal Ca²⁺ stores underlies RP replenishment. SV dynamics is involved in synaptic plasticity and DNMJs are suitable for further studies.     

SAD: a presynaptic kinase associated with synaptic vesicles and the active zone cytomatrix that regulates neurotransmitter release

A serine/threonine kinase SAD-1 in C. elegans regulates synapse development. We report here the isolation and characterization of mammalian orthologs of SAD-1, named SAD-A and SAD-B, which are specifically expressed in the brain. SAD-B is associated with synaptic vesicles and, like the active zone proteins CAST and Bassoon, is tightly associated with the presynaptic cytomatrix in nerve terminals. A short conserved region (SCR) in the COOH-terminus is required for the synaptic localization of SAD-B. Overexpression of SAD-B in cultured rat hippocampal neurons significantly increases the frequency of miniature excitatory postsynaptic current but not its amplitude. Introduction of SCR into presynaptic superior cervical ganglion neurons in culture significantly inhibits evoked synaptic transmission. Moreover, SCR decreases the size of the readily releasable pool measured by applying hypertonic sucrose. Furthermore, SAD-B phosphorylates the active zone protein RIM1 but not Munc13-1. These results suggest that mammalian SAD kinase presynaptically regulates neurotransmitter release.     

Pre- and postsynaptic mechanisms in Hebbian activity-dependent synapse modification

We have used a three compartment tissue culture system that involved two separate populations of cholinergic neurons in the side compartments that converged on a common target population of myotubes in the center compartment. Activation of the axons from one population of neurons produced selective down-regulation of the synaptic inputs from the other neuronal population (when the two inputs innervated the same myotubes). The decrease in heterosynaptic inputs was mediated by protein kinase C (PKC). An activity-dependent action of protein kinase A (PKA) was associated with the stimulated input and this served to selectively stabilize this input. These changes associated with PKA and PKC activation were mediated by alterations in the number of acetylcholine receptors at the neuromuscular junction. These results suggest that neuromuscular electrical activity produces postsynaptic activation of both PKA and PKC, with the latter producing generalized synapse weakening and the former a selective synapse stabilization. Treatment of the neuronal cell body and axon to increase PKC activity by putting phorbal ester (PMA) in the side chamber did not affect synaptic transmission (with or without stimulation). By contrast, PKA blockade in the side compartment did produce an activity-dependent decrease in synaptic efficacy, which was due to a decrease in quantal release of neurotransmitter. Thus, when the synapse is activated, it appears that presynaptic PKA action is necessary to maintain transmitter output.     

Control of excitatory synaptic transmission by capsaicin is unaltered in TRPV1 vanilloid receptor knockout mice

Several studies have shown that capsaicin could effectively regulate excitatory synaptic transmission in the central nervous system, but the assumption that this effect is mediated by TRPV1 vanilloid receptors (TRPV1Rs) has not been tested directly. To provide direct evidence, we compared the effect of capsaicin on excitatory synapses in wild type mice and TRPV1R knockouts. Using whole-cell patch-clamp techniques, excitatory postsynaptic currents (EPSCs) were recorded in granule cells of the dentate gyrus. First, we investigated the effect of capsaicin on EPSCs evoked by focal stimulation of fibers in the stratum moleculare. Bath application of 10 microM capsaicin reduced the amplitude of evoked EPSCs both in wild type and TRPV1R knockout animals to a similar extent. Treatment of the slices with the TRPV1R antagonist capsazepine (10 microM) alone, or together with the agonist capsaicin, also caused a decrease in the EPSC amplitude both in wild type and TRPV1R knockout animals. Both drugs appeared to affect the efficacy of excitatory synapses at presynaptic sites, since a significant increase was observed in paired-pulse ratio of EPSC amplitude after drug treatment. Next we examined the effect of capsaicin on spontaneously occurring EPSCs. This prototypic vanilloid ligand increased the frequency of events without changing their amplitude in wild type mice. Similar enhancement in the frequency without altering the amplitude of spontaneous EPSCs was observed in TRPV1R knockout mice. These data strongly argue against the hypothesis that capsaicin modulates excitatory synaptic transmission by activating TRPV1Rs, at least in the hippocampal network.     

Ethanol-induced increase in Fyn kinase activity in the dorsomedial striatum is associated with subcellular redistribution of protein tyrosine phosphatase α

In vivo exposure of rodents to ethanol leads to a long-lasting increase in Fyn kinase activity in the dorsomedial striatum (DMS). In this study, we set out to identify a molecular mechanism that contributes to the enhancement of Fyn activity in response to ethanol in the DMS. Protein tyrosine phosphatase α (PTPα) positively regulates the activity of Fyn, and we found that repeated systemic administration or binge drinking of ethanol results in an increase in the synaptic localization of PTPα in the DMS, the same site where Fyn resides. We also demonstrate that binge drinking of ethanol leads to an increase in Fyn activity and to the co-localization of Fyn and PTPα in lipid rafts in the DMS. Finally, we show that the level of tyrosine phosphorylated (and thus active) PTPα in the synaptic fractions is increased in response to contingent or non-contingent exposure of rats to ethanol. Together, our results suggest that the redistribution of PTPα in the DMS into compartments where Fyn resides is a potential mechanism by which the activity of the kinase is increased upon ethanol exposure. Such neuroadaptations could be part of a mechanism that leads to the development of excessive ethanol consumption.     

Pre‐ and postsynaptic mechanisms in Hebbian activity‐dependent synapse modification

We have used a three compartment tissue culture system that involved two separate populations of cholinergic neurons in the side compartments that converged on a common target population of myotubes in the center compartment. Activation of the axons from one population of neurons produced selective down‐regulation of the synaptic inputs from the other neuronal population (when the two inputs innervated the same myotubes). The decrease in heterosynaptic inputs was mediated by protein kinase C (PKC). An activity‐dependent action of protein kinase A (PKA) was associated with the stimulated input and this served to selectively stabilize this input. These changes associated with PKA and PKC activation were mediated by alterations in the number of acetylcholine receptors at the neuromuscular junction. These results suggest that neuromuscular electrical activity produces postsynaptic activation of both PKA and PKC, with the latter producing generalized synapse weakening and the former a selective synapse stabilization. Treatment of the neuronal cell body and axon to increase PKC activity by putting phorbal ester (PMA) in the side chamber did not affect synaptic transmission (with or without stimulation). By contrast, PKA blockade in the side compartment did produce an activity‐dependent decrease in synaptic efficacy, which was due to a decrease in quantal release of neurotransmitter. Thus, when the synapse is activated, it appears that presynaptic PKA action is necessary to maintain transmitter output. Published 2002 Wiley Periodicals, Inc. J Neurobiol 52: 241–250, 2002     

Opposite effects of two estrogen receptors on tau phosphorylation through disparate effects on the miR‐218/PTPA pathway

The two estrogen receptors (ERs), ERα and ERβ, mediate the diverse biological functions of estradiol. Opposite effects of ERα and ERβ have been found in estrogen‐induced cancer cell proliferation and differentiation as well as in memory‐related tasks. However, whether these opposite effects are implicated in the pathogenesis of Alzheimer's disease (AD) remains unclear. Here, we find that ERα and ERβ play contrasting roles in regulating tau phosphorylation, which is a pathological hallmark of AD. ERα increases the expression of miR‐218 to suppress the protein levels of its specific target, protein tyrosine phosphatase α (PTPα). The downregulation of PTPα results in the abnormal tyrosine hyperphosphorylation of glycogen synthase kinase‐3β (resulting in activation) and protein phosphatase 2A (resulting in inactivation), the major tau kinase and phosphatase. Suppressing the increased expression of miR‐218 inhibits the ERα‐induced tau hyperphosphorylation as well as the PTPα decline. In contrast, ERβ inhibits tau phosphorylation by limiting miR‐218 levels and restoring the miR‐218 levels antagonized the attenuation of tau phosphorylation by ERβ. These data reveal for the first time opposing roles for ERα and ERβ in AD pathogenesis and suggest potential therapeutic targets for AD.     

Modulation of Synaptic Vesicle Exocytosis in Muscle-Dependent Long-Term Depression at the Amphibian Neuromuscular Junction

We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry.     

Calcium/calmodulin-dependent protein kinase II expression in motor neurons: Effect of axotomy

Although Ca²⁺/calmodulin-dependent (CaM) protein kinase II isoforms are present in the nervous system in high amounts, many aspects of in vivo expression, localization, and function remain unexplored. During development, CaM kinase IIα and IIβ are differentially expressed. Here, we examined CaM kinase II isoforms in Sprague-Dawley rat sciatic motor neurons before and after axotomy. We cut the L4-5 spinal nerves unilaterally and exposed the proximal nerve stumps to a fluoroprobe, to retrogradely label the neurons of origin. Anti-CaM kinase IIβ antibody showed immunoreactivity in motor neurons, which decreased to low levels by 4 days after axotomy. We found a similar response by in situ hybridization with riboprobes. The decrease in expression of mRNA and protein was confined to fluorescent motor neurons. For CaM kinase IIα, in situ hybridization showed that the mRNA was in sciatic motor neurons, with a density unaffected by axotomy. However, these neurons were also enlarged, suggesting an up-regulation of expression. Northern blots confirmed an mRNA increase. We were unable to find CaM kinase IIα immunoreactivity before or after axotomy in sciatic motor neuron cell bodies, suggesting that CaM kinase IIα is in the axons or dendrites, or otherwise unavailable to the antibody. Using rats with crush lesions, we radiolabeled axonal proteins being synthesized in the cell body and used two-dimensional polyacrylamide gel electrophoresis with Western blots to identify CaM kinase IIα as a component of slow axonal transport. This differential regulation and expression of kinase isoforms suggests separate and unique intracellular roles. Because we find CaM kinase IIβ down-regulates during axonal regrowth, its role in these neurons may be related to synaptic transmission. CaM kinase IIα appears to support axonal regrowth. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 796–810, 1997     

Neuromodulatory changes in short-term synaptic dynamics may be mediated by two distinct mechanisms of presynaptic calcium entry

Although synaptic output is known to be modulated by changes in presynaptic calcium channels, additional pathways for calcium entry into the presynaptic terminal, such as non-selective channels, could contribute to modulation of short term synaptic dynamics. We address this issue using computational modeling. The neuropeptide proctolin modulates the inhibitory synapse from the lateral pyloric (LP) to the pyloric dilator (PD) neuron, two slow-wave bursting neurons in the pyloric network of the crab Cancer borealis. Proctolin enhances the strength of this synapse and also changes its dynamics. Whereas in control saline the synapse shows depression independent of the amplitude of the presynaptic LP signal, in proctolin, with high-amplitude presynaptic LP stimulation the synapse remains depressing while low-amplitude stimulation causes facilitation. We use simple calcium-dependent release models to explore two alternative mechanisms underlying these modulatory effects. In the first model, proctolin directly targets calcium channels by changing their activation kinetics which results in gradual accumulation of calcium with low-amplitude presynaptic stimulation, leading to facilitation. The second model uses the fact that proctolin is known to activate a non-specific cation current I _MI . In this model, we assume that the MI channels have some permeability to calcium, modeled to be a result of slow conformation change after binding calcium. This generates a gradual increase in calcium influx into the presynaptic terminals through the modulatory channel similar to that described in the first model. Each of these models can explain the modulation of the synapse by proctolin but with different consequences for network activity.