Temporal synaptic tagging by I(h) activation and actin: involvement in long-term facilitation and cAMP-induced synaptic enhancement

Presynaptic I(h) channels become activated during a tetanus through membrane hyperpolarization resulting from Na(+) accumulation and electrogenic Na(+)/K(+) exchange. I(h) activation is obligatory for inducing long-term facilitation (LTF), a long-lasting synaptic strengthening. cAMP-induced synaptic enhancement also requires I(h) activation, and both processes are sensitive to actin depolymerization. Other mechanisms are responsible for expression of the responses. Once initiated, continued response to cAMP is I(h) and actin independent. Moreover, LTF-induced activation of I(h) renders subsequent cAMP enhancement insensitive to both I(h) blockers and actin depolymerization. This actin-stabilized "temporal synaptic tagging" set by I(h) activation is prolonged when I(h) is activated concurrent with an elevation in presynaptic calcium concentration ([Ca(2+)]i), permitting the further strengthening of synapses given appropriate additional stimuli.     

Essential role of somatic and synaptic protein synthesis and axonal transport in long-term synapse-specific facilitation at distal sensorimotor connections in Aplysia

To investigate further the cellular mechanisms underlying long-term facilitation (LTF) and long-term synapse-specific facilitation (LTSSF), we studied the role of axonal transport and somatic and synaptic protein synthesis at proximal and distal synapses of Aplysia siphon sensory neurons (SNs). The long soma-synapse distances (2.5 to 3 cm) of the SN distal synapses impose important temporal and mechanistic constraints on long-term facilitation and on intracellular signaling. Excitatory postsynaptic potentials (EPSPs) evoked by SNs in central and peripheral siphon motor neurons were used to assay LTF 24-30 h after various pharmacological treatments. Inhibition of protein synthesis via anisomycin application at either the SN soma or distal synapses blocked the induction of LTF and LTSSF normally produced by synaptic application of the facilitating transmitter serotonin (5-hydroxytryptamine). Further, disruption of axonal transport by application of nocodazole to the isolated siphon nerve completely blocked LTF at distal synapses. These results indicate an essential role for somatic and synaptic protein synthesis and active axonal transport in LTSSF at distal synapses, and raise intriguing questions for current synaptic marking/capture models of synapse specificity and LTF.     

Protein synthesis at synapse versus cell body: enhanced but transient expression of long-term facilitation at isolated synapses

Protein synthesis at synaptic terminals contributes to LTP in hippocampus and to the formation of new synaptic connections by sensory neurons (SNs) of Aplysia. Here we report that after removal of the SN cell body, isolated SN synapses of Aplysia in culture express protein-synthesis dependent long-term facilitation (LTF) produced by 5-HT that decays rapidly. Changes in expression of a SN-specific neuropeptide sensorin in isolated SN varicosities parallel the changes in synaptic efficacy. At 24 h after 5-HT the magnitude of LTF produced at isolated SN synapses was significantly greater than that produced when SN cell bodies were present. LTF was maintained at 48 h at connections with SN cell bodies, but not at isolated SN synapses. The increase in synaptic efficacy at isolated SN synapses at 24 h was blocked by the protein synthesis inhibitor anisomycin. LTF was accompanied by changes in expression of sensorin. The increase in sensorin level at isolated SN varicosities with 5-HT was blocked by anisomycin or was reversed 48 h after 5-HT treatment alone. The results suggest that, as is the case for initial synapse formation between SNs and L7, changes in protein synthesis at synaptic terminals may contribute directly to LTF of stable synapses. Changes in expression within the cell body provide additional contributions for long-term maintenance of the new level of synaptic efficacy that was initiated directly by local changes in protein synthesis at or near synaptic terminals.     

Protein synthesis at synapse versus cell body: Enhanced but transient expression of long‐term facilitation at isolated synapses

Protein synthesis at synaptic terminals contributes to LTP in hippocampus and to the formation of new synaptic connections by sensory neurons (SNs) of Aplysia. Here we report that after removal of the SN cell body, isolated SN synapses of Aplysia in culture express protein‐synthesis dependent long‐term facilitation (LTF) produced by 5‐HT that decays rapidly. Changes in expression of a SN‐specific neuropeptide sensorin in isolated SN varicosities parallel the changes in synaptic efficacy. At 24 h after 5‐HT the magnitude of LTF produced at isolated SN synapses was significantly greater than that produced when SN cell bodies were present. LTF was maintained at 48 h at connections with SN cell bodies, but not at isolated SN synapses. The increase in synaptic efficacy at isolated SN synapses at 24 h was blocked by the protein synthesis inhibitor anisomycin. LTF was accompanied by changes in expression of sensorin. The increase in sensorin level at isolated SN varicosities with 5‐HT was blocked by anisomycin or was reversed 48 h after 5‐HT treatment alone. The results suggest that, as is the case for initial synapse formation between SNs and L7, changes in protein synthesis at synaptic terminals may contribute directly to LTF of stable synapses. Changes in expression within the cell body provide additional contributions for long‐term maintenance of the new level of synaptic efficacy that was initiated directly by local changes in protein synthesis at or near synaptic terminals. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 275–286, 2003     

Roles of PKA and PKC in facilitation of evoked and spontaneous transmitter release at depressed and nondepressed synapses in Aplysia sensory neurons.

Two second messenger pathways, one that uses the cAMP-dependent protein kinase A (PKA), the other that uses protein kinase C (PKC), have been found to contribute to the short-term presynaptic facilitation of the connections between the sensory neurons in Aplysia and their target cells, the interneurons and motor neurons of the gill-withdrawal reflex. To study their relative contributions as a function of the previous history of the neuron's activity, we have examined the effects of inhibiting PKA (using Rp-cAMPS) and PKC (using H7) on the short-term facilitation of spontaneous release as well as of the evoked release induced by serotonin at nondepressed, partially depressed, and highly depressed synapses. Our results suggest that whereas activation of PKA is sufficient to trigger the facilitation of nondepressed synapses, activation of both PKA and PKC is required to facilitate depressed synapses, with the contribution of PKC becoming progressively more important as synaptic transmission becomes more depressed.     

Behavioral, Cellular, and Molecular Analysis of Memory in Aplysia II: Long-Term Facilitation

Long-term facilitation (LTF) of Aplysia tail sensory neuron–motorneuron (SN–MN) synapses provides a synaptic correlateof memory for long-term behavioral sensitization of the tail-siphonwithdrawal reflex. LTF can be induced by repeated exposuresof serotonin (5HT) in the isolated pleural-pedal ganglion preparation.In addition, we have shown previously (Sherff and Carew, 1999)that LTF can also be induced by coincident 5HT exposure comprisedof a single 25-min exposure of 5HT at the SN cell body and a5 min pulse of 5HT at the SN-MN synapses. If synaptic 5HT isapplied either 15 min before or after somatic 5HT, LTF is significantlyreduced or is not induced at all. These results show that twoanatomically remote cellular compartments can functionally interactwithin a surprisingly short time period. In this chapter, wediscuss some of the mechanistic implications of this temporalconstraint. We also find that coincident LTF and LTF inducedby repeated pulses of 5HT differ (1) in whether they induceanother temporal phase of facilitation (intermediate-term facilitation,ITF, expressed up to 1.5 hr after 5HT), and (2) in their requirementsfor protein synthesis. The results described both in this paperand in the preceding companion paper show that there are multipleforms of both ITF and LTF that differ in their induction andexpression requirements, and at least in some instances, thedifferent temporal phases of facilitation, and perhaps comparablephases of memory, can be induced independently of each other.     

Serotonin regulates the secretion and autocrine action of a neuropeptide to activate MAPK required for long-term facilitation in Aplysia

In Aplysia, long-term facilitation (LTF) of sensory neuron synapses requires activation of both protein kinase A (PKA) and mitogen-activated protein kinase (MAPK). We find that 5-HT through activation of PKA regulates secretion of the sensory neuron-specific neuropeptide sensorin, which binds autoreceptors to activate MAPK. Anti-sensorin antibody blocked LTF and MAPK activation produced by 5-HT and LTF produced by medium containing sensorin that was secreted from sensory neurons after 5-HT treatment. A single application of 5-HT followed by a 2 hr incubation with sensorin produced protein synthesis-dependent LTF, growth of new presynaptic varicosities, and activation of MAPK and its translocation into sensory neuron nuclei. Inhibiting PKA during 5-HT applications and inhibiting receptor tyrosine kinase or MAPK during sensorin application blocked both LTF and MAPK activation and translocation. Thus, long-term synaptic plasticity is produced when stimuli activate kinases in a specific sequence by regulating the secretion and autocrine action of a neuropeptide.     

The role of cyclic AMP in the modulation of synaptic efficacy.

1. Heterosynaptic facilitation (modification of synaptic transmission by a neuron influencing the terminals of the presynaptic neuron) was studied in the pleural ganglion of Aplysia. Among several identified synapses, heterosynaptic facilitation was observed only in one type (EIPSP synapses) when repetitive stimulation was applied to the tentacular nerve or to a particular identified neuron. 2. Serotonin was shown to increase the amplitude of the EIPSP at this synapse; this facilitatory effect was prolonged in the presence of theophylline and mimicked by cyclic AMP. 3. When transmission was abolished by calcium-free solution, calcium injected in the region of the synapse caused partial recovery of the EIPSP; when calcium injection was preceded by serotonin injection near the same terminal, the EIPSP was much larger than with calcium injection alone. 4. It was concluded that the activation of one neuron (the heterosynaptic neuron) caused it to release serotonin, which activated an adenylate cyclase in the pre-synaptic terminals of another neuron. Consequent accumulation of cyclic AMP in these terminals is supposed to have increased their voltage-dependent calcium conductance and hence the amount of transmitter released during an action potential.     

Molecular mechanisms underlying a unique intermediate phase of memory in aplysia

Short- and long-term synaptic facilitation induced by serotonin at Aplysia sensory-motor (SN-MN) synapses has been widely used as a cellular model of short- and long-term memory for sensitization. In recent years, a distinct intermediate phase of synaptic facilitation (ITF) has been described at SN-MN synapses. Here, we identify a novel intermediate phase of behavioral memory (ITM) for sensitization in Aplysia and demonstrate that it shares the temporal and mechanistic features of ITF in the intact CNS: (1) it declines completely prior to the onset of LTM, (2) its induction requires protein but not RNA synthesis, and (3) its expression requires the persistent activation of protein kinase A. Thus, in Aplysia, the same temporal and molecular characteristics that distinguish ITF from other phases of synaptic plasticity distinguish ITM from other phases of behavioral memory.     

Trans-synaptic plasticity: presynaptic initiation, postsynaptic memory

A novel mechanism of persistent facilitation induced by serotonin at Aplysia synapses depends upon rapid postsynaptic protein synthesis and increased responsiveness to glutamate; whereas the memory for this synaptic change is postsynaptic, the initiating signal may be an increase in spontaneous release of glutamate from the presynaptic terminals.     

Synaptic plasticity in a regenerated crayfish phasic motoneuron.

Crustacean neuromuscular systems provide many advantages for the study of synaptic transmission and plasticity. The present study examines aspects of synaptic transmission in the phasic, fast closer excitor (FCE) motoneuron of regenerated crayfish claws. Excitatory postsynaptic potentials (EPSPs) fatigued rapidly and showed poor long-term facilitation (LTF) in the smallest of regenerating claws. EPSPs in larger regenerating claws fatigued less and showed pronounced facilitation. These observations were not the same as those previously made during primary development of this motoneuron (Lnenicka and Atwood, 1985a, J. Neuroscience 5:459-467). Hence, regeneration is not the recapitulation of primary development. In situ stimulation of the FCE is known to lead to long-lasting adaptation of synaptic performance. This adaptation is age dependent; it is expressed in young but not old animals. In the regenerated FCE of old animals, we observed a novel form of long-lasting adaptation to imposed activity: EPSPs showed large initial EPSPs and did not exhibit resistance to fatigue during maintained stimulation. This indicates that aged motoneurons can express adaptive changes to increased activity following axonal regeneration, but that the adaptive changes are the opposite to what is observed in nonregenerated motoneurons.     

Invited review: Intermittent hypoxia and respiratory plasticity.

Intermittent hypoxia elicits long-term facilitation (LTF), a persistent augmentation (hours) of respiratory motor output. Considerable recent progress has been made toward an understanding of the mechanisms and manifestations of this potentially important model of respiratory plasticity. LTF is elicited by intermittent but not sustained hypoxia, indicating profound pattern sensitivity in its underlying mechanism. During intermittent hypoxia, episodic spinal serotonin receptor activation initiates cell signaling events, increasing spinal protein synthesis. One associated protein is brain-derived neurotrophic factor, a neurotrophin implicated in several forms of synaptic plasticity. Our working hypothesis is that increased brain-derived neurotrophic factor enhances glutamatergic synaptic currents in phrenic motoneurons, increasing their responsiveness to bulbospinal inspiratory inputs. LTF is heterogeneous among respiratory outputs, differs among experimental preparations, and is influenced by age, gender, and genetics. Furthermore, LTF is enhanced following chronic intermittent hypoxia, indicating a degree of metaplasticity. Although the physiological relevance of LTF remains unclear, it may reflect a general mechanism whereby intermittent serotonin receptor activation elicits respiratory plasticity, adapting system performance to the ever-changing requirements of life.     

Synaptic plasticity in a regenerated crayfish phasic motoneuron

Crustacean neuromuscular systems provide many advantages for the study of synaptic transmission and plasticity. The present study examines aspects of synaptic transmission in the phasic, fast closer excitor (FCE) motoneuron of regenerated crayfish claws. Excitatory postsynaptic potentials (EPSPs) fatigued rapidly and showed poor long-term facilitation (LTF) in the smallest of regenerating claws. EPSPs in larger regenerating claws fatigued less and showed pronounced facilitation. These observations were not the same as those previously made during primary development of this motoneuron (Lnenicka and Atwood, 1985a, J. Neuroscience 5:459–467). Hence, regeneration is not the recapitulation of primary development. In situ stimulation of the FCE is known to lead to long-lasting adaptation of synaptic performance. This adaptation is age dependent; it is expressed in young but not old animals. In the regenerated FCE of old animals, we observed a novel form of long-lasting adaptation to imposed activity: EPSPs showed large initial EPSPs and did not exhibit resistance to fatigue during maintained stimulation. This indicates that aged motoneurons can express adaptive changes to increased activity following axonal regeneration, but that the adaptive changes are the opposite to what is observed in nonregenerated motoneurons. © 1992 John Wiley & Sons, Inc.     

Mechanisms for generating the autonomous cAMP-dependent protein kinase required for long-term facilitation in Aplysia

The formation of a persistently active cAMP-dependent protein kinase (PKA) is critical for establishing long-term synaptic facilitation (LTF) in Aplysia. The injection of bovine catalytic (C) subunits into sensory neurons is sufficient to produce protein synthesis-dependent LTF. Early in the LTF induced by serotonin (5-HT), an autonomous PKA is generated through the ubiquitin-proteasome-mediated proteolysis of regulatory (R) subunits. The degradation of R occurs during an early time window and appears to be a key function of proteasomes in LTF. Lactacystin, a specific proteasome inhibitor, blocks the facilitation induced by 5-HT, and this block is rescued by injecting C subunits. R is degraded through an allosteric mechanism requiring an elevation of cAMP coincident with the induction of a ubiquitin carboxy-terminal hydrolase.