Samuel Schacher

Inhibitors of protein and RNA synthesis block structural changes that accompany long-term heterosynaptic plasticity in Aplysia

Synaptic connections between the sensory and motor neurons of Aplysia in culture undergo long-term facilitation in response to serotonin (5-HT) and long-term depression in response to FMRFamide. These long-term functional changes are dependent on the synthesis of macromolecules during the period in which the transmitter is applied and are accompanied by structural changes. There is an increase and a decrease, respectively, in the number of sensory neuron varicosities in response to 5-HT and FMRFamide. To determine whether macromolecular synthesis is also required for the structural changes, we examined in parallel the effects of inhibitors of protein (anisomycin) or RNA (actinomycin D) synthesis on the structural and functional changes. We have found that anisomycin and actinomycin D block both the enduring alterations in varicosity number and the long-lasting changes in synaptic potential. These results indicate that macromolecular synthesis is required for expression of the long-lasting structural changes in the sensory cells and that this synthesis is correlated with the long-term functional modulation of sensorimotor synapses.

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.

Identified target motor neuron regulates neurite outgrowth and synapse formation of aplysia sensory neurons in vitro

To determine the influence that an appropriate target cell has on the axonal structure of a presynaptic neuron in vivo, we examined the morphologies of individual Aplysia sensory neurons in dissociated cell culture in the presence or absence of identified target motor neurons. We find that an appropriate target, the motor cell L7, regulates the morphological differentiation of the presynaptic sensory neurons in two ways: the target induces the axons of the sensory neurons to develop a more elaborate structure and to form active zones, and the target guides the outgrowth of the sensory neurons. The influence of the appropriate target, L7, on the morphological differentiation of sensory neurons appears to be related to the formation of chemical synaptic connections between the sensory neurons and L7, since sensory neurons co-cultured with an inappropriate target motor neuron do not exhibit a comparable elaboration of their axonal processes.

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.

cAMP and arachidonic acid simulate long-term structural and functional changes produced by neurotransmitters in aplysia sensory neurons

The efficacy of the synapses between the sensory and motor cells of Aplysia, as well as the number of presynaptic sensory cell varicosities in vitro, can undergo long-term increases and decreases, respectively, following application of the facilitatory modulator serotonin or the inhibitory modulator FMRFamide. We here report that cAMP and arachidonic acid, two second messenger systems mediating some of the short-term actions of serotonin and FMRFamide on sensory cells, reproduce some of the long-term changes in the structure of the sensory cells, and these structural changes in turn parallel the long-term changes in the functional effectiveness of the synapses. cAMP enhances the strength of the connections between the sensory and motor cells and increases the number of sensory varicosities. Conversely, arachidonic acid decreases the strength of the connections and decreases the number of sensory varicosities. Thus, each of the modulatory neurotransmitters may activate the same intracellular second messenger system to establish both short and long lasting functional changes in synaptic efficacy, as well as to produce enduring structural changes in neuron connectivity.

Target-dependent structural changes in sensory neurons of aplysia accompany long-term heterosynaptic inhibition

FMRFamide evokes both short-term and long-term inhibition of synapses between mechanosensory and motor neurons in Aplysia. We report here, using dissociated cell culture and low-light epifluorescence video microscopy, that depression lasting 24 hr of sensorimotor synapses evoked by four brief applications of FMRFamide is accompanied by a significant loss of sensory cell varicosities and neurites. These structural changes in the sensory cells require the presence of the target motor cell L7. Because the loss of structures known to contain transmitter release sites correlates significantly with the changes in the amplitude of the excitatory postsynaptic potential in L7, our results suggest that the structural changes evoked by FMRFamide reflect a loss of synaptic contacts. Thus, long-term depression parallels long-term facilitation of the sensorimotor synapse produced by serotonin in that both forms of heterosynaptic plasticity involve target-dependent modulation of the number of presynaptic varicosities.

Two Signaling Pathways Regulate the Expression and Secretion of a Neuropeptide Required for Long-Term Facilitation in Aplysia

Activation of several signaling pathways contributes to long-term synaptic plasticity, but how brief stimuli produce coordinated activation of these pathways is not understood. In Aplysia, the long-term facilitation (LTF) of sensory neuron synapses by 5-hydroxytryptamine (serotonin; 5-HT) requires the activation of several kinases, including mitogen-activated protein kinase (MAPK). The 5-HT-enhanced secretion of the sensory neuron-specific neuropeptide sensorin mediates the activation of MAPK. We find that stimulus-induced activation of two signaling pathways, phosphoinositide 3-kinase (PI3K) and type II protein kinase A (PKA), regulate sensorin secretion and responses. Treatment with 5-HT produces a rapid increase in sensorin synthesis, especially at varicosities, which precedes the secretion of sensorin. PI3K inhibitor and rapamycin block LTF and the rapid synthesis of sensorin at varicosities even in the absence of sensory neuron cell bodies. Secretion of the newly synthesized sensorin from the varicosities and activation of the autocrine responses of sensorin to produce LTF require type II PKA interaction with AKAPs (A-kinase anchoring proteins). Thus, long-term synaptic plasticity is produced when multiple signaling pathways that are important for regulating distinct cellular functions are activated in a specific sequence and recruit the secretion of a neuropeptide to activate additional critical pathways.

Some principles emerging from the study of short- and long-term memory

Recent studies indicate that in invertebrates short-term memory for various forms of learning involves covalent modifications of pre-existing proteins. By contrast, long-term memory utilizes genes and proteins not required for short-term memory.

The Two Regulatory Subunits of Aplysia cAMP-Dependent Protein Kinase Mediate Distinct Functions in Producing Synaptic Plasticity

Activation of the cAMP-dependent protein kinase (PKA) is critical for both short- and long-term facilitation in Aplysia sensory neurons. There are two types of the kinase, I and II, differing in their regulatory (R) subunits. We cloned Aplysia RII; RI was cloned previously. Type I PKA is mostly soluble in the cell body whereas type II is enriched at nerve endings where it is bound to two prominent A kinase-anchoring-proteins (AKAPs). Disruption of the binding of RII to AKAPs by Ht31, an inhibitory peptide derived from a human thyroid AKAP, prevents both the short- and the long-term facilitation produced by serotonin (5-HT). During long-term facilitation, RII is transcriptionally upregulated; in contrast, the amount of RI subunits decreases, and previous studies have indicated that the decrease is through ubiquitin-proteosome-mediated proteolysis. Experiments with antisense oligonucleotides injected into the sensory neuron cell body show that the increase in RII protein is essential for the production of long-term facilitation. Using synaptosomes, we found that 5-HT treatment causes RII protein to increase at nerve endings. In addition, using reverse transcription-PCR, we found that RII mRNA is transported from the cell body to nerve terminals. Our results suggest that type I operates in the nucleus to maintain cAMP response element-binding protein-dependent gene expression, and type II PKA acts at sensory neuron synapses phosphorylating proteins to enhance release of neurotransmitter. Thus, the two types of the kinase have distinct but complementary functions in the production of facilitation at synapses of an identified neuron.

Protein Kinase C Regulates Local Synthesis and Secretion of a Neuropeptide Required for Activity-Dependent Long-Term Synaptic Plasticity

Long-term facilitation (LTF) of sensory neuron synapses in Aplysia is produced by either nonassociative or associative stimuli. Nonassociative LTF can be produced by five spaced applications of serotonin (5-HT) and requires a phosphoinosotide 3-kinase (PI3K)-dependent and rapamycin-sensitive increase in the local synthesis of the sensory neuron neuropeptide sensorin and a protein kinase A (PKA)-dependent increase in the secretion of the newly synthesized sensorin. We report here that associative LTF produced by a single pairing of a brief tetanus with one application of 5-HT requires a rapid protein kinase C (PKC)-dependent and rapamycin-sensitive increase in local sensorin synthesis. This rapid increase in sensorin synthesis does not require PI3K activity or the presence of the sensory neuron cell body but does require the presence of the motor neuron. The secretion of newly synthesized sensorin by 2 h after stimulation requires both PKA and PKC activities to produce associative LTF because incubation with exogenous anti-sensorin antibody or the kinase inhibitors after tetanus plus 5-HT blocked LTF. The secreted sensorin leads to phosphorylation and translocation of p42/44 mitogen-activated protein kinase (MAPK) into the nuclei of the sensory neurons. Thus, different stimuli activating different signaling pathways converge by regulating the synthesis and release of a neuropeptide to produce long-term synaptic plasticity.