Whereas short-term (minutes) facilitation at Aplysia sensory-motor neuron synapses is presynaptic, long-term (days) facilitation involves synaptic growth, which requires both presynaptic and postsynaptic mechanisms. How are the postsynaptic mechanisms recruited, and when does that process begin? We have been investigating the possible role of spontaneous transmitter release from the presynaptic neuron. In the previous paper, we found that spontaneous release is critical for the induction of long-term facilitation, and this process begins during an intermediate-term stage of facilitation that is the first stage to involve postsynaptic as well as presynaptic mechanisms. We now report that increased spontaneous release during the short-term stage acts as an orthograde signal to recruit postsynaptic mechanisms of intermediate-term facilitation including increased IP3, Ca 2+ , and membrane insertion and recruitment of clusters of AMPA-like receptors, which may be first steps in synaptic growth during long-term facilitation. These results suggest that the different stages of facilitation involve a cascade of pre-and postsynaptic mechanisms, which is initiated by spontaneous release and may culminate in synaptic growth.synaptic plasticity | metabotropic glutamate receptor | octopamine | latrotoxin | botulinum toxin W hereas short-term (minutes) facilitation in Aplysia involves covalent modifications restricted to the presynaptic neuron, long-term (days) facilitation is accompanied by the growth of new synapses, which involves coordinated pre-and postsynaptic structural changes (1-4). Those findings raise two questions that are applicable to other forms of plasticity that can involve synaptic growth, such as the late phase of long-term potentiation (LTP) in the hippocampus (5-9) and other brain areas: (i) What signaling mechanisms coordinate the pre-and postsynaptic changes, and (ii) when is that signaling first engaged?To address these questions, we have been investigating the possible role of spontaneous transmitter release from the presynaptic neuron in recruiting the postsynaptic mechanisms of long-term facilitation in Aplysia. We previously found that spontaneous transmitter release is critical for the induction of longterm facilitation (10) (in this issue of PNAS). In addition, we found that this signaling is engaged quite early, during an intermediate-term stage that incorporates elements of both shortand long-term plasticity (11, 12), and therefore might form part of a bridge or cascade connecting them. The intermediate stage is also the first stage to involve both presynaptic molecular mechanisms and postsynaptic mechanisms including IP3, Ca 2+ , protein synthesis, and membrane insertion of AMPA-like receptors (1, 2, 13, 14). Postsynaptic Ca 2+ and protein synthesis are in turn necessary for long-term facilitation, in part through retrograde signaling (15).We have now investigated mechanisms downstream of spontaneous release, and have found that it plays a critical role in recruitment of the postsynaptic mechan...

Long-term plasticity can differ from short-term in recruiting the growth of new synaptic connections, a process that requires the participation of both the presynaptic and postsynaptic components of the synapse. How does information about synaptic plasticity spread from its site of origin to recruit the other component? The answer to this question is not known in most systems. We have investigated the possible role of spontaneous transmitter release as such a transsynaptic signal. Until recently, relatively little has been known about the functions of spontaneous release. In this paper, we report that spontaneous release is critical for the induction of a learning-related form of synaptic plasticity, long-term facilitation in Aplysia. In addition, we have found that this signaling is engaged quite early, during an intermediate-term stage that is the first stage to involve postsynaptic as well as presynaptic molecular mechanisms. In a companion paper, we show that spontaneous release from the presynaptic neuron acts as an orthograde signal to recruit the postsynaptic mechanisms of intermediate-term facilitation and initiates a cascade that can culminate in synaptic growth with additional stimulation during long-term facilitation. Spontaneous release could make a similar contribution to learning-related synaptic plasticity in mammals.serotonin | cell culture | miniature excitatory postsynaptic current | octopamine | botulinum toxin S pontaneous transmitter release was discovered 60 y ago by Fatt and Katz (1), who found that it represents the quantal unit of transmitter release evoked by a presynaptic action potential. However, until recently, relatively little has been known about other possible functions of spontaneous release. In the last few years, we have learned that spontaneous release can contribute to postsynaptic firing (2, 3), regulation of postsynaptic kinase pathways (4), and maintenance of postsynaptic dendritic spines and receptors (5, 6). Spontaneous release has also been found to contribute to some cases of homeostatic scaling of synaptic strength (7-14). Here we report a role of spontaneous transmitter release in the induction of a learning-related form of synaptic plasticity, longterm facilitation produced by serotonin (5HT) in Aplysia.Long-term plasticity can differ from short-term in recruiting the growth of new synaptic connections, a process that requires the participation of both the presynaptic and postsynaptic components of the synapse (15-21). Because short-term plasticity often involves only one component of the synapse (22-24), the question arises: How does information about synaptic plasticity spread from its site of origin to recruit the other component of the synapse? Studies of synaptic growth during development have revealed a fairly elaborate program of pre-and postsynaptic changes involving a variety of orthograde and retrograde messengers (25), including activity-dependent or spontaneous release of the transmitter itself (26-30). We have now investigated the possible role of spo...

There is a pressing need to improve approaches for drug discovery related to neuropsychiatric disorders (NSDs). Therapeutic discovery in neuropsychiatric disorders would benefit from screening assays that can measure changes in complex phenotypes linked to disease mechanisms. However, traditional assays that track complex neuronal phenotypes, such as neuronal connectivity, exhibit poor scalability and are not compatible with high-throughput screening (HTS) procedures. Therefore, we created a neuronal phenotypic assay platform that focused on improving the scalability and affordability of neuron-based assays capable of tracking disease-relevant phenotypes. First, using inexpensive laboratory-level automation, we industrialized primary neuronal culture production, which enabled the creation of scalable assays within functioning neural networks. We then developed a panel of phenotypic assays based on culturing of primary neurons from genetically modified mice expressing HTS-compatible reporters that capture disease-relevant phenotypes. We demonstrated that a library of 1,280 compounds was quickly screened against both assays using only a few litters of mice in a typical academic laboratory setting. Finally, we implemented one assay in a fully automated high-throughput academic screening facility, illustrating the scalability of assays designed using this platform. These methodological improvements simplify the creation of highly scalable neuron-based phenotypic assays designed to improve drug discovery in CNS disorders.

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