James H. Schwartz

Integration of Long-Term-Memory-Related Synaptic Plasticity Involves Bidirectional Regulation of Gene Expression and Chromatin Structure

Excitatory and inhibitory inputs converge on single neurons and are integrated into a coherent output. Although much is known about short-term integration, little is known about how neurons sum opposing signals for long-term synaptic plasticity and memory storage. In Aplysia, we find that when a sensory neuron simultaneously receives inputs from the facilitatory transmitter 5-HT at one set of synapses and the inhibitory transmitter FMRFamide at another, long-term facilitation is blocked and synapse-specific long-term depression dominates. Chromatin immunoprecipitation assays show that 5-HT induces the downstream gene C/EBP by activating CREB1, which recruits CBP for histone acetylation, whereas FMRFa leads to CREB1 displacement by CREB2 and recruitment of HDAC5 to deacetylate histones. When the two transmitters are applied together, facilitation is blocked because CREB2 and HDAC5 displace CREB1-CBP, thereby deacetylating histones.

Ubiquitin C-Terminal Hydrolase Is an Immediate-Early Gene Essential for Long-Term Facilitation in Aplysia

The switch from short-term to long-term facilitation of the synapses between sensory and motor neurons mediating gill and tail withdrawal reflexes in Aplysia requires CREB-mediated transcription and new protein synthesis. We isolated several downstream genes, one of which encodes a neuron-specific ubiquitin C-terminal hydrolase. This rapidly induced gene encodes an enzyme that associates with the proteasome and increases its proteolytic activity. This regulated proteolysis is essential for long-term facilitation. Inhibiting the expression or function of the hydrolase blocks induction of long-term but not short-term facilitation. We suggest that the enhanced proteasome activity increases degradation of substrates that normally inhibit long-term facilitation. Thus, through induction of the hydrolase and the resulting up-regulation of the ubiquitin pathway, learning recruits a regulated form of proteolysis that removes inhibitory constraints on long-term memory storage.

Facilitatory transmitter causes a selective and prolonged increase in adenosine 3':5'-monophosphate in sensory neurons mediating the gill and siphon withdrawal reflex in Aplysia

Sensitization of the gill and siphon withdrawal reflex in the marine mollusc, Aplysia california, is a simple form of learning Underlying this behavioral changes is a cascade of biochemical events. The first step in this cascade is postulated to be an increase in cAMP within the sensory neurons of the abdominal ganglion. We have developed a labeling protocol with 32Pi which permits us to measure the synthesis of cAMP within a single sensory neurons. Application of serotonin for 5 min was found to triple the content of [32P]cAMP in sensory neurons. The response is specific to serotonin: dopamine, a transmitter that does not produce sensitization, did not increase cAMP. Physiological stimulation of facilitator neurons also resulted in a 3.5-fold increase of cAMP in sensory neurons but not in other cells of the ganglion. We studied the time course of the increase of cAMP in sensory cells stimulated with serotonin and found that it parallels closely the time course of the short term form of presynaptic facilitation. We also have determined the effects of transmitters on the synthesis of cAMP in other identified neurons of the ganglion. The bag cells responded specifically to serotonin. R15, which has been shown to be hyperpolarized both the serotonin and by dopamine, responded to both transmitters by increased synthesis synthesis of cAMP. Thus, the dopamine- and serotonin-sensitive cyclase can be localized to both the same and different cells. Other cells did not respond to serotonin or to dopamine, indicating that a transmitter-sensitive adenylate cyclase is a specific property and is not present in all neurons.

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.

A family of genes that codes for ELH, a neuropeptide eliciting a stereotyped pattern of behavior in Aplysia

We describe a particularly advantageous experimental system for studying gene structure, expression and modulation in the nervous system. In the marine mollusc Aplysia, the bag cells, two discrete clusters of neurons, secrete a peptide of known behavioral function. This neuroactive peptide, egg-laying hormone (ELH), produces a characteristic and stereotypic behavioral repertoire, consisting first of a cessation of walking and inhibition of feeding, followed by head waving and egg laying. We have cloned the genes encoding ELH and characterized their organization and expression. At least five distinct genes for ELH exist within the chromosome. Sequence analysis of one recombinant clone unambiguously identifies a contiguous stretch of nucleotides that encodes the 36 amino acids of ELH. Transcription of this small multigene family results in the expression of at least five distinct RNA transcripts encoding ELH. The pattern of transcripts differs strikingly in different tissues: bag cells express three distinct mRNA species, whereas the atrial gland, a secretory reproductive gland, expresses two distinct mRNAs. Several other neuronal and nonneuronal tissues do not express ELH RNA. In vitro these mRNAs produce a series of long polypeptide precursors that must be processed to generate the active ELH peptide. This processing event is likely to generate several additional neuroactive peptides. Thus the same peptide, ELH, may be released in association with different combinations of other neuroactive peptides. The concept of combinatorial sets of neuropeptides, each bearing one overlapping peptide ELH, and each directing a differing pattern of behavior, greatly expands the information potential of a small set of genes.

Distribution of serotonin-immunoreactivity in juvenile Aplysia

Serotonin-immunocytochemistry has been applied to whole mounts of the central nervous system and of several peripheral tissues from stage 12 juvenile Aplysia californica. The small size of animals at this stage permits visualization of the three-dimensional distribution of structures containing serotonin-immunoreactivity in unsectioned tissues. Many neuronal cell bodies are stained in addition to the giant cerebral neuron of the cerebral ganglion and cells in the RB cluster of the abdominal ganglia which previously had been characterized biochemically and pharmacologically as being serotoninergic. Neuronal cell bodies, both in central ganglia and in the wall of the gut, are encircled by plexuses of serotoninergic varicosities. The neuropil of ganglia and the eye also contain fine, immunoreactive axons bearing varicosities. Intraganglionic connectives and nerves contain many stout fluorescent axons. Serotoninergic varicosities are also observed in the connective tissue sheath surrounding central ganglia and nerves, as well as in heart and body muscle, blood vessels and gut.

The many dimensions of cAMP signaling

In the previous issue of PNAS, Rich et al. (1) provide dynamic evidence that cAMP is produced in a restricted microdomain near the surface membrane of human embryo kidney cells transformed with adenovirus containing cyclic nucleotide-gated (CNG) channels. When activated by cAMP, the channels conduct Ca2+ into the cell, thereby offering an on-line assay for the cyclic nucleotide through Ca2+ imaging. When the cells were stimulated with prostaglandin E1, the Ca2+ signal increased abruptly, then rapidly fell back to baseline. In contrast, total cAMP (assayed biochemically in the cells prelabeled with [3H]adenine) rose rapidly to a plateau and then remained elevated for some time. The rapidly declining cAMP measured by Ca2+ influx through CNG channels was taken to be “membrane localized”, whereas the radiolabeled cAMP represents the “total cellular” pool. These two pools are present within distinct compartments that are separated by diffusion barriers. Although subcellular compartmentalization of cAMP action was recognized at least 20 years ago (2), only recently has there been direct evidence for the idea that cAMP can act in special cellular domains rather than uniformly and everywhere (for recent general reviews, see refs. 3–5). An understanding of the molecular basis of compartmentalization should be achievable because of the extensive information now available concerning the molecular components of cAMP signaling pathways. Only recently has there been direct evidence for the idea that cAMP can act in special cellular domains rather than uniformly and everywhere.

Axonal transport of eukaryotic translation elongation factor 1α mRNA couples transcription in the nucleus to long-term facilitation at the synapse

Long-term synaptic plasticity requires both gene expression in the nucleus and local protein synthesis at synapses. The effector proteins that link molecular events in the cell body with local maintenance of synaptic strength are not known. We now show that treatment with serotonin (5-HT) that produces long-term facilitation induces the Aplysia eukaryotic translation elongation factor 1α (Ap-eEF1A) as a late gene that might serve this coupling function in sensory neurons. Although the translation factor is induced, it is not transported into axon processes when the stimulation with 5-HT was restricted to the cell body. In contrast, its mRNA is transported when 5-HT was applied to both cell body and synapses. Intracellular injection of antisense oligonucleotides or antibodies that block the induction and expression of Ap-eEF1A do not affect the initial expression of long-term facilitation but do block its maintenance beyond 24 h. The transport of eEF1A protein and its mRNA to nerve terminals suggests that the translation factor plays a role in the local protein synthesis that is essential for maintaining newly formed synapses.

Activation and Degradation of the Transcription Factor C/EBP During Long-Term Facilitation in Aplysia

Abstract : Long‐term facilitation (LTF) of the sensory‐to‐motor synapses that mediate defensive reflexes in Aplysia requires induction of the transcription factor Aplysia CCAAT/enhancer binding protein (ApC/EBP) as an early response gene. We examined the time course of ApC/EBP DNA binding during the induction of LTF : Binding activity was detected within 1 h of the sensitization treatment with serotonin, reached a maximum at 2 h, and decreased after 6 h. How are DNA binding and the turnover of ApC/EBP regulated ? We find that phosphorylation of ApC/EBP by mitogen‐activated protein (MAP) kinase is essential for binding. MAP kinase appears to be activated through protein kinase C. We also showed that ApC/EBP is degraded through the ubiquitin‐proteasome pathway but that phosphorylation by MAP kinase renders it resistant to proteolysis. Thus, phosphorylation by MAP kinase is required for ApC/EBP to act as a transcription activator as well as to assure its stability early in the consolidation phase, when genes essential for the development of LTF begin to be expressed.

Ubiquitin-mediated proteolysis in learning and memory

Sensitization of defensive reflexes inAplysia is a simple behavioral paradigm for studying both short- and long-term memory. In the marine mollusk, as in other animals, memory has at least two phases: a short-term phase lasting minutes and a long-term phase lasting several days or longer. Short-term memory is produced by covalent modification of pre-existing proteins. In contrast, long-term memory needs gene induction, synthesis of new protein, and the growth of new synapses. The switch from short-term (STF) to long-term facilitation (LTF) inAplysia sensory neurons requires not only positive regulation through gene induction, but also the specific removal of several inhibitory proteins. One important inhibitory protein is the regulatory (R) subunit of the cAMP-dependent protein kinase (PKA). Degradation of R subunits, which is essential for initiating long-term stable memory, occurs through the ubiquitin-proteasome pathway.