Phosphoproteins associated with the regulation of a specific potassium channel in the identified Aplysia neuron R15

The neurotransmitter serotonin (5HT) activates a specific K+ conductance in the identified Aplysia neuron R15. This response to 5HT has been shown previously to be mediated by cAMP and cAMP-dependent protein phosphorylation. We have measured protein phosphorylation within neuron R15 in vivo, following the intracellular injection of [gamma-32P]ATP, and have demonstrated that 5HT modulates the phosphorylation of a number of proteins in R15. The present study was undertaken to determine which of these phosphoproteins are closely associated with, and may be responsible for, the K+ conductance increase. Treatment of neuron R15 with a cAMP analog produces some but not all of the 5HT-induced phosphoprotein changes, indicating that some are not cAMP-dependent and thus can be dissociated from the cAMP-dependent K+ conductance increase. Similar results are obtained by intracellular injection of the adenylate cyclase inhibitor guanosine 5'-O-(2-thiodiphosphate), which completely blocks the 5HT-evoked K+ conductance increase but fails to block some of the 5HT-induced phosphorylation changes. Examination of the phosphoprotein pattern at short times after 5HT application has demonstrated that some of the phosphoprotein changes, but not others, are closely associated in time with the appearance of the physiological response. These and other pharmacological and kinetic experiments have allowed the identification of two phosphoproteins, of Mr = 29,000 and 70,000, which cannot be dissociated from the 5HT-induced K+ conductance increase whatever the experimental manipulation. Thus, one or both of these phosphoproteins may be involved in the regulation of the 5HT-sensitive K+ channel in neuron R15.     

A single gene encodes multiple neuropeptides mediating a stereotyped behavior

Egg laying in Aplysia is characterized by a stereotyped behavioral array which is mediated by several neuroactive peptides. We have sequenced two genes encoding the A and B peptides thought to initiate the egg-laying process, as well as a gene encoding egg-laying hormone (ELH) which directly mediates the behavioral array. The three genes share 90% sequence homology and are representatives of a small multigene family. Each gene encodes a protein precursor in which the active peptides are flanked by internal cleavage sites providing the potential to generate multiple small peptides. Each of the three genes consists of sequences homologous to A or B peptide as well as ELH. Although these genes share significant nucleotide homology, they have diverged such that different member genes express functionally related but nonoverlapping sets of neuroactive peptides in different tissues.     

Generation of variants of a motor act in a modular and hierarchical motor network

BACKGROUND: Most motor systems can generate a variety of behaviors, including categorically different behaviors and variants of a single motor act within the same behavioral category. Previous work indicated that many pattern-generating interneuronal networks may have a modular organization and that distinct categories of behaviors can be generated through flexible combinations of a small number of modules or building blocks. However, it is unclear whether and how a small number of modules could possibly generate a large number of variants of one behavior. RESULTS: We show that the modular feeding motor network of Aplysia mediates variations in protraction duration in biting-like programs. Two descending commands are active during biting behavior and trigger biting-like responses in a semiintact preparation. In the isolated CNS, when activated alone, the two commands produce biting-like programs of either long or short protraction duration by acting specifically on two modules that have opposite effects on protraction duration. More importantly, when coactivated at different frequencies, the two commands produce biting programs with an intermediate protraction duration. CONCLUSIONS: It was previously hypothesized that behavioral variants may be produced by combining different activity levels of multiple descending commands. Our data provide direct evidence for such a scheme and show how it is implemented in a modularly organized network. Thus, within a modular and hierarchical architecture, in addition to generating different categories of behavior, a small number of modules also efficiently implements variants of a single behavior.     

Structural plasticity at identified synapses during long-term memory in Aplysia

We have used the gill- and siphon-withdrawal reflex of Aplysia californica to determine the morphological basis of the prolonged changes in synaptic effectiveness that underlie long-term habituation and sensitization. We have found that clear structural changes accompany behavioral modification and have demonstrated that these can be detected at the level of identified sensory neuron synapses, a critical site of plasticity for the short-term forms of both types of learning. These alterations occur at two different levels of synaptic organization and include (1) changes in focal regions of synaptic membrane specialization--the number, size and vesicle complement of sensory neuron active zones are larger in sensitized animals and smaller in habituated animals compared with controls--and (2) a parallel but more dramatic and global trend involving modulation of the total number of presynaptic varicosities per sensory neuron. Quantitative analysis of the time course over which these structural alterations occur during sensitization has further demonstrated that changes in the number of varicosities and active zones persist in parallel with the behavioral retention of the memory. This increase in the number of sensory neuron synapses during long-term sensitization in Aplysia is similar to changes in the number of synapses in the mammalian brain following various forms of environmental manipulations and learning (Greenough, 1984). Therefore learning may involve a form of neuronal growth across a broad segment of the animal kingdom, thereby suggesting a role for structural synaptic plasticity during long-term behavioral modifications.     

Are different proteins synthesized in distinct domains in a neuron?

Are different messenger RNA species translated in distinct, limited domains within a cell? For the particular case of the giant R2 neuron from Aplysia californica an answer to this question is possible for the more abundantly synthesized proteins. After brief labeling with 35S-methionine, the R2 neuron soma was frozen and divided into two or three parts. The newly synthesized proteins were analyzed following two-dimensional polyacrylamide gel electrophoresis. No evidence for limited domains of synthesis for 26 abundantly synthesized polypeptides in the R2 neuron was found.     

Intracellular injection of guanyl nucleotides alters the serotonin- induced increase in potassium conductance in Aplysia neuron R15

The effects of the adenylate cyclase inhibitor GDP beta S on the response of Aplysia neuron R15 to serotonin (5HT) were investigated. Previous studies have demonstrated that 5HT causes an increase in K+ conductance in R15 and that the response is mediated by cAMP. At concentrations in the micromolar range, GDP beta S inhibits the stimulation of adenylate cyclase by 5HT in particulate fractions from Aplysia ganglia. When micromolar concentrations of GDP beta S are injected into neuron R15, there is no effect on the resting membrane conductance, but the increase in K+ conductance normally elicited by 5HT is completely inhibited. Furthermore, the decrease in inward current normally elicited by dopamine (DA), which does not appear to involve cAMP, is not affected by micromolar concentrations of GDP beta S. In addition, application of 8-benzylthio cAMP to R15 can evoke an increase in K+ conductance even after the injection of GDP beta S, which indicates that events subsequent to the activation of adenylate cyclase are not inhibited by the GDP analogue. In contrast, when millimolar concentrations of GDP beta S are injected into R15, direct effects on membrane conductance are observed and the response of R15 to 5HT is enhanced. Although these effects of high concentrations of GDP beta S are only poorly understood, the results with micromolar concentrations are consistent with the hypothesis that stimulation of adenylate cyclase is necessary for the 5HT-induced increase in K+ conductance in neuron R15.     

Decreased Response to Acetylcholine during Aging of Aplysia Neuron R15

How aging affects the communication between neurons is poorly understood. To address this question, we have studied the electrophysiological properties of identified neuron R15 of the marine mollusk Aplysia californica. R15 is a bursting neuron in the abdominal ganglia of the central nervous system and is implicated in reproduction, water balance, and heart function. Exposure to acetylcholine (ACh) causes an increase in R15 burst firing. Whole-cell recordings of R15 in the intact ganglia dissected from mature and old Aplysia showed specific changes in burst firing and properties of action potentials induced by ACh. We found that while there were no significant changes in resting membrane potential and latency in response to ACh, the burst number and burst duration is altered during aging. The action potential waveform analysis showed that unlike mature neurons, the duration of depolarization and the repolarization amplitude and duration did not change in old neurons in response to ACh. Furthermore, single neuron quantitative analysis of acetylcholine receptors (AChRs) suggested alteration of expression of specific AChRs in R15 neurons during aging. These results suggest a defect in cholinergic transmission during aging of the R15 neuron.     

Central actions of R15, a putative peptidergic neuron in Aplysia

We report that the bursting pacemaker neuron R15 has central actions on other identified neurons in the abdominal ganglion of Aplysia california. The follower cells are located on the dorsal surface of the left lower quadrant of the ganglion and include members of the LC cell cluster. A spontaneous burst of impulses in R15 produces a slow, graded, excitatory potential of up to 8 mV in follower cells. The response begins about 2-3 s after the first impulse in an R15 burst, and reaches its peak at about 4-6 s (corresponding approximately to the end of the R15 burst). In some preparations a biphasic response was seen composed of the early depolarization followed by a slower excitatory or inhibitory phase. All the responses were blocked when R15 was hyperpolarized to prevent spiking. The magnitude of the response was reduced in a graded fashion by prematurely terminating the R15 burst with hyperpolarizing current and was increased when depolarizing current was injected into R15 during a burst. Central actions of R15 were observed in only 28% of our preparations, and their presence may depend on unknown physiological factors. The effects are likely to be mediated by R15 neuropeptides. The accessibility of both R15 and its targets in this preparation should facilitate further analysis of this interaction.     

Serotonin-induced regulation of the actin network for learning-related synaptic growth requires Cdc42, N-WASP, and PAK in Aplysia sensory neurons

Application of Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, at the Aplysia sensory to motor neuron synapse blocks long-term facilitation and the associated growth of new sensory neuron varicosities induced by repeated pulses of serotonin (5-HT). We have isolated cDNAs encoding Aplysia Rho, Rac, and Cdc42 and found that Rho and Rac had no effect but that overexpression in sensory neurons of a dominant-negative mutant of ApCdc42 or the CRIB domains of its downstream effectors PAK and N-WASP selectively reduces the long-term changes in synaptic strength and structure. FRET analysis indicates that 5-HT activates ApCdc42 in a subset of varicosities contacting the postsynaptic motor neuron and that this activation is dependent on the PI3K and PLC signaling pathways. The 5-HT-induced activation of ApCdc42 initiates reorganization of the presynaptic actin network leading to the outgrowth of filopodia, some of which are morphological precursors for the learning-related formation of new sensory neuron varicosities.     

Predicting adaptive behavior in the environment from central nervous system dynamics

To generate adaptive behavior, the nervous system is coupled to the environment. The coupling constrains the dynamical properties that the nervous system and the environment must have relative to each other if adaptive behavior is to be produced. In previous computational studies, such constraints have been used to evolve controllers or artificial agents to perform a behavioral task in a given environment. Often, however, we already know the controller, the real nervous system, and its dynamics. Here we propose that the constraints can also be used to solve the inverse problem--to predict from the dynamics of the nervous system the environment to which they are adapted, and so reconstruct the production of the adaptive behavior by the entire coupled system. We illustrate how this can be done in the feeding system of the sea slug Aplysia. At the core of this system is a central pattern generator (CPG) that, with dynamics on both fast and slow time scales, integrates incoming sensory stimuli to produce ingestive and egestive motor programs. We run models embodying these CPG dynamics--in effect, autonomous Aplysia agents--in various feeding environments and analyze the performance of the entire system in a realistic feeding task. We find that the dynamics of the system are tuned for optimal performance in a narrow range of environments that correspond well to those that Aplysia encounter in the wild. In these environments, the slow CPG dynamics implement efficient ingestion of edible seaweed strips with minimal sensory information about them. The fast dynamics then implement a switch to a different behavioral mode in which the system ignores the sensory information completely and follows an internal "goal," emergent from the dynamics, to egest again a strip that proves to be inedible. Key predictions of this reconstruction are confirmed in real feeding animals.     

Water regulation by a presumptive hormone contained in identified neurosecretory cell R15 of Aplysia

Injection of an homogenate of identified neuron R15 into the hemocele of Aplysia produced a weight increase of 3-10% within 90 min. Control injections of several other identified neurons or of seawater, were ineffective. The weight increase occurred even when the animals were maintained in 5% hyperosmotic seawater. The activity of the R15 homogenate was retained after acidification to pH 2 and heating to 100 degrees C; but activity was destroyed by proteolytic digestion with Pronase. Dialysis in cellulose dialysis tubing resulted in a significant loss of aion on Sephadex G-50 (nominal exclusion limits 1,500-30,000 daltons), activity was present in the partially included volumes, but was absent in the totally excluded or totally included volumes. The data support the notion that R15 contains one or more hormones involved in ionic regulation or water balance. The results of bioassays of R15 extracts subjected to different treatments are consistent with the hypothesis that activity is due to one or more stable polypeptides of relatively low molecular weight.     

DSL-Notch signaling in the Drosophila brain in response to olfactory stimulation

Neurexin and neuroligin, which undergo heterophilic interactions with each other at the synapse, are mutated in some patients with autism spectrum disorder, a set of disorders characterized by deficits in social and emotional learning. We have explored the role of neurexin and neuroligin at sensory-to-motor neuron synapses of the gill-withdrawal reflex in Aplysia, which undergoes sensitization, a simple form of learned fear. We find that depleting neurexin in the presynaptic sensory neuron or neuroligin in the postsynaptic motor neuron abolishes both long-term facilitation and the associated presynaptic growth induced by repeated pulses of serotonin. Moreover, introduction into the motor neuron of the R451C mutation of neuroligin-3 linked to autism spectrum disorder blocks both intermediate-term and long-term facilitation. Our results suggest that activity-dependent regulation of the neurexin-neuroligin interaction may govern transsynaptic signaling required for the storage of long-term memory, including emotional memory that may be impaired in autism spectrum disorder.     

Cyclic AMP enhances calcium-dependent potassium current inAplysia neurons

The effect on the Ca-dependent potassium current, IK(Ca), of procedures that increase intracellular cAMP levels was studied in Aplysia neurons using three different pharmacological approaches. Exposure to cAMP analogues which were either resistant to or protected from phosphodiesterase hydrolysis caused an increase in IK(Ca) from 30 to 50% in 10 min. The degree of reversibility of this effect varied from complete with db cAMP to very little with pcpt cAMP. Exposure to cholera toxin, which stimulates the synthesis of endogenous cAMP, increased IK(Ca) 25% in 10 min and the effect was not reversible. Both approaches were effective in all seven neuron types studied. Application of serotonin plus phosphodiesterase inhibitor caused an increase in IK(Ca) in neuron R15 but not in the other neuron types. Application of pentylene tetrazole (PTZ) led to a decrease in IK(Ca). It is proposed that elevation of cyclic AMP mediates an increased sensitivity of the IK(Ca) channel to Ca ions.     

Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins

We developed a method for expression in Arabidopsis of a transgene encoding a cleavable chimeric polyprotein. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), DmAMP1 originating from Dahlia merckii seeds and RsAFP2 originating from Raphanus sativus seeds, which are linked by an intervening sequence ("linker peptide") originating from a natural polyprotein occurring in seed of Impatiens balsamina. The chimeric polyprotein was found to be cleaved in transgenic Arabidopsis plants and the individual AMPs were secreted into the extracellular space. Both AMPs were found to exert antifungal activity in vitro. It is surprising that the amount of AMPs produced in plants transformed with some of the polyprotein transgene constructs was significantly higher compared with the amount in plants transformed with a transgene encoding a single AMP, indicating that the polyprotein expression strategy may be a way to boost expression levels of small proteins.     

Alteration of protein metabolism in individual,identified neurons from Aplysia

A search for control mechanisms governing protein metabolism in neurons from Aplysia californica has uncovered two examples of altered patterns of newly synthesized proteins: (1) The pattern of newly synthesized proteins in the R2 neuron is altered when protein synthesis occurs at elevated temperatures (22–30°C as compared with 13–15°C). (2) The processing of newly synthesized 12,000 dalton (12k) material to 6–9,000 dalton (6–9k) size in the R15 neuron (Strumwasser, F. and Wilson, D. F. [1976], J. Gen. Physiol., in press) can be blocked by certain ion replacements. If acetate replaces chloride in the incubation medium during the synthesis of 12k material, an early step in the processing, prior to the actual breakdown of 12k material, is blocked. Experiments with RNA-synthesis inhibitors indicate that none of the mRNAs which code for abundantly synthesized protein species in the R2 or R15 neurons have short (less than 4 hr) half-lives. This result has implications for an earlier report of regulation of protein synthesis in the R15 neuron.     

Patterns of proteins synthesized in the R15 neuron of Aplysia. Temporal studies and evidence for processing

The time-course of changes in the pattern of newly synthesized proteins in the R15 neuron of the parietovisceral ganglion of Aplysia californica has been studied at 14 degrees C. 5% polyacrylamide gels containing sodium dodecyl sulfate (SDS) have been used to separate newly synthesized (leucine-labeled) proteins from the neuron. We have demonstrated that the pattern of newly synthesized proteins from the R15 neuron does not change significantly if 5-h pulses of labeled leucine are given during the first 72 h of in vitro incubation of the excised ganglion. However, the level of leucine incorporation begins to decline somewhere between 17 and 43 h after the ganglion is isolated; at 43 and 69 h the levels of incorporation fell to 29 and 10% of the initial level, respectively. A number of conclusions have been drawn from the use of a sequential, double-label type of experiment in the same cell. There is processing of SDS-soluble, 12,000-dalton (12k) material to 6,000-9,000-dalton (6-9k) material. These materials are the two major peaks on gels after long labeling periods and together account for about 35% of all newly synthesized proteins. After synthesis of 12k material, there is a gradual disappearance of 12k (half-life about 8 h) and simultaneous appearance of 6-9k material on the gels, as the postsynthesis "chase" period of ganglia incubation is increased. The processing of 12k to 6-9k material occurs even in the presence of anisomycin, a protein syntehsis inhibitor, during the chase period. While the rate of 12k to 6-9k conversion can vary from cell to cell, it appears to remain consistent within, and is characteristic of, any individual R15. We detect no circadian rhythm in either the rate of 12k synthesis or the rate of 12k to 6-9k processing with 5-h label periods. These results are discussed in relation to the roles of 12k and 6-9k material in the R15 neuron.     

Aplysia synapse associated protein (APSAP): identification, characterization, and selective interactions with Shaker-type potassium channels

The vertebrate post-synaptic density (PSD) is a region of high molecular complexity in which dynamic protein interactions modulate receptor localization and synaptic function. Members of the membrane-associated guanylate kinase (MAGUK) family of proteins represent a major structural and functional component of the vertebrate PSD. In order to investigate the expression and significance of orthologous PSD components associated with the Aplysia sensory neuron-motor neuron synapse, we have cloned an Aplysia Dlg-MAGUK protein, which we identify as Aplysia synapse associated protein (ApSAP). As revealed by western blot, RT-PCR, and immunocytochemical analyses, ApSAP is predominantly expressed in the CNS and is located in both sensory neuron and motor neurons. The overall amino acid sequence of ApSAP is 55–61% identical to Drosophila Dlg and mammalian Dlg-MAGUK proteins, but is more highly conserved within L27, PDZ, SH3, and guanylate kinase domains. Because these conserved domains mediate salient interactions with receptors and other PSD components of the vertebrate synapse, we performed a series of GST pull-down assays using recombinant C-terminal tail proteins from various Aplysia receptors and channels containing C-terminal PDZ binding sequences. We have found that ApSAP selectively binds to an Aplysia Shaker-type channel AKv1.1, but not to (i) NMDA receptor subunit AcNR1-1, (ii) potassium channel AKv5.1, (iii) receptor tyrosine kinase ApTrkl, (iv) glutamate receptor ApGluR1/4, (v) glutamate receptor ApGluR2/3, or (vi) glutamate receptor ApGluR7. These findings provide preliminary information regarding the expression and interactions of Dlg-MAGUK proteins of the Aplysia CNS, and will inform questions aimed at a functional analysis of how interactions in a protein network such as the PSD may regulate synaptic strength.     

Long-term change of viability of neuron functioning and its possible behavioral consequences in the adult Aplysia

1. In Aplysia of different ages, three well defined behavioral responses and their substrates were examined. 2. Two of the behaviors and their substrates are age-sensitive, the gill withdrawal reflex, and osmoregulation. They are sensory-dependent for their activation. The third, the gill respiratory pumping movements, GPM, and its substrates is age-invariant. It is initiated by a network in the CNS, and modulated by sensory input. 3. Age-sensitivity of a neuron appears dependent on its pathway: the pathways mediating sensory-initiated behavior are more vulnerable to aging than the pathway mediating CNS-initiated behavior. 4. a) The age-sensitive, GWR, gill withdrawal reflex, in unrestrained animals and in reduced preparations conforms to the age-dependent reduced functioning of L7, the major mediator of the reflex. b) Whereas, the age-invariant GPM conforms to the age-independent functioning of LDG1, a major contributor to GPMs. c) The age-related slowing of osmoregulation is consistent with the lack of response in the putative water balance neuron, R15, to stimulation of osmoreceptors by dilute seawater. 5. Age-sensitivity of L7 is defined by its reduced function, decreased facilitation at its terminals, reduced input resistance, and remodeling of junctions in the muscles it innervates. This is in contrast to the age-invariance of these properties in LDG1 and of the junctions in muscles it innervates. Thus far, the age-sensitivity of R15 is revealed by its reduced responsiveness to synaptic input, reduced input resistance, and little response to osphradial stimulation. 6. Age-sensitivity of the GWR, osmoregulation and of their substrates is not necessarily maladaptive.(ABSTRACT TRUNCATED AT 250 WORDS)