Emergence of posttetanic potentiation as a distinct phase in the differentiation of an identified synapse in Aplysia.

The developmental time course of posttetanic potentiation was studied at an identified chemical synapse. In stage 11 juveniles (3 weeks after metamorphosis), the synaptic connections made by cholinergic neuron L10 onto postsynaptic neurons L2 to L6 were present but showed no posttetanic potentiation. In stage 13 adults (12 weeks after metamorphosis), the same tetanus resulted in an increase of 300 percent in the synaptic potential. A similar pattern was observed at two other identified synapses in the abdominal ganglion. Thus, the initial steps in synapse formation do not include the expression of this plastic capability. Rather, at least 10 weeks is required between the onset of synaptic function and the final expression of mature synaptic properties.

Epileptogenic agents enhance transmission at an identified weak electrical synapse in Aplysia.

To examine the possibility that alterations in the effectiveness of electrical synapses might participate in epileptogenesis, the effects of several convulsants on an identified weak electrical synapse in Aplysia were examined. Application of pentylenetetrazole, strychnine, or tetraethylammonium led to a dramatic increase in the size of the electrical postsynaptic potential mediated by the synapse; penicillin was considerably less effective. In a number of animals, the increased electrical synaptic effectiveness led to the abnormal conduction of spikes across the synapse. If convulsants have a similar action in mammalian cortex, enhanced transmission at weak electrical synapses may provide abnormal pathways for the flow of seizure activity and contribute in part to the synchronous firing of neurons characteristic of epileptic activity.

Development of plastic mechanisms related to learning at identified chemical synaptic connections in Aplysia.

In the marine snail Aplysia californica learned changes in behavior have been traced to alterations in synaptic efficacy. With the ability to raise the animals in the laboratory, we have explored the development of four types of plastic mechanisms at identified synapses: post-tetanic potentiation and pre-synaptic inhibition, which do not as yet have known behavioral functions, and homosynaptic depression, the cellular mechanism of short-term habituation, and heterosynaptic facilitation, the basis of short-term sensitization. Homosynaptic depression and pre-synaptic inhibition are present early in juvenile life. In contrast, post-tetanic potentiation and heterosynaptic facilitation appear only later, after a discrete interval. The step-wise ontogeny of synaptic plastic mechanisms in Aplysia parallels the gradual emergence of behavior in successive developmental stages. Interestingly, senescence reverses the developmental sequence for habituation and sensitization mechanisms. To pursue further an understanding of the relationship between synapse formation and plasticity underlying learning it will be necessary to extend these studies to dissociated cell culture where mechanisms can be explored on the molecular level. Cell culture may also permit examination of the development of cellular mechanisms underlying classical and operant conditioning which may clarify differences between associative and non-associative mechanisms.

Differential emergence of cellular mechanisms mediating habituation and sensitization in the developing Aplysia nervous system.

The development of the cellular substrates underlying habituation and sensitization, two simple forms of learning, was examined at a polysynaptic sensory-to-motor connection in the neural circuit mediating defensive mucus release in the marine mollusc, Aplysia californica. Animals were studied throughout juvenile life, stages 9 (40 days of development) to 12 (95 days), and into adulthood, stage 13 (120 days), starting just after metamorphosis when mucus release first becomes evident. Homosynaptic depression, which mediates habituation, was already present in its adult form in stage 9. Heterosynaptic facilitation, which mediates sensitization, appeared in stage 10 and reached maturity during stages 11 and 12. Thus, the development of synaptic plasticity in this circuit occurs in discrete phases in which the gradual emergence of heterosynaptic facilitation occurs only after homosynaptic depression is well established.

Synaptic plasticity in vitro: cell culture of identified Aplysia neurons mediating short-term habituation and sensitization.

The gill withdrawal reflex of the marine mollusk, Aplysia californica, shows habituation and sensitization, two simple forms of learning. In order to extend the cellular studies on synaptic plasticity underlying the changes in the reflex behavior, and to explore further the development of synaptic plasticity during synapse formation, we have sought to establish the neural circuit of the gill withdrawal reflex in vitro. We report here the reconstruction of the elementary gill withdrawal circuit in cell culture and find that the cells show short-term homosynaptic depression and heterosynaptic facilitation, the cellular mechanisms of habituation and sensitization, respectively.

Giant Aplysia neuron R2 has distal dendrites: evidence for protein sorting and a second spike initiation site.

Because of its anatomy, the neuron R2 of Aplysia has been used to study how proteins are distributed to their appropriate destinations within the cell. The R2 cell body resides in the abdominal ganglion, while its axons terminate on glands in the skin. Using intracellular injection of HRP and intraxonal recordings, we found that R2 has a dendritic (receptive) arborization in the pleural ganglion. The structure of these dendrites was examined after injecting the soma with 3H-L-fucose, thereby labeling glycoproteins that are transported to all regions of the cell. Light- and electron-microscope autoradiography show that the openings to the dendrites are not on the periphery, but are suspended inside the axon by glial cell infoldings. All of the organelles seen in the axon are found in the dendrites, including 2 types of vesicles. Neither the axon nor the dendrites contain ribosomes. Thus, R2 has 3 functionally distinct regions--cell body, dendrites, presynaptic terminals--that are separated from each other by at least 4 cm. This implies that pre- and postsynaptic proteins made in the cell body are transported along the axon to the pleural ganglion, where they are sorted. To investigate this idea, we exposed the abdominal ganglion to 35S-methionine to label R2's proteins. Analyses by SDS-PAGE of the rapidly transported labeled proteins from R2 consistently showed a 78 kDa band that accumulated in the pleural ganglion and did not move into the peripheral nerves. This then is a putative dendritic constituent.

Identified cholinergic neurons R2 and LPl1 control mucus release in Aplysia.

1. R2 and LPl1 are homologous giant cholinergic neurons in the nervous system of Aplysia with overlapping and almost symmetrical axonal trees extending over most of the body wall. In spite of much experimental study, the behavioral role of the cells has remained unknown. 2. After intrasomatic injection of R2 and LPl1 with horseradish peroxidase (HRP), the giant cell axons were traced to the periphery and found to contact subepidermal glands in the body wall exclusively. 3. The axons penetrated the glandular basal lamina, indenting the gland cell cytoplasm, and expanded into varicosities containing putative cholinergic transmissive sites. 4. Histochemical characterization of the contents of the glands showed that they contain mucus, suggesting that the giant cells control mucus release from the body wall. 5. Stimulation of R2 or LPl1 resulted in glandular discharge, as measured both by an increase in the appearance of protein and of mucus on the body wall. 6. R2 and LPl1 control mucus release from the body wall, thus providing a new system for investigations of neuroglandular control as well as a behavioral context for cellular studies using these two neurons.

Giant Aplysia neuron R2 reliably forms strong chemical connections in vitro.

The giant cholinergic neuron R2 of Aplysia was cultured in combination with identified neurons L11 and R15 and members of a group of left upper quadrant (LUQ) cells L2 to L6 from the abdominal ganglion. All of these neurons receive cholinergic input from other cells in vivo, but not from R2. In vitro, R2 reliably formed unidirectional chemical connections with these cells. Single action potentials in R2 produced a dual fast and slow inhibitory response in LUQ cells (L2 to L6), a dual fast inhibitory-slow excitatory response in L11, and a slow inhibitory response in R15. The connections formed on LUQ cells were characteristic of their cholinergic input, but the R2-L11 and the R2-R15 connections also had noncholinergic properties. Thus, unlike L10 which forms connections only with its normal targets in vitro, R2 forms strong chemical connections with other neurons which are not found in vivo. The properties of the R2 connections also suggest that it may release another neurotransmitter besides acetylcholine.

Proteins rapidly transported to the synapses of a single identified neuron of Aplysia californica.

The cell body of R2, a giant cholinergic neuron of Aplysia californica, resides in the abdominal ganglion, whereas its synapses are on thousands of unicellular mucus glands located in the skin. Due to the great spatial separation between the site of macromolecular synthesis and the presynaptic terminals, rapid axonal transport can be used to segregate synaptic proteins from those to be used elsewhere in the cell. The proteins of R2 were labeled by incubating the abdominal ganglion in [35S]methionine for 5 hr in a chamber separated from the rest of the isolated central nervous system. After 50 hr, 28 radiolabeled proteins were reproducibly found by one- and two-dimensional polyacrylamide gel electrophoresis to be transported to the distal regions of peripheral nerves P6, P7, and P8 that innervate the parapodia and middle body wall. We are sure that R2 is the source of these proteins since radioautography of sections taken throughout the nervous system, complemented by cobalt tracings, showed that R2 is the only neuron in the abdominal ganglion with axons in these nerves. Nine of the 28 transported proteins are glycoproteins since they were also labeled after injecting R2's cell body with [3H]-L-fucose. There is evidence that the proteins and glycoproteins are destined for R2's presynaptic terminals. For example, in experiments in which the body wall and parapodium remained attached to the nerves, the proteins were transported to the skin region that contains the glands. Moreover, analyses of the distribution of the rapidly transported proteins by qualitative radioautography and by extrusion of axoplasm indicated that none are constituents of the axolemma.(ABSTRACT TRUNCATED AT 250 WORDS)