SECOND MESSENGER MECHANISMS IN APLYSIA NEURONS 
James H. Schwartz, M.D., Ph.D., Investigator 
Central to Dr. Schwartz's work is the identifica- 
tion of molecular events that control synaptic trans- 
mission. Signal transduction mechanisms are funda- 
mental to understanding synaptic plasticity (the 
change of a neuron's activities as a result of prior 
stimulation or training), which is thought to under- 
lie learning and memory. The principal second mes- 
senger systems that have been studied in identified 
Aplysia neurons are the cAMP cascade, the activa- 
tion of protein kinase C, and the newly discovered 
action of lipoxygenase metabolites or arachidonic 
acid. Identification of a modulatory cascade and 
specification of its target substrates are not, how- 
ever, sufficient for explaining memory and learning. 
Dr. Schwartz and his co-workers have concentrated 
on understanding the mechanisms by which the 
modulatory molecular events are made to endure 
in the neuron and thus how they might operate to 
change the behavior of the animal. 
I. Cyclic AMP 
One molecular mechanism for persistent protein 
phosphorylation had previously been demon- 
strated in Dr. Schwartz's laboratory: as a conse- 
quence of long-term training, regulatory (R) sub- 
units of the cAMP-dependent protein kinase 
diminish in sensory neurons, whereas catalytic (C) 
subunits remain constant. To determine how this 
change in ratio of R-to-C subunits is produced, anti- 
bodies to both subunits are being raised (using se- 
quence information obtained from last year's work 
on the cloning of these molecules), a synaptosome 
preparation from Aplysia ganglia has been de- 
veloped, and it has been shown that synthesis of 
new protein is required for maintenance of the 
change in subunit ratios that occurs with long-term 
training. 
The turnover of R does not occur after short-term 
training. This change in the R-to-C ratio provides a 
mechanism for the persistent phosphorylation ob- 
served by Dr. Eric R. Kandel (HHMI, Columbia Uni- 
versity) in long-term, trained sensory neurons in 
which the level of cAMP is baseline, since decreas- 
ing the amount of R relative to C should increase 
kinase activity at any subsaturating concentration of 
cAMP (left shift). Dr. Kandel and his co-workers re- 
ported, however, that inhibitors of protein synthe- 
sis prevent the development of long-term sensitiza- 
tion and block persistent protein phosphorylation. 
In collaboration with Dr. Kandel, Dr. Schwartz and 
his colleagues examined whether the lasting disap- 
pearance of R subunits depends on new protein 
synthesis, with isolated sensory neuron clusters ex- 
posed to serotonin for 2 h, a protocol that pro- 
duces facilitation lasting 24 h. This treatment repro- 
duces the diminution of R subunits in sensory 
neuron clusters found in the intact animal when 
the sensory cells are tested 24 h after the exposure. 
(The isolated nervous tissue provides a more con- 
venient experimental preparation to examine this 
phenomenon than does the intact animal.) If the 
diminution of R subunits is involved in producing 
long-term sensitization, their enduring loss would 
be expected to be blocked by the same inhibitors. 
Inclusion of anisomycin, an inhibitor of protein 
synthesis, during the exposure to serotonin 
blocked this effect, indicating that new protein 
must be made to maintain the diminished ratio of 
R-to-C subunits. 
Although new protein synthesis is required for 
the sustained decrease in R-to-C ratio, the biochem- 
ical mechanism by which R subunits are made to di- 
minish is post-translational, because it occurs in 
Aplysia synaptosomes (which do not make new 
protein) during continuous exposure to a permeat- 
ing and effective analogue of cAMP, with no degra- 
dation of the C subunit or any other major protein. 
Dr. Schwartz now will determine the mechanisms 
by which the R-to-C ratio is altered and how that al- 
teration is maintained. Preliminary experiments 
indicate that there is no change in messenger RNA 
for R subunits after long-term treatments, and this 
suggests that the alteration may be maintained by a 
change in RNA processing or the induction of a spe- 
cific protease. 
II. Protein Kinase C. 
Behavioral training of the animal, as well as ap- 
plication of serotonin, produces the same translo- 
cation of protein kinase C from cytosol to mem- 
brane seen with phorbol esters, which Dr. Kandel 
and his co-workers have found to produce short- 
term facilitation. This translocation, which occurs 
directly in sensory cells, has been confirmed by 
measuring the kinase both by its enzymatic activity 
and by phorbol ester binding. Kinase activity on the 
membrane is constitutively active; that is, it no 
longer requires exogenous lipid activators. Dr. 
Continued 
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