CELL AND MOLECULAR MECHANISMS OF LEARNING 
Eric R. Kandel, M.D., Senior Investigator 
I. Molecular Mechanisms of Long-Term Memory. 
In a variety of learning tasks, long-term memory 
differs from short-term memory in requiring 
protein synthesis during training. Sensitization of 
the gill- and siphon-withdrawal reflex in Apfysia 
californica is a simple form of learning in which 
this requirement can be studied in individual 
identified sensory and motor neurons that partici- 
pate in the storage of both the short- and long-term 
memory. Behavioral training for sensitization in 
the intact animal or application of serotonin (a 
transmitter that mediates sensitization) to indi- 
vidual sensory and motor cells in cultures leads to 
presynaptic facilitation, an enhancement of trans- 
mitter release from the sensory neurons. A single 
tail stimulus or a single application of serotonin (5- 
HT) or cAMP (the second messenger activated by 
5-HT) causes a transient increase in synaptic 
strength lasting minutes that is independent of new 
protein and RNA synthesis. In contrast, five tail 
stimuli or five repeated applications of 5-HT (or 
cAMP) give rise to long-term enhancement lasting 
one day or more that requires both translation and 
transcription. 
A. Injection of the cAMP-responsive element into 
the nucleus o/Aplysia sensory neurons blocks long- 
term facilitation. To gain insight into the molecu- 
lar events that lead to long-term presynaptic facili- 
tation in sensory neurons, A. Barzilai, T. E. Kennedy, 
J. D. Sweatt, and Kandel used analytical two-dimen- 
sional gels to analyze 5-HT-stimulated ['^Sjmethio- 
nine incorporation into proteins. 5-HT rapidly stim- 
ulated transcriptionally dependent changes in 15 
early proteins that peaked within 15-30 min and 
subsided within 1-3 h. Of the early proteins, 10 
showed increases and 5 decreases in net incorpora- 
tion of label. The same 15 early proteins were also 
induced by cAMP In these features— rapid and tran- 
sient induction, transcriptional dependence, and 
second messenger mediation— these early proteins 
resemble the immediate early gene products in- 
duced in vertebrate cells by growth factors. 
Most cAMP-inducible genes so far studied are ac- 
tivated by specific transcription factors that bind to 
the enhancer sequence TGACGTCA, called the 
cAMP-responsive element (CRE). The CRE binds as 
a dimer of 43 kDa protein subunits, the enhancer- 
binding protein (CREBP). P. Dash, B. Hochner, and 
Kandel have now found that extracts of Aplysia 
sensory neurons contain proteins that specifically 
bind to a mammalian (somatostatin) CRE sequence. 
The Aplysia protein resembles the mammalian 
CREB protein, in that it is —45 kDa and serves as a 
substrate for the A kinase. Microinjection of oligo- 
nucleotides containing the CRE sequence into the 
nucleus of a sensory neuron selectively blocks the 
long-term increase in synaptic strength without af- 
fecting short-term facilitation. Injection of control 
enhancer sequences fails to block the increase in 
synaptic strength. These results indicate that tran- 
scription of cAMP-inducible genes is required for 
the expression of long-term facilitation. 
B. Acquisition of long-term facilitation in Aplysia 
sensory neurons leads to a novel mechanism for 
the persistent activation of the A kinase: protein 
synthesis-dependent loss of regulatory subunits. 
One of the consequences of the transcriptionally 
dependent alterations, produced in the sensory 
neurons by repeated application of 5-HT, is the 
persistent phosphorylation of a set of substrates 
also phosphorylated transiently after one pulse 
of 5-HT. To explore possible mechanisms under- 
lying sustained phosphorylation, P Bergold, J. H. 
Schwartz (HHMI, Columbia University), Sweatt, 
and Kandel examined changes in the properties of 
the Aplysia cAMP-dependent protein kinase (A 
kinase), a heterodimer of two regulatory (R) 
subunits that inhibit two catalytic (C) subunits. The 
A kinase is activated during both short- and long- 
term sensitization, and the amount of R is lowered 
as compared with C in the sensory cells of long- 
term behaviorally sensitized animals. Bergold, 
Schwartz, Sweatt, and Kandel found that facilitatory 
stimuli (5-HT or cAMP) also diminish the R-to-C 
ratio in the sensory neurons and that this reduction 
in R requires new protein synthesis. Thus one of 
the functions of the macromolecular synthesis re- 
quired for long-term facilitation is to synthesize 
proteins that regulate the cAMP-dependent ki- 
nase in a long-term manner. This long-term regula- 
tion may account for the persistent increase in pro- 
tein phosphorylation observed in long-term 
facilitation. 
Continued 
