Cell Biological Studies of Memory 
Aplysia sensory neurons and measured the levels 
of expression driven by the CRE in response to 
stimulation by 5-HT. We next exposed the neu- 
rons to five repeated (5-min) pulses of 5-HT, the 
protocol that produces long-term synaptic facili- 
tation, and found a fourfold induction of the re- 
porter gene. Similarly, cAMP induced expression 
by 3-4-fold. Thus repeated pulses or prolonged 
exposure to 5-HT stimulates transcription, and 
this stimulation can be simulated by elevating lev- 
els of cAMP. The increase in lacZ expression is 
mediated specifically through the CRE sequence. 
A plasmid (ACRE-/«cZ) from which the CRE ele- 
ment is deleted shows no expression. 
Does the stimulation of transcription mediated 
by 5-HT depend on the binding of transcription 
factors to the CRE sequence? To address this ques- 
tion, we co-injected the CRE reporter construct 
with either an oligonucleotide that encodes the 
wild-type CRE sequence and binds CREB, or with 
a mutant oligonucleotide that does not bind 
CREB-like factors. The wild-type oligonucleo- 
tide, which binds to and titrates out CREB, 
blocked the 5-HT-induced expression of the re- 
porter construct, whereas the mutant oligonucle- 
otide produced no inhibition. 
These experiments suggest that the induction 
mediated by 5-HT requires positively acting 
CREB-like cellular factors that interact with the 
CRE, and provide direct evidence that 5-HT can 
stimulate transcription of genes containing the 
CRE sequences. This is consistent with results 
that Pramod Dash, Binyamin Hochner, and I ob- 
tained last year in physiological experiments, in 
which we microinjected the CRE into the nucleus 
of sensory neurons and selectively blocked the 
long-term increase in synaptic effectiveness with- 
out affecting short-term facilitation. Both these 
sets of results suggest that CREB-like transcrip- 
tional activators are required for the induction of 
long-term facilitation. 
Does the activation of transcription by 5-HT 
have a clear threshold or is it graded? To assess 
this question, Kaang, Grant, and I were in- 
fluenced by our earlier findings discussed above, 
that a single (5-min) pulse of 5-HT will only 
elicit short-term facilitation. To generate signifi- 
cant long-term facilitation, four or five pulses are 
required. Using the same pulse protocol, we 
found that a single pulse of 5-HT does not, in fact, 
stimulate expression of the reporter gene, 
whereas four to five pulses of 5-HT/IBMX pro- 
duced a sevenfold stimulation. An intermediate 
number of pulses (two or three) gave an interme- 
diate level (twofold) of stimulation. This correla- 
tion, between the number of 5-HT trials and the 
level of transcriptional induction and facilita- 
tion, suggests that the graded nature of long-term 
facilitation may reflect the graded nature of tran- 
scriptional induction. 
Does activation by 5-HT require that CREB be 
phosphorylated? If so, must activation of CREB in 
the sensory neuron be mediated by protein kinase 
A (PKA)? To address these questions, Kaang, 
Grant, and I next microinjected two constructs 
into the sensory neurons: a reporter gene as well 
as a chimeric transactivation plasmid that ex- 
presses CREB. Following co-injection, exposure 
to 5-HT produced a 10-fold stimulation of tran- 
scription. By contrast, injection of either trans- 
activation or reporter plasmid alone showed 
no expression. This transcriptional stimulation 
depends on phosphorylation of the PKA consen- 
sus site at Ser' in CREB. Microinjecting a transac- 
tivation plasmid (pSAl 19) containing a mutation 
that converts Ser"^ to Ala"^ showed no stimula- 
tion with 5-HT. 
Thus long-term facilitation induced by 5-HT 
leads to activation of CRE-inducible genes. The 
induction of these genes by the cAMP cascade is 
graded and can be initiated by PKA through phos- 
phorylation of CREB. A conventional modulatory 
transmitter, 5-HT, can therefore select either a 
cytoplasmic or a genomic program of cellular ac- 
tion, depending on the number of presentations 
of 5-HT. By being able to activate a nuclear signal 
(through the phosphorylation of CREBs and its 
action on the CREs) , modulatory transmitters can 
activate transcription and thereby take on the 
properties of growth factors. In the case of sen- 
sory neurons, transcriptional activation of CREB- 
related proteins seemed to represent one compo- 
nent of the switch, which extends the short-term 
cytoplasmic process for synaptic facilitation into 
the genomic changes characteristic of the long- 
term facilitatory process — one that includes the 
growth of new synaptic connections. 
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