Proc. Nati. Acad. Sci.
USA
Vol. 84, pp. 6948-6952, October 1987
Neurobiology
Inhibition of protein synthesis prolongs Ca2 -mediated reduction of
K+ currents in molluscan neurons
(Hermissenda crasswornis/memory/protein kinase C/Ca2+-activated phospborylation/anisomycin)
D. L. ALKON*, B. BANK*, S. NAITO*, C. CHENt,
*Section on Neural Systems, Laboratory of Biophysics, Intramural Research Program, National Institute of Neurological and Communicative Disorders and
Stroke, National Institutes of Health at the Marine Biological Laboratory, Woods Hole, MA 02543; and tBoston University Marine Program, Marine
Biological Laboratory, Woods Hole, MA 02543
Communicated by Mark R. Rosenzweig, June 16, 1987 (received for review March 4, 1987)
ABSTRACT Elevated intracellular Ca2l concentration
within the Hermissenda type B cell has previously been shown
to cause transient reduction of both the early K+ current IA and
the delayed, Ca2+-dependent K+ current ICaZ+ K, a reduction
that is more permanent with classical conditioning. Other
earlier experiments suggested that Ca2+-mediated reduction of
K+ currents initially involves the dual activation of Ca2+/
calmodulin-dependent and Ca2+/lipid-dependent protein ki-
nases. In the present study, voltage-clamp conditions that cause
substantial increases in intracellular Ca`+ concentration (i.e.,
a Ca2+ "load") were used to produce IA and ICa2+.K+ reduction
with and without the protein synthesis inhibitor anisomycin or
cycloheximide or the control substance deacetylanisomycin in
the bathing medium. Anisomycin (100 !LM) and cycloheximide
(100 ,uM) caused no significant change of resting membrane
potential, holding current, or the non-voltage-dependent
"leak" current. However, inhibition of protein synthesis
prevented recovery from Ca2+-mediated K+-current reduc-
tion. This effect resembled the effect of ih'ecting purified
Ca2+-dependent kinases and was blocked by the presence of
trifluoperazine in the bathing medium. Activation of protein
kinase C with a water-soluble phorbol ester caused marked
reduction of protein synthesis in Herinissenda neurons as
monitored by two-dimensional gel electrophoresis. Synthesis of
new proteins therefore may be important for reversal of initial
steps during memory storage, and Ca2 -activated phosphoryl-
ation pathways may initiate long-term changes by turning off
(as well as by turning on) the synthesis of particular proteins.
Molecular mechanisms of memory storage have to provide a
means for cellular information to survive the normal turnover
of subcellular constituents. Such mechanisms could involve
the production of molecules with very long half-lives, alter-
ation of the protein synthetic apparatus (i.e., DNA and/or
RNA) (1-6), or as yet undescribed self-perpetuating cycles of
change in signaling between the cytoplasm and the nucleus
(7-11). Many previous studies have shown an association
between the disruption of long-term memory storage and
inhibition of protein synthesis during a critical period of
memory consolidation (1-6). These studies suggest that the
synthesis of new proteins is required for initiating events in
the long-term storage process. A crucial initiating step in the
acquisition of a classically conditioned response of the
mollusc Hermissenda crassicornis is Ca2"-mediated reduc-
tion of K+ currents (12-16). Specific K+ currents (an early
K+ current, IA, and a delayed Ca2+-dependent K+ current,
ICa2+-K+) remain reduced for at least 2-3 days during the
retention period of the classical conditioning (15, 16). This
K+-current reduction has, by a number of criteria, been
causally implicated in the actual storage of the learned
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AND J. RAM*t
association between the conditioned and unconditioned stim-
uli (13). Evidence for similar persistent modification of K+
currents has been obtained for CA1 pyramidal cells in the
hippocampi of classically conditioned rabbits (17, 18).
Elevation of intracellular Ca2+ within the Hermissenda
type B cells was shown to cause reduction of both IA and
ICa2+-K+ as occurred with classical conditioning (12-14).
Similarly, iontophoresis of inositol trisphosphate (but not
inositol monophosphate) caused IA and ICa2+-K+ reduction
(19). A variety of other experiments suggests that elevated
Ca2` concentration reduces the K+ currents by a dual
activation of Ca2+/calmodulin-dependent kinase and Ca2+/
lipid-dependent protein kinase (kinase C) (20-22).
We asked in the present study whether Ca2+-mediated
reduction of K+ currents might in some way involve the
synthesis of new proteins. Voltage-clamp conditions that
produce substantial increases in intracellular Ca2' concen-
tration (i.e., a Ca2+ "load") were used to cause IA and
ICa2+-K+ reduction with and without protein synthesis inhib-
itors in the bathing medium. Inhibition of protein synthesis is
shown here to prevent recovery from Ca2+-mediated K+
current reduction. Once the K+ currents are reduced by a
Ca2+ load, they remain reduced, and with additional Ca2+
loads, they are reduced further. This effect-i.e., prevention
of recovery from Ca2+-mediated reduction of K+ current-is
similar to the effect of injection into the cell of purified Ca2+/
calmodulin-dependent kinase and of kinase C activation,
following a Ca2+ load (20-22). Furthermore, activation of the
latter enzyme with a phorbol ester was found to reduce
protein synthesis as measured by gel electrophoretic tech-
niques. The results of this study suggest, therefore, that the
synthesis of new proteins is important for reversal of initial
steps during memory storage and that Ca2+-activated
phosphorylating pathways (e.g., via the Ca2 /lipid-depen-
dent kinase C) may initiate long-term changes by turning off
(as well as by turning on) the synthesis of particular proteins.
METHODS
Voltage Clamp. The type B cell soma was isolated by
axotomy from all synaptic interactions and impulse-generat-
ing membrane as previously described (23). The membrane
potential of the cell was then clamped to -60 mV (absolute)
with a two-microelectrode voltage clamp as described in
detail elsewhere (12, 14, 24).
Type B Soma Currents. There are four major voltage-
dependent ionic currents across the soma membrane. As
previously described (12, 14, 24), these are IA, an early,
rapidly inactivated outward K+ current sensitive to 4-
aminopyridine (3 mM); ICa2+-K+, a delayed, voltage-depen-
dent outward K+ current dependent on the level of cytosolic
tPresent address: Department of Physiology, Wayne State Univer-
sity Medical School, Detroit, MI 48201.
6948
 Neurobiology: Alkon et A Proc. Natl. Acad. Sci. USA 84 (1987) 6949

Ca2+; IK, a delayed outward K+ current eliminated by pLM deacetylanisomycin also caused substantial inhibition of
tetraethylammonium ion (100 mM) and minimally activated protein synthesis (Table 1). Minimal inhibition resulted,
at potentials <0 mV (absolute); and Ica2+, a sustained however, if the deacetylanisomycin had been left to stand 5
voltage-dependent inward Ca2+ current. There are also a hr prior to the incubation. Thus, deacetylanisomycin previ-
light-induced inward Na' current (INa+) and a smaller, ously standing at 220C for .5 hr was used as a control solution
light-induced outward K+ current (ICa2+-K+)(14). for the recording experiments. Protein kinase C activation by
Values of IA obtained here were peak amplitudes (occur- bath application of 10 AM deoxyphorbol 13-isobutyrate
ring within 30-40 msec) after correction for the voltage- 20-acetate in the presence of [14C]leucine had an effect on
independent "leak" current (12, 14). Values of ICa2+-K+ were protein synthesis similar to that of anisomycin: a marked
"leak"-corrected peak amplitudes occurring within 300-400 overall reduction of protein synthesis (Fig. 1).
msec after onset of the depolarizing command. Depolarizing Inhibition of Protein Synthesis Decreases IA and ICa2+-K+.
commands were given regularly at 30-sec intervals. Following a "Ca2+-load," effected by a 25-sec depolarization
Measurement of Protein Synthesis in the Circumesophageal to 0 mV (from a holding potential of -60 mV, absolute) paired
Nervous System. To assess the efficacy of inhibitor and with a 2.0-sec light step (1035 ergs-cm-2Esec-1), IA and
control solutions, Hermissenda circumesophageal nervous ICa2+-K+ are transiently reduced. Thus, by z90 sec after the
systems were dissected out and isolated from the animals. light paired with depolarization, a 1.0-sec depolarizing step to
They were pretreated with 100 gM anisomycin or deacetyl- 0 mV (presented at 30-sec intervals) elicits approximately the
anisomycin, which, when indicated, were left to stand at same magnitude of IA and ICa2+-K+ as was elicited before the
room temperature in artificial sea water for 5 hr. The nervous light and depolarization (Fig. 2C). Prior exposure of the type
systems were exposed to one of these two drugs (both from
Pfizer) in artificial sea water at 150C for 45 min. Protein B cell for >20 minutes to 100 ,uM anisomycin caused IA and
synthesis was measured by adding [14C]leucine (Dupont- ICa2+-K+ to remain reduced after the Ca2+ load (Fig. 2). For
New England Nuclear, 0.5 ,uCi per 200 Al; 1 Ci = 37 GBq) to example, the third test depolarization elicited significantly
the bathing media. 14C incorporation into newly synthesized less IA [mean difference, -1.75 ± 0.61 nA (mean ± SD, n =
proteins was terminated at 60 min by homogenizing each 5, P < 0.002)] and ICa2+-K+ [mean difference, - 1.35 ± 0.49 nA
nervous system in 50 1ul of ice-cold Hepes buffer (50 mM, pH (n = 5, P < 0.002)] as tested by a direct two-tailed comparison
7.4) containing 1 mM phenylmethylsulfonyl fluoride. Homog-
enate proteins were precipitated by 10%6 trichloroacetic acid pH -,
with 100 ug of bovine serum albumin as a carrier. Solutions Mr
were centrifuged at 10,000 x g for 5 min, and precipitates
were washed twice with 10%o trichloroacetic acid by resus-
pension and centrifugation. Precipitates were dissolved in
200 Al of 0.1 M NH40H, and their radioactivities were
determined in 10 ml of Aquassure (New England Nuclear) by
scintillation counting.
In order to examine the effects of protein kinase C
activation on protein synthesis, the above [14C]leucine incor-
poration assays were also performed in the presence of 10 ,/M
deoxyphorbol 13-isobutyrate 20-acetate. The reaction was
terminated by homogenizing each nervous system in 50 ,ul of
O'Farrell's (25) NaDodSO4 sample buffer (62.5 mM Trist
HCl, pH 7.4/2.3% NaDodSO4/5 mM 2-mercaptoethanol).
Incorporation of [14C]leucine into protein was visualized by
two-dimensional (isoelectric focusing/NaDodSO4) gel anal- CONTROL
ysis (25). The isoelectric focusing gradient was constructed
by a combination of pH 3.5-5, pH 5-7, and pH 7-9 (1:3:1)
ampholytes. pH ,
Mr
RESULTS
Effects of Inhibitors on Protein Synthesis. Incubation of
Hermissenda nervous systems in 100 jLM anisomycin for 45
min resulted in .95% inhibition of protein synthesis (Table
1). This was true even after the anisomycin had been left to
stand in artificial sea water for 5 hr at 220C. Incubation in 100
Table 1. Inhibition of protein synthesis
['4C]Leucine
incorporation,*
Treatment cpm % inhibition
Control 4283 + 370 (n = 4)
Anisomycin (100 AIM)
Fresh 198+ 15 (n = 4) 95 DPBA
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Standing at 220C for 5 hr 164 + 35 (n = 6) 96

Deacetylanisomycin (100 AM) FIG. 1. The protein kinase C activator deoxyphorbol 13-isobutyr-
Fresh 535 +213 (n = 4) 88 ate 20-acetate (DPBA, 20 .M) decreases incorporation of [14C]leu-
Standing at 220C for 5 hr 4372 + 225 (n = 4) 0 cine into newly synthesized protein. DPBA caused an overall
reduction in protein synthesis as indicated by two-dimensional gel
*Mean ± SEM; n = number of nervous systems, with each nervous autoradiography. This marked reduction was also consistently (n =
system representing one independent experiment. 4) apparent with one-dimensional gel analysis.
 6950 Neurobiology: Alkon et al. Proc. Natl. Acad. Sci. USA 84 (1987)
A A
sec sec B sec
IA sec

ANISOMYCIN
+ TFP
1.5' After

I 5nA
t 0 150 mV
Co - LOAD Co2+- LOAD

C sec

Co2+_ LOAD

Light =-- -
__-j
FIG. 2. Anisomycin effects on Hermissenda K+ currents. (A) IA and ICa2+.K+ elicited by 1.0-sec command depolarizations to 0 mV (absolute)
at 30-sec intervals remain reduced 1.5 min after a Ca2+ lQad (as in C). Stippled area shows the difference between current magnitude 1.5 min
after the Ca2+ load compared to the magnitude before the Ca2+ load. (B) IA and ICa2+-K+ do not remain reduced after a Ca2+ load if trifluoperazine
(TFP) is present together with anisomycin in the bathing medium. (C) Ca2+-loading conditions are effected by a 25-sec command depolarization
to 0 mV paired with a 2-sec light step (104 ergs-cm 2 sec-1) whose onset begins 5.0 sec after the onset of the depolarizing command. Broken
lines in A and B represent the level of the non-voltage-dependent (or "leak") current. Broken line in C shows level of outward current in absence
of the light stimulus.
t test. This persistent decrease of IA and ICa2+K+ was difference, -0.25 ± 0.43 nA (n = 6, P > 0.2)] nor ICa2+-K+
observed for every cell tested. Furthermore, repeated Ca2+ [mean difference, 0.5 ± 0.87 nA (n = 6, P > 0.2)] showed a
loads were followed by progressive reduction of IA and persistent decrease (1.5 min later) as shown above (Fig. 2). IA
ICa2+-K+ (Fig. 3). Some reduction, even after a single Ca2+ did show a small, persistent decrease in one of five cases; in
load, persisted for the duration of the recording period (30-90 none of the cases did ICt2+-K+ show a persistent decrease.
min). Prior exposure to another protein synthesis inhibitor, These results demonstrate that anisomycin specifically alters
cycloheximide (100 ,uM), for >10 min produced the same recovery from Ca2+-mediated reduction of IA and ICa2+-K+
persistent reduction of IA [mean difference, -3.25 + 0.66 nA without affecting other biophysical properties of the cell.
(n = 5, P < 0.01)] and ICa2+-K+ [mean difference: -2.33 + 0.29
nA (n = 5, P < 0.005)] for each case examined. DISCUSSION
Exposure to either of the protein synthesis inhibitors The results show that the synthesis of new proteins is
caused no significant alteration of resting membrane poten- necessary for recovery from Ca2+-mediated reduction of K+
tial, holding current, or the non-voltage-dependent "leak" currents. In the absence of other obvious effects on the type
current (Table 2). B membrane properties, anisomycin and cycloheximide, but
Control-Solution Effects on IA and ICa2+-K+. By contrast, not deacetylanisomycin inactivated by preincubation in ar-
exposure (-30 min) to an analogue of anisomycin, deacetyl- tificial sea water, caused the reduction of IA and Ica2+-K+
anisomycin, whose ability to block protein synthesis (Table following a Ca2+ load to persist. This effect was similar to the
1) was almost eliminated after the drug stood in artificial sea effect of activation of protein kinase C (21) or injection of
water at 220C for -5 hr, did not cause the persistent reduction purified Ca2+/calmodulin-dependent kinase (22) following
of IA and ICa2+-K+ observed above. The third test depolariza- Ca2+-loading conditions. The possibility that the effects of
tion following a Ca2' load elicited essentially the same IA enzymatic activation on K+ currents lead to or must precede
[mean difference, -0.67 ± 2.75 nA (n = 6, P > 0.25)] and the effects of protein synthesis inhibition was suggested by
ICa2+-K+ [mean difference, 0.17 ± 0.52 nA (n = 6, P > 0.2)] as the findings with trifluoperazine. Addition of trifluoperazine
elicited before the Ca2+ load. Thus, deacetylanisomycin did to the artificial sea water during exposure to the protein
not block protein synthesis and did not enhance or prolong IA synthesis inhibitor anisomycin prevented persistent reduc-
and ICa2+-K+ reduction following a Ca2+ load. tion of IA and ICa2+-K+. This hypothesis was further substan-
Trifluoperazine Blocks Reduction of IA and IC.2+.K+ by tiated by the finding that protein kinase C activation (by
Anisomycin. Since both inhibition of protein synthesis and phorbol ester) causes an overall reduction in the incorpora-
Ca2+-dependent kinase activation (20-22) caused prolonged tion of [14C]leucine into newly synthesized proteins. It is
reduction of IA and ICa2+-K+ following a Ca2+ load, we decided possible, therefore, that protein kinase C may exert some or
to test for evidence of any interaction of these effects. all of its previously observed biophysical effects (21, 26) by
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Trifluoperazine blocks Ca2+/calmodulin-dependent protein inhibiting protein synthesis.

kinase and, to a lesser extent, protein kinase C. Type B cells Brief mention should be made of the conditions necessary
were exposed (.30 min) to anisomycin together with triflu- for the control agent deacetylanisomycin. Most likely be-
operazine (10 ,uM). Following the Ca2+ load neither 'A [mean cause of diffusion barriers within the Hermissenda central
 Neurobiology: Alkon et al. Proc. Natl. Acad. Sci. USA 84 (1987) 6951

sec The results reported here and in previous studies suggest

that Ca2"-loading conditions cause activation of Ca2+/cal-
ANISOMYCIN modulin-dependent and Ca2+/lipid-dependent kinase path-
ways, which, in turn, may regulate protein synthesis. Facil-
itation of these pathways by artificial means (e.g., enzyme
A injection) may simulate what occurs during associative learn-
ing of intact Hermissenda: namely, stimulus pairing activates
the Ca2+-dependent kinases, which then turn off the synthe-
sis of specific proteins that regulate or are part of the IA and
ICa2+-K+ channels. These proteins have not been identified,
nor is it known that Ca2 -dependent kinases and protein
synthesis inhibitors are acting on the same proteins. One
possibility not yet ruled out is that anisomycin prevents the
synthesis of phosphatase(s) and/or proteases that would
normally reverse the effects of activation of Ca2+/calmodu-
lin- and Ca2+/lipid-dependent kinases. This would result in
protracted states of phosphorylation of the ion channels
responsible for the Ca2+-mediated current reductions and,
hence, prolong the effects of protein kinase activation by
CYCLOHEXI
MIDE paired light and depolarization (or rotation) in situ.
A common view in neurobiology is that protein synthesis
is required for long-term memory lasting on the order of days
A D
or more. This view is based primarily on the finding that
protein synthesis inhibitors block consolidation of memory in
a variety of animals and tasks (3-6). More recently, use of a
tissue culture analogue of a non-associative learning para-
digm (sensitization) has led to the suggestion that protein
synthesis is required for long-term serotonin-induced
changes (27); however, the immediate effects of protein
I 5 nA synthesis inhibition on membrane currents were not con-
trolled for.
By contrast, in the present study, inhibition of protein
L * LJ~L L I 50 mV synthesis was shown to result in the prolongation of biophys-
ical changes similar to those measured for associative learn-
FIG. 3. Protein synthesis inhibitors cause progressive, persistent ing. Calcium-activated kinase regulation of protein synthesis
reduction of K+ current with successive Ca2+ loads. Type B cell may be relevant for the immediate induction of processes that
somata were incubated .30 min prior to penetration in artificial sea prolong the effects of acquisition into a time domain that
water containing 100 ,uM anisomycin (upper records) or cyclohexi- precedes the synthesis of new proteins. Turning-off of protein
mide (lower records). Ca2+-loading conditions (25-sec depolarization
to 0 mV paired with light as in Fig. 1) produced persistent reduction synthesis is well-suited for this role due to its rapid kinetics,
of IA or ICa2+-K+ measured 1.5 min later as in Fig. 1. With successive since much more time is required for the synthesis, packag-
Ca21 loads the persistent reductions summed to produce progres- ing, and intracellular transport of new proteins. In an inter-
sively larger reduction, which did not reverse for the recording mediate time domain, a mechanism is required whereby
period (>1.5 hr). Broken lines represent the level of the non-voltage- learning-induced alterations of the biophysical characteris-
dependent (or "leak") current. tics of cells can be maintained. Conditioning-specific trans-
location of protein kinase C has been found to span this
nervous system, a relatively high concentration (100 ,uM) of temporal domain (28). Therefore, as hypothesized by some
anisomycin was needed to effectively block protein synthesis (7-9, 29), increased synthesis of proteins may not be causally
(Table 1). The same concentration of deacetylanisomycin involved in some examples of memory lasting 1-2 days.
also blocked protein synthesis (Table 1), but for unknown However, the longest temporal domain of memory (many
reasons not after the drug solution stood for 5 hr. Since days and longer) may well be subserved by increases in
anisomycin left to stand for 5 hr still blocked protein protein synthesis that would result in permanent modification
synthesis, such a condition (standing before use) was incor- of neuronal properties.
porated into our protocol.
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