J. Exp. Biol. (1973), 58, 411-421
With 7 text-figures
Printed in Great Britain
411
PROGRESSIVE DECREMENTS IN THE
ACTIVITY OF APLYSIA NEURONES FOLLOWING
REPEATED INTRACELLULAR STIMULATION:
IMPLICATIONS FOR HABITUATION
BY CATHY LAMAR STEPHENS
Department of Physiology, Center for the Health Sciences,
University of California, Los Angeles, California 90024
{Received 14 August 1972)
INTRODUCTION
Behaviourally, habituation can be denned as a centrally mediated, progressive and
reversible decrement of a response to a constant, repeated stimulus. It is possible to
demonstrate habituation in very simple forms such as single-cell organisms (Wood,
1970; Applewhite & Gardner, 1971), multicellular invertebrates (Bruner & Tauc,
1966 a, b; Krasne & Woodsmall, 1969; Pinsker, Kupfermann, Castellucci & Kandel,
1970; Roberts, 1962) and in reduced neural systems of vertebrates (Buchwald, Halas &
Schramm, 1965; Griffin & Pearson, 1967; Thompson & Spencer, 1966; Wickelgren,
1967a, b).
The physiological basics of habituation is not known, although in some cases a
progressive decrement of the excitatory post-synaptic potential (EPSP) in neurones
receiving repeated trans-synaptic stimulation shows a temporal course similar to
behavioural habituation (Krasne, 1969; Kupfermann, Castellucci, Pinsker & Kandel,
1970). Partly because of the correlation between these EPSP decrements and behavioural habituation, there has been considerable interest in studying the phenomenon
of synaptic depression following low-frequency stimulation, and several current reviews
deal with synaptic depression in invertebrates and its hypothesized role in habituation
(Bruner & Kehoe, 1970; Kandel, Castellucci, Pinsker & Kupfermann, 1970; Horn,
1970).
Some of the studies of synaptic depression in invertebrates have suggested that there
is no change in the membrane properties of the post-synaptic cell during repeated
trans-synaptic stimulation (Bruner & Kennedy, 1970; Bruner & Tauc, 1966 a, b;
Castellucci, Pinsker, Kupfermann & Kandel, 1970). However, the possibility that small
changes in post-synaptic neurone properties may contribute to the habituation phenomenon has never been critically studied. Because modulation of excitability in the
post-synaptic cell^er se could potentially make a significant contribution to phenomena
of response decrements, it seemed important to examine this issue in more detail.
Using the probability of action potential discharge to a repeated intracellular stimulus
as a measure of excitability, the work reported here will show that neurones of Aplysia
can exhibit the parametric characteristics of habituation independent of any synaptic
modification.
412
C. L.
STEPHENS
METHODS
The animals used were Aplysia California ranging in size from about 50 to over
500 g. The connectives and nerves of the abdominal ganglion were cut and the ganglion
was removed and placed in a chamber filled with artificial sea water. The connective
tissue surrounding the ganglion was pinned down into resin (Sylgard 182 Encapsulating
resin, Dow Chemical) which lined the bottom of the chamber. Neurones were exposed
by using very fine forceps and iridectomy scissors to dissect away the connective
tissue above the cell bodies of interest.
Glass pipettes filled with 3 M-KC1 were used for intracellular recording and stimulation. The electrodes were pulled so as to have a resistance of about 8 megohms in
sea water. For most experiments stimulation and recording were carried out with the
same electrode using a WPI amplifier (W-P Instruments, Inc.). An Ag-AgCl wire
electrode served as ground. Current was delivered to the WPI by a Grass S44 stimulator (Grass Instruments) through a Grass SIV5 stimulus-isolation unit. The preamplifier-stimulator circuit balance was checked repeatedly during each experiment
by observing the initial rapid displacement of potential at the start of a stimulus. No
adjustment of the balance was required during most experiments.
To ensure that a constant current was being delivered, several experiments were
performed using separate electrodes for stimulating and recording. In these cases the
recording electrode and ground electrode (a sea water-Ag-AgCl agar bridge) were
connected to high-impedance pre-amplifiers (Elsa-2, Electronics for Life Sciences)
and the difference in pre-amplifier outputs was displayed on the oscilloscope. The
stimulus was delivered through a 20 megohm resistor to the stimulating electrode.
During stimulation current was monitored across a resistor between a silver wire in
the bath and ground.
The intracellular stimulus used to demonstrate response decrements consisted of a
current pulse intensity and duration sufficient to initiate an action potential, but close
to the firing threshold of the cell (about 15-20 mV more positive than resting). The
current utilized ranged from 6 to 800 nA with pulse durations from 50 to 200 msec,
depending upon the responsiveness of the particular cell and experimental procedure
being utilized. The individual stimulus pulses were delivered at intervals which in
different experimental sequences ranged from 2 to 10 sec.
In some experiments repeated extracellular stimulation was delivered to the right
connective. The connective was placed across a bipolar silver-wire stimulating electrode
positioned just above the surface of the sea water. The connective was stimulated at
intervals which were the same as those used in the intracellular stimulation experiments, i.e. 2-10 sec.
Forty-two abdominal ganglion cells were studied. Each was naturally silent, had a
resting potential of at least 50 mV and an action potential of 90-110 mV. Nineteen of
these cells were identified as the right dorsal giant cell (Frazier, Kandel, Kupfermann,
Waziro & Coggeshall, 1967); the others were located on the surface of the dorsal side
of the ganglion. All experiments were performed at approximately 23 °C.
Activity of Aplysia neurones
4*3
10
lOminresti
&
6
o
4
\20 min rest
B
Z
j
l
Blocks of ten trials
Fig. i. Decreased probability of action potential discharge from a single neurone during
repeated intracellular stimulation (3 sec interstimulus interval) and spontaneous recovery. A
pulse of depolarizing current produced either one action potential or no action potential. The
trials are grouped in blocks of ten. The first ten stimulations induced action potential discharge
every time; stimulations 81-90 (9th block) produced only three action potentials. Following an
initial ' habituation' sequence, the neurone was ' rehabituatd' after rest eperiods of various
durations. Only one 'habituation' curve is shown as in the four sequences illustrated,
'habituation' curves essentially overlapped. A 30-min rest period intervened between each
complete ' habituation-rehabituation' sequence. The order in which effects of the different rest
periods were tested was 5 min, 20 min, 1 min and 10 min.
RESULTS
The initial depolarizing stimulus was adjusted so as to produce one action potential
per pulse. In all of the cells studied the probability of action potential discharge
progressively decreased as the same stimulus was repeatedly presented. In a typical
experiment at least eight to ten action potentials occurred during the first block of ten
stimulations, and the number of action potentials in successive blocks always progressively decreased. A typical example of this response decrement is illustrated in Fig. 1.
During the first block of ten stimuli each stimulus produced an action potential, while
during the ninth block of ten stimuli (or the 81-o.oth trials) only three action potentials
were produced. The decremental process took place while the current remained constant throughout the experimental tests; thus changes in the resistance at the tip of
the electrode did not account for the effects.
In all cases the response decrement which developed during repeated intracellular
stimulation was reversible if the cell was allowed to rest. With sufficient rest periods
there was complete recovery and the 'rehabituation' curve had essentially the same
form as the original 'habituation' curve (Fig. 1, 20 min rest). In general, recovery was
complete only after 10-20 min.
In order to test the time course of recovery, multiple experiments of ' habituation'
and 'rehabituation' were conducted. Fig. 1 shows the results of one experiment in
which the time course of recovery was sampled at different periods of time after
C. L.
STEPHENS
101-
&
4
0
2
4
Blocks of ten trials
6
Fig. 2. Effect of interstimulus interval (ISI) upon the probability of action potential discharge
in a single neurone. A 30-min rest period intervened between each stimulation sequence
'habituation'. The cell was 'habituated', tested for recovery by 'rehabituation' after a
rest period of a particular length, and then the neurone was allowed to rest for 30 min.
This rest period was followed, again, by a 'habituation' run with a 'rehabituation'
test for recovery after a different time lapse from that previously used. The initial
'habituation' sequences produced response curves which essentially overlapped,
indicating that the neurone had not deteriorated during the experiment. Fig. 1
indicates the amount of spontaneous recovery after different time periods.
At any point during the 'habituation' or recovery period a stimulus somewhat
stronger than the' habituating' stimulus could initiate action potentials. The decreased
responsiveness was, therefore, not due to any total inactivation of the mechanisms
responsible for the production of the action potential.
Parametric studies
Parametric studies of habituation typically show greater habituation with short'
rather than long, interstimulus intervals (Buchwald & Humphrey, 1971; Thompson &
Spencer, 1966). To determine whether this feature of habituation also resulted from
intracellular stimulation a variety of interstimulus intervals was used, e.g. 2, 3, and
5 sec. The neurones clearly showed the greatest decrements when the shortest interstimulus intervals were used (Fig. 2).
Another parametric characteristic of habituation is the greater response decrement
which develops during repeated presentations of a weak, rather than a strong, stimulus
(Buchwald & Humphrey, 1971; Thompson & Spencer, 1966). Aplysia neurones
Activity of Aplysia neurones
4*5
10
O
I
0
I
I
I
j_
2
4
6
8
Blocks of ten trials
Fig. 3. Effect of stimulus intensity upon response decrement. A 3-sec ISI was utilized; current
is expressed in nanoamperes (nA). A 30-min rest period intervened between each stimulation
sequence.
subjected to repeated intracellular stimulation typically showed this inverse relationship between stimulus intensity and degree of ' habituation', i.e. the decrements
appeared greatest with the weakest stimulation (Fig. 3).
Control experiments
In examining the decrement described above it is important to consider whether the
injection of current per se through the intracellular electrode could cause the decreased
responsiveness. A positive intracellular pulse, such as that used in the present experiments to elicit an action potential, might inject some potassium into the cell; if a
significant amount were released, more than just the slow passive leakage of potassium
chloride out of the electrode, this could conceivably affect the production of action
potentials during repeated intracellular stimulation. Any increase in internal potassium
concentration should increase outward potassium current, which in turn would raise
the threshold for action potential production, since threshold is defined as the point at
which inward current just equals outward current. However, the stimulus per se was
apparently not causing the effects, as was demonstrated by the two control experiments
described below.
If injection of potassium into the cell body with just-threshold stimuli was causing
the decreased responsiveness, then a slightly subthreshold stimulus should have a
similar effect. That this was not the case is shown in Fig. 4. Fifteen presentations of a
subthreshold stimulus made no difference in the decrement of response probability to
an 'habituating' stimulus. Thus, either (1) the production of action potentials is
416
C. L.
STEPHENS
10 i -
10
\ Prior subthreshold stimulation
\\
8 -
Control \
\
\
o 6 -
8.
\
\
4 --
B
2 -
1
1
i
V.
1
2
4
Blocks of ten trials
1
I4
Z
I
4
6
Blocks of ten trials
Fig. S
Fig. 4
Fig. 4. Effect of prior repeated subthreshold intracellular stimulation upon the decremental
process during intracellular threshold stimulation. The intensity of repeated subthreshold
stimuli was 21 nA. This prior stimulation had no effect on action potential probabilities induced
by subsequent threshold stimulation (27 nA).
Fig. 5. Action potential discharge induced by repeated antidromic stimulation. With successive
extracellular stimulation of the giant cell axon, decrements in action potential discharge
developed with a time course similar to that of the action potential decrements which developed
during repeated intracellular stimulation. The interstimulus interval was 3 sec.
necessary to the decremental process, or (2) stimuli which are subthreshold for action
potential production are also below the threshold for the decremental mechanism to
be effective.
A second confirmation of the lack of any spurious effects of direct intracellular
stimulation was provided by stimulating the giant cell antidromically through the
right connective. The resultant action potentials decreased in firing probability during
repeated extracellular stimulation of the axon just as they did with repeated intracellular stimulation (Fig. 5). The amplitude of all antidromic spikes recorded from the
dorsal giant cell remained constant, which indicated that spike transmission along the
axon was not blocked; had such a block occurred, one would expect to record a greatly
diminished spike in the soma (Tauc, 1962). Thus the spikes recorded in the soma probably were elicited by the spikes occurring in the axon. However, the decremental
process could have originated in the axon at the site of stimulation, rather than in the
neurone soma. These experiments indicate that the form of intracellular stimulation
used did not cause 'habituation' if applied at just-subthreshold levels, and was not
necessary to produce the phenomenon.
Activity of Aplysia neurones
417
Before repeated intracellular stimulation
After
lsec
"""
10 mV
I
Fig. 6. Typical membrane response to a hyperpolarizing pulse before and after a ' habituation'
series. In this case there were 50 trials and the ISI was 2 sec.
Possible mechanisms affecting decrements in activity
Three principal mechanisms might cause the 'habituation' observed with postsynaptic stimulation of Aplysia neurones: (1) the membrane potential might be
hyperpolarized progressively, so that a larger stimulus was required to reach the
threshold depolarization; (2) the membrane conductance might increase, producing a
small depolarization for a given outward-current stimulus; (3) the critical depolarization, or theshold, might rise to more positive levels.
The membrane resting potential was measured at the start and end of each block of
ten stimuli. In all of the cells examined action potential decrements occurred without a
measurable concurrent change in the resting potential. Sometimes a gradual hyperpolarization developed during the course of several hours of recording, but such drifts
in the resting potential were independent of decremental and recovery processes.
Brodwick & Junge (1972) and Connor & Stevens (1971) have reported a long-lasting
(order of seconds) increase in potassium conductance in molluscan nerve cells following depolarizations sufficient to produce action potentials. Such an increase in
membrane conductance could account for the decremental phenomenon with very
small changes in conductance, if the stimulus applied is close to the threshold level. In
order to determine whether any change in membrane conductance occurred during
the process of 'habituation', hyperpolarizing pulses were delivered immediately
before and after each decremental sequence (Fig. 6). The current intensity was the same
for each hyperpolarizing test pulse, so any increase in membrane conductance should
appear as a decreased amount of hyperpolarization. With the accuracy afforded by this
method no change in conductance could be seen as a result of the decremental process.
However, even a very small increase in membrane conductance might cause a decrease
in effectiveness of the stimulus sufficient to produce the observed decrements.
Alternatively, the decremental process could result from an accumulation of sodium
4i8
6
2
Blocks of ten trials
Fig. 7. Hyperpolarization of the membrane following repeated intracellular stimulation. The
giant cell was repeated stimulated at an ISI of 5 sec to produce action-potential decrements.
After response decrements had developed the membrane was hyperpolarized for about 4 sec
during one ISI. The subsequent block of ten ' habituation' stimuli induced a larger number of
action potentials than had occurred prior to hyperpolarization.
inactivation (Hodgkin & Huxley, 1952; Geduldig & Gruener, 1970; Chandler &
Meves, 1970) resulting in an increase in the threshold depolarization for action potential
production. It is possible to obtain an indication as to which of these mechanisms is
operating by imposing hyperpolarizing stimuli after the decremental process has
developed and examining the effect on the subsequence response level. Brodwick &
Junge (1972) reported that hyperpolarizing pulses had no effect on the post-stimulus
conductance, while it is well known that hyperpolarization can remove sodium
inactivation (Hodgkin & Huxley, 1952).
To determine whether a build-up of sodium inactivation might be the mechanism
underlying the decrement in firing probability, a hyperpolarizing pulse was delivered
intracellularly after a significant degree of 'habituation' had developed. The hyperpolarization extended for about 4 sec during the 5 sec interval which intervened
between two depolarizing, 'habituating' stimuli. As demonstrated by Fig. 7, such
intracellular hyperpolarization resulted in a partial reversal of the decreased action
potential discharge to the depolarizing 'habituation' stimuli. These experiments
suggest that sodium inactivation might be a primary cause of the decreased firing
probability resulting from repeated stimulation.
DISCUSSION
The present research indicates that repeated intracellular stimulation of Aplysia
neurones induces a decremental process which has the parametric characteristics of
habituation. When Aplysia nerve cells were stimulated intracellularly close to their
firing threshold the probability that an action potential would occur decreased with
Activity of Aplysia neurones
419
repeated stimulation. After a period of rest the neurones showed spontaneous recovery
such that a subsequent series of depolarizing stimuli resulted in a decremental process
with the same general time course as the initial series. Other parametric characteristics of habituation displayed in these experiments were the relationship of the strength
of the stimulus and interstimulus interval to the amount of 'habituation1. Thus, a
stronger stimulus or a longer interstimulus interval produced less decrement than a
weaker stimulus or a shorter interstimulus interval.
The fact that the excitability of an individual neurone can decrease in response to a
constant, repeated stimulus indicates that the possibility of changes in excitability of
neurones independently of synaptic modifications should be considered in any
physiological analysis of habituation. It should be emphasized that the neuronal
excitability changes demonstrated in this report have not as yet been related to
behaviour. However, previous studies have shown that this decremental process is of
significant importance during trans-synaptic depression (Stephens, 1972).*
The mechanism responsible for these changes is not obvious in the present experiments; there was no experimentally related change in the resting potential and the
passive membrane response to stimulation was constant. This does not, however,
completely rule out steady-state conductance changes. Since the stimulus is near
threshold, a small effect, such as a light increase in potassium conductance, could
decrease firing probability. Such a small change might not be detected by either a
change in the resting potentials or in the conductance as measured by a hyperpolarizing
pulse.
Previous studies (Bruner & Tauc, 1966a, b; Castellucci et al. 1970) of habituationlike phenomena in Aplysia neurones have typically measured the decrements of EPSP
following repeated stimulation. These studies have focused upon synaptic alternations
in an attempt to develop an intercellular model of habituation. Since an EPSP
decrement could be caused either by conductance changes in the membrane of the
post-synaptic cell or by changes taking place at the synaptic site, the response of the
post-synaptic cell to a hyperpolarizing pulse before the start of stimulation was compared
with the response to a hyperpolarizing pulse after the EPSP had decreased. Because
there was no change in the response to the hyperpolarizing pulse, it was concluded
that the EPSP decrement resulted from changes at the synaptic site. Although small
changes in conductance might not be detected by a hyperpolarizing pulse, changes
which were large enough to significantly affect EPSP amplitude would probably have
been detected. Therefore, it is reasonable to conclude that the EPSP was probably
decreasing because of changes at the synapse, e.g. transmitter release. However, the
results of the experiments reported here demonstrate that the post-synaptic neurone
could also undergo a decrease in excitability independent of synaptic modulation.
These results suggest that, in considering possible mechanisms of habituation, it is
important not only to look at alterations of the synaptic potential but also at changes
of the membrane properties that develop only as a result of repeated stimulation and
independent of any synaptic activity.
• A detailed paper is in preparation comparing the effects of synaptic depression with the changes
in the excitability of of the post-synaptic neurone occurring independently of synaptic modification,
but during trans-synaptic stimulation.
420
C. L. STEPHENS
SUMMARY
1. Repeated intracellular stimulation of nuerones from the isolated abdominal
ganglion of Aplysia californica produced progressive response decrements with
parametric features common to trans-synaptic models of habituation.
2. The probability that a constant intracellular pulse of depolarizing current would
produce an action potential decreased with repeated stimulation, the response
decrements developed with interstimulus intervals ranging from 2 to 10 sec.
3. In all cases, the response decrements were reversible with prolonged rest.
4. In some cases complete recovery did not occur for up to 20 min, which indicated
that the spontaneous recovery process was long-term in nature.
5. As is typical of parametric studies of habituation, short, rather than long interstimulus intervals, and a weak, rather than a strong stimulus, produced greater
response decrements.
6. These results demonstrate that an individual neurone shows response decrements
as a function of repeated stimulation, which suggests that there are at least two
processes responsible for the response decrements seen during trans-synaptic
stimulation: (a) synaptic depression and (b) a depressive process originating in the
post-synaptic neurone.
This work was supported by NS 05434 a n o ' GM 00448. It is a pleasure to thank
Dr Jennifer Buchwald for her encouragement and support during all phases of this
work. I would also like to thank Dr Douglas Junge for clarifying various theoretical
aspects of neurophysiology and for his helpful comments on the manuscript. In
addition, I wish to thank Dr Susumu Hagiwara for his critical reading of a preliminary
draft of the manuscript and for his helpful suggestions.
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