Download Slow depolarization produced by associative conditioning of Aplysia

Brain Research, 280 (1983) 165-168
1.65
Elsevier
Slow depolarization produced by associative conditioning of Aplysia
sensory neurons may enhance C a 2÷ entry
EDGAR T. WALTERS and JOHN H. BYRNE
Department oJ Physiology and ('ell Biology, The University of Texas Medical School, P.O. Box 20708, Houston, TX 77225 (U. S. A. )
(Accepted August 9th, 1983)
Key words: Ca 2+ conductance - - depolarizing response - - hyperpo[arizing response - -
associative conditioning - - voltage-dependence
Sensory neurons activated by intracellular stimulation immediately before sensitizing tail shock displayed a slow depolarization after the shock. By contrast, sensory neurons exposed to the effects of tail shock alone or unpaired activation and tail shock showed a
slow hyperpolarizing response to the shock. A voltage-sensitive Ca 2+ conductance that is activated near the resting potential may be
modulated by these opposite effects of associative and non-associative training.
We recently showed that a classical conditioning
protocol applied directly to individual tail sensory
neurons in A p l y s i a resulted in associative modification of monosynaptic connections to tail m o t o r neurons 14. Sensory neurons receiving a conditioned stimulus (CS, intracellular activation of the sensory neuron) immediately before the unconditioned stimulus
(US, tail shock) showed significantly more synaptic
facilitation than sensory neurons exposed to the US
alone or to unpaired CS and US applications. It is of
interest to examine changes in the cell soma during
associative conditioning since these changes may
parallel changes in the synapses 9 and might also be
important for signalling the genetic apparatus of the
cell during learning s . We now r e p o r t that associative
conditioning of the tail sensory neurons is accompanied by a slow depolarization of the sensory neuron
soma that contrasts strikingly with the slow hyperpolarizing responses to unpaired US application. In addition, we show that the changes in m e m b r a n e potential produced by paired and unpaired conditioning
may modulate v o l t a g e - d e p e n d e n t Ca 2+ conductances in the sensory neuron. A preliminary report of
some of these results has a p p e a r e d in abstract form~.
The results described below on the modulation of
m e m b r a n e potential during conditioning were obtained by further analysis of the experiments on associative conditioning r e p o r t e d by Walters and
Bvrne H. Thus. the semi-intact tail withdrawal prepaO(~Ofi-899Y83/$03.00~ 1983 Elsevier Science Publishers B.V.
ration 14,15 was utilized, which allowed concurrent
measurement of responses in the sensory and m o t o r
neurons. Details of the conditioning protocol have
been described previously 1~. To examine the possible
effect of changes in m e m b r a n e potential on Ca 2+ conductances in the sensory neuron soma we used a second p r e p a r a t i o n , the isolated pedal-pleural ganglion,
in which the tail sensory neurons were axotomized
near their somata by cutting across the root of the
pleural-pedal connective 15. This p r o c e d u r e eliminated axon spikes normally observed when voltageclamping the intact neuron. Individual somata were
voltage-clamped using techniques described below
and by Byrne et al. 2.
Examples of the long-lasting changes in m e m b r a n e
potential typically p r o d u c e d by the US (tail shock delivered outside of the excitatory receptive fields of
the monitored cells) are illustrated bv the responses
of two tail sensory neurons recorded simultaneously
(Fig. 1). The US alone p r o d u c e d a slow hyperpolarizing response that lasted nearly 5 rain (Fig. 1A~,
see also ref. 15). Similar hyperpolarizing responses
were produced by the unpaired US (not shown). By
contrast, the cell receiving a burst of 10 action potentials (20 Hz) immediately prior to (paired with) the
same US exhibited a slow depolarization that persisted for at least 5 min (Fig. 1A2).
A quantitative comparison of these effects was obtained by measuring the "peak depolarization' during
166
A1
US
ALONE A
A2
P A I R ~
I
20sec
B
j4•v
3
I
E
Z
0
I,-,
N
m,,,.
5
0
a.
1
5
PAIRED
UNPAIRED
U S ALONE
1
-1
Fig. 1. A: slow polarizing responses recorded simultaneously
in two tail sensory neurons in response to the tail shock US. The
arrow indicates the 'peak depolarization' (see text) in each record. The dashed line extends from the resting potential prior
to the US. A~: prolonged hyperpolarization in the neuron exposed to the effects of the US alone. A2: slow depolarization
produced by pairing intracellular activation (10 spikes] with the
US. The spikes have been clipped to allow enlargement of the
slow response. B: mean peak depolarizations ( ± standard error
of the mean) in sensory neurons trained with paired, unpaired.
and US-alone protocols (n = 10 cells in each group) after the
first and fifth training trials.
a defined interval on the first and fifth training trials.
This interval began 5 s after and e n d e d 2 min after US
application. W e first d e t e r m i n e d the point of peak
depolarization in each record (,arrows in Fig. 1A) by
finding the most d e p o l a r i z e d (or least hyperpolarized) value in the interval. W e then expressed this
value as the difference from the resting potential recorded i m m e d i a t e l y before the US. Fig. 1B shows
the m e a n p e a k depolarization (+_ "S.E.) of the 3
groups of sensory neurons whose synaptic responses
were previously analyzedi4. On both the first and
fifth trials an analysis of variance ievealed signfficanl
overall effects ( P < (I.()111I, and ,m both trials the
paired cells showed greater peak depolarizations
than did cells exposed to the US alone or to unpaired
CS and US presentations (P ~" (~l)l In each case,
N e w m a n - K e u l s test I. There w.eic ll~ significant differences within each group between responses on the
first and fifth trials,
Although the function of fhe associative and nonassociative modulations of m e m b r a n e potential m
the sensory neurons is not yet known, it s e e m e d possible that these responses could contribute to cellular
modifiability by altering Ca -'+ influx through voltaged e p e n d e n t channels in the sensor~ neurons "1"(~examine this possibility we voltage-clamped the axotomized sensory neuron soma under conditions m
which all voltage-sensitive conduclances except the
Ca2~ conductance should have been blocked or reduced. The ganglion was superfused with a Na ~ arid
Ca z+ free solution containing 11 m M B a :+. till) mM
t e t r a e t h y l a m m o n i u m ( T E A ) , lfl inM 4-aminopyridine (4-AP), and 10-4 M serotonm (5-HTI. B a : - was
used to replace Ca 2+ since Ba 2 is thought to pass
through Ca2+ channels without aclivating C a > - d e pendent K ~ currents 5. 5-HT was used to block the
steady-state 5-HT-sensitive K + currentkL The possibility that residual K + currents remained cannot be
entirely ruled out. Fig. 2 A illustrates the currents
produced by 2 s voltage-clamp steps from a holding
potential of -55 inV. A t - - 3 5 mV a clear steady
state inward current was produced. The steady state
c u r r e n t - v o l t a g e relationship for this cell (Fig, 2B)
shows that even at - - 4 5 mV the current was less outward than predicted by extrapolating from the linear
part of the curve. Since the normal resting potential
of these cells ranges between ---411 and
5(1 mV in
the semi-intact p r e p a r a t i o n i L these results suggest
that a Ca e' conductance ma) be activated at or near
the resting potential (assuming that Ca e+ and Ba-"
conductances have similar voltage d e p e n d e n a e s t .
Therefore. this Ca-'-- conductance might be modulated by the slow changes in potential p r o d u c e d bv
associative and non-associative conditioning,
Previously we p r o p o s e d that the associative conditioning of the tail sensory neurons m~ght involve the
amplification by Ca 2+ of cyclic A M P - m e d i a t e d ell
fectsl4 (see also refs. 8 and 11 ) The present results indicate that a v o l t a g e - d e p e n d e n t Ca -z" conductance i~
167
B
A
#
+0.3 -
l
#
#
-,,mV
!
tD
+O.1
-45mY _~L
-85
F
-0. I
-65 1 -45%
-25
-5
Vm (mV)
f-v
E
-0.3
-65mY -~
j~
-75mV
I~
-0.5
llnA -0.7
lsec
Fig. 2. Evidence for a voltage-dependent Ca 2+ conductance in the tail sensory neuron soma that is activated near resting potential.
The ganglion was superfused with a solution that blocks or reduces all currents cxcept those flowing through Ca > channels (see text).
A: currents in response to voltage clamp steps from a holding potential of--55 mV. B: current voltage relationship for the cell illustrated in A. The curve is non-linear for depolarizations of 10 mV or more from the holding potential ( 55 mVI.
present in these cells and thus that Ca 2+ probably enters during the brief burst of spikes that serves as the
CS. F u r t h e r m o r e , the observation that this conductance can be activated at or near the resting potential
suggests that the small but prolonged depolarization
following each pairing of the CS with the US may result in additional Ca z+ influx. This additional influx
might prolong the interaction of Ca 2+ with the subcellular machinery underlying the associative
changes in the soma and possibly also in the presynaptic terminals, and thus might be involved in the
'consolidation" of the associative memory. Conversely, the slow hyperpolarizing response to the unpaired
US may reduce Ca z+ influx and thus decrease the degree of modifiability following the US. These considerations suggest that the opposite modulations of
m e m b r a n e potential by paired and unpaired stimula-
tion may enhance the contrast between the associative and non-associative effects of arousing stimulation. Although these ideas are speculative, they are
consistent with 4 general observations. First, longlasting polarizing responses are being reported in an
increasing number of systems 7. Second, long-lasting
depolarization is p r o d u c e d by associative conditioning in at least one other preparation~,~. Third, voltage-dependent Ca 2+ conductances are activated at or
near the resting potential in many neurons >. Fourth,
Ca 2+ is a potent second messenger and has been implicated in many forms of cellular reguhitionl-'.
This work was supported by NIH Fellowship F32
NS06455 to E . W . , bv N I H G r a n t NS19895 to J.B.
and by University of Texas Biomedical Research
Support Grants to E . W . and J.B.
168
1 Alkon, D. L., Membrane depolarization accumulates during acquisition of an associative behavioral change, Science, 210 (1980) 1375-1376.
2 Byrne, J. H., Shapiro, E., Dieringer, N. and Koester, J.,
Biophysical mechanisms contributing to inking behavior in
Aplysia, J. NeurophysioL, 42 (1979) 1233-1250.
3 Byrne, J. H. and Waiters, E. T., Associative conditioning
of single sensory neurons in Aplysia. II. Activity-dependent modulation of membrane responses, Soc. Neurosci.
Abstr,, 8 (1982) 836.
4 Cohen, M. J., A comparison of invertebrate and vertebrate
central neurons. In F. O. Schmitt (Ed.), The Neurosciences'. Second Study Program, Rockefeller Univ. Press,
New York, 1970, pp. 798-812.
5 Connor, J.A., Calcium current in molluscan neurones:
measurement under conditions which maximize its visibility, J. Physiol. (Lond.). 286 (1979) 41-60.
6 Crow, T. J. and Alkon, D. L., Associative behavioral modification in Hermissenda: cellular correlates, Science, 209
(1980) 412-414.
7 Hartzell, H. C., Mechanisms of slow postsynaptic potentials, Nature (Lond.), 291 (1981) 539-544.
8 Hawkins, R. D., Abrams, T., Carew, T. J. and Kandel, E.
R., A cellular mechanism of classical conditioning in Aplv-
9
10
11
12
13
14
15
sia: activity-dependent amplification o1 presynaptic facilitation, Science, 219 (1983) 400-405:
Kandel, E. R. and Schwartz, J. H., Molecular biology of
learning I modulation of transmitter retease, Science; 218
(1982) 433-443.
Koester, J. and Byrne, J. H. (Eds.), Molluscan Nerve Cell,s:
From Biophysics to Behavior. Cold Spring Harbor Laboratory, New York, 1980.
Ocorr, K., Walters, E. T. and Byrnc, J. H., Associative
conditioning analog in Aplysia tail sensory neurons selectively increases cAMP content. S o , NeuroscL Abstr., 9
(1983) in press.
Rasmussen, H., Calcium and cAMP as Synarchic Messengers, John Wiley and Sons, New York, 1981.
Siegelbaum, S. A., Camardo, J. S. and Kandel, E. R., Serotonin and cyclic AMP close single K ~ Channels in Aplysia
sensory neurones, ,Nature (Lond. ~, 299 (1982) 4 t 3-417.
Waiters, E. T. and Byrne, .1. H., Associative conditioning
of single sensory neurons suggests a cellular mechanism for
learning, Science, 219 (1983) 405-408.
Waiters, E. T., Byrne, J. H.. Carew, T~ C. and Kandel, E
R., Mechanoafferent neurons innervating the tail of Aplys'ia: I. Response properties and synaptic connections, J.
Neurophysiol., in press.