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Serotonin acts in the synaptic region of sensory neruons in Aplysia to enhance transmitter release
Neuroscience Letters, 104 (1989) 235-240
235
Elsevier Scientific Publishers Ireland Ltd.
NSL 06332
Serotonin acts in the synaptic region of sensory neurons in Aplysia to enhance transmitter release M. Hammer 2, L.J. C l e a r y 1 a n d J.H. B y r n e 1 ~Department of Neurobiology and Anatomy, University of Texas Medical School, Houston, TX 77225 (U.S.A.) and 21nstitutfiir Neurobiologie, Freie Universitdt Berlin, Berlin (F.R.G.) (Received 3 April 1989; Revised version received 25 May 1989; Accepted 26 May 1989)
Key word~: Facilitation; Synaptic release; Sensitization; Serotonin; Aplysia; Spike broadening
An important mechanism that contributes to sensitization in Aplysia is heterosynaptic facilitation of the synaptic connections between sensory neurons (SNs) and motor neurons (MNs). Heterosynaptic facilitation, in turn, is associated with broadening of the spike in the SN. Spike broadening is readily observed in recordings from somata of SNs, and from growth cones of SNs in culture, but broadening in synaptic terminals has only been inferred. Intracellular recordings were made from somata of SNs and from somata of follower MNs. Additional recordings were made from the axons of SNs as they enter the neuropil in the pedal ganglion. Serotonin (5-HT) broadened action potentials in axons of SNs and enhanced excitatory postsynaptic potentials (EPSPs) in the MNs, even after the axons of SNs were surgically separated from their somata. These results indicate that both heterosynaptic facilitation and spike broadening in the axon are due to the local action of 5-HT and can occur independently of modulation of membrane properties in the soma.
One behavioral modification in Aplysh~ that has been studied in great detail is sensitization, a simple form of non-associative learning. Sensitization appears to be due to the enhanced release of neurotransmitter from sensory neurons (SNs) produced by activation of a network of facilitatory interneurons [18]. Facilitation appears to be due, at least in part, to broadening of spikes in the SNs [2, 14, 16, 17, 21, 22, 31]. Although spike broadening, as a result of either sensitizing stimuli or application of serotonin (5-HT; a transmitter that mimics the effects of sensitizing stimuli), is readily observed in recordings from somata of SNs, and from growth cones of SNs in culture [3a], but broadening in the synaptic terminals has only been inferred. In the pleural ganglion, the somata of SNs that innervate the tail are located 2-3 mm away from their synaptic contacts with motor neurons (MNs) in the pedal ganglion [8]. By recording directly from the axons of SNs near their targets in the pedal ganglion, the kinetics of spikes near synapses can be compared with those far away in the soma. Correspondence." L. Cleary, Dept. of Neurobiology and Anatomy, University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77225, U.S.A 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
236
The sensory and motor neurons innervating the tail can be readily identified on the basis of several anatomical and electrophysiological characteristics [30]. The tail SNs are located in a discrete cluster on the ventral surface of the pleural ganglion (Fig. 1). The tail M N s are located along a tract on the dorsomedial surface of the pedal ganglion [30]. Axons of SNs were impaled with an electrode in the neuropil of the pedal ganglion adjacent to the somata of the tail MNs. This region is close to the site of synaptic contacts between tail SNs and MNs as determined histologically [8]. Recordings were also made from the somata of a SN and a follower MN. Axons that run into the neuropil of the pedal ganglion from the pleural-pedal connective can be readily penetrated with fine-tipped microelectrodes (Fig. 2). Axons from SNs were distinguished from those of other neurons on the basis of several electrophysiological properties characteristic of the SN somata [5, 30]. While these criteria are not absolutely definitive, we are confident that they allowed us to distinguish SN axons since only a small percentage of the impaled axons met these criteria. Moreover, on two occasions we were able to definitively identify SN axons by recording simultaneously from the soma of the same cell in the pleural ganglion (e.g. Fig. 2). In general, the kinetics of axon spikes are quite similar to those of soma spikes, although axon spikes have higher amplitudes and in some cases are slightly broader. Since the effects of spike broadening are most important at sites of synaptic release, we wanted to establish that the recording sites in the axon were electrotonically close to the terminals. To do this, we took advantage of the fact that the membrane potential of a presynaptic neuron determines the effectiveness of transmitter release (e.g.
MN
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@ Fig. 1. Diagrammatic representation of the experimental preparation. Electrodes were placed in somata of both sensory neurons (SN) in the pleural ganglion and motor neurons (MN) in the pedal ganglion. A separate electrode was used to impale the axon o f a SN in the pedal ganglion. In two experiments, simultaneous recordings were made fiom the axon and soma of the same SN, as shown, but this was not generally the case. To verify the electrophysiological properties of the axon, the posterior pedal nerve (P9) and the pleural-abdorninal connective (P1-A) were stimulated. In some experiments, the pleural-pedal connective was cut before the axon was impaled (arrows). Also indicated for orientation is the pleural-cerebral connective (P I-C).
237 refs. 19, 28, 29). A simple protocol was used to determine the effect o f m e m b r a n e potential o n t r a n s m i t t e r release. While s i m u l t a n e o u s l y recording EPSPs in the M N , 5 spikes were triggered at intervals o f 60 s by passing a brief (1-3 ms) intracellular depolarizing pulse. O n alternate cycles, the m e m b r a n e potential o f the axon was hyperpolarized by 10 m V for 5 s, at which time the spike was triggered. A single series of 5 spikes was analyzed as 2 series of 3 spikes. Thus, the effect o f h y p e r p o l a r i z a t i o n was assessed twice in each series. Because the a m p l i t u d e o f excitatory postsynaptic potentials (EPSPs) decrements when spikes are triggered at regular intervals [6, 7], the effectiveness o f h y p e r p o l a r i z a t i o n was assessed by c o m p a r i n g it to the a m p l i t u d e of the following EPSP rather t h a n to the preceding one. W h e n the c u r r e n t passing electrode was in the axon, the a m p l i t u d e of the EPSP elicited with the SN at hyperpolarized m e m b r a n e potentials was reduced to 71 __+26% of the first EPSP. The third EPSP recovered to 88 _ 34% of the first EPSP. The difference between the second a n d third EPSPs is statistically significant ( t = 2 . 3 2 , 25 dE
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Fig. 2. Effects of 5-HT on spike width and EPSP amplitude. Simultaneous recordings were made from the axon and soma of the same SN and from a follower MN. Recordings were made before (solid line) and after (dotted line) addition of 5-HT. The baselines of all traces were aligned. 5-HT increased the spike duration in both the soma and the axon. Spike broadening is better illustrated at the faster time base shown in the inset (arrow). Both the amplitude and the rate of rise of the EPSP were increased after addition of 5-HT. Similar results were obtained in 5 other experiments. Fig. 3. Effects of 5-HT on spike width and EPSP amplitude persist after SN axons have been cut. Simultaneous recordings were made from the axon of a SN and from a follower MN after the pleural-pedal connective had been severed. Recordings were made before (solid line) and after (dotted line) addition of 5-HT. 5-HT increased the duration of the axonal spike and the amplitude of the EPSP. Spike broadening is better illustrated at the faster time base shown in the inset (arrow).
238 P < 0.05, paired t-test). Thus, the recording site is close enough to the synapse to reduce transmitter release to a level below that expected from synaptic depression alone. These results confirm those reported previously [28] in other neurons of Aplysia indicating that hyperpolarization of a presynaptic neuron reduces synaptic transmission. When the current passing electrode was located in the soma, however, the amplitude of EPSP was unaffected by membrane potential. The amplitude of the EPSP elicited when the soma was hyperpolarized decreased to an average of 92 + 15% of the first EPSP. The third EPSP, elicited with the soma at the resting membrane potential, decreased further to 8 8 + 13% of the first ( n = 14). The small reduction in EPSP amplitude was probably due to homosynaptic depression. The regional effects of 5-HT, were examined by recording simultaneously from SN axons and somata. Recordings were usually made from different neurons, but in two cases we were able to record from the same neuron. When recording from different cells, spikes were triggered separately in the somata and axons at intervals of 60 s and offset by 2 s. When recording from the same cell, the spike was triggered at 60 s intervals by firing the soma. After 5 min, small concentrated aliquots (20/tl) of 5-HT (Sigma) were added to the static bath for a final concentration of 50-300/tM. Within 5 rain spike broadening was observed. The effects of serotonin on the axon spikes were partially reversed by washing out the 5-HT ( n = 4 of 4), but we could not record from the axons long enough to observe complete reversal. Bath applications of 5-HT enhanced the EPSP in the M N and broadened the spike in both the soma and the axon (Fig. 2). However, the degree of spike broadening is clearly less in the axon than in the soma. Nevertheless, the amount of broadening we observed in the axon is of approximately the same magnitude as that shown in the somata of abdominal SNs [16, 23]. The above experiments demonstrated that spike broadening could be observed in recordings from the axon. Because the somata were electrotonically far, it was unlikely that modulation of membrane properties in the soma contributed to this phenomenon. To test further this hypothesis, we surgically isolated axons from their somata by cutting the pleural-pedal connective (see arrows in Fig. 1). The protocol analyzing the effects of 5-HT was identical to that described above. Serotonin produced spike broadening in the axon and facilitated the EPSP recorded from the M N (Fig. 3). The spike recorded from a soma in the pleural ganglion proximal to the cut was also broadened (data not shown). The degree of broadening in the axon was not distinguishable from that observed in intact cells. Results similar to those shown in Fig. 3 were obtained in three other experiments. Thus, it appears that 5-HT does have direct effects in the synaptic region of SNs. This indicates that the somata of SNs are not necessary for short-term modulation of spike kinetics and synaptic transmission by 5-HT. Moreover, the axons themselves, which generally function as signal-conducting elements, may also have receptors for 5-HT. These results do not rule out the possibility that the action of 5-HT in the synaptic terminal itself is more like that in the soma than that in the axon. 5-HT-sensitive channels may be more concentrated in the terminals than the axon, resulting in more
239
prominent spike broadening in the terminals themselves. Calcium channels which are essential for broadening are also more concentrated in terminals than axons. Alternatively, these results may indicate that the cellular mechanisms active in the soma are less prominent in the terminal and that other mechanisms such as mobilization [13-15, 17] or altered Ca 2+ handling [4] may be critical for the enhancement of synaptic transmission. Although the broadening of spikes in somata is unlikely to affect synaptic transmission at the sensory-to-motor synapse in the pedal ganglion, it may have significant effects on synaptic transmission between SNs and their followers in the pleural ganglion [9]. In addition, responses in the somata may reflect other functions of modulation. For example, a plexus of serotonergic fibers infiltrates the cluster of pleural SNs, making contacts with their somata [32]. These serotonergic inputs could produce generalized changes in excitability [3, 10, 20]. In addition, serotonergic input to somata could activate mechanisms that are localized to the soma such as those regulating protein synthesis [1, 11, 12] that are likely to contribute to the changes underlying long-term sensitization [10, 24-27]. While our results demonstrate a local effect of 5-HT on heterosynaptic facilitation and show that spike broadening occurs near the release sites, the experiments do not address the extent to which the broadening accounts quantitatively for facilitation of the EPSP. Further experiments are necessary to examine the relative contribution of spike broadening and other processes such as mobilization of neurotransmitter [14, 17] to facilitation at the pleural sensory to pedal motor neuron synapse. 1 Barzilai, A., Kennedy, T.E., Kandel, E.R. and Sweatt, J.D., Serotonin causes changes in protein synthesis in pleural sensory neurons ofAplysia, Soc. Neurosci. Abstr., 14 (1988) 909. 2 Baxter, D.B. and Byrne, J.H., Serotonin-modulated membrane currents in Aplysia tail sensory neurons, Soc. Neurosci. Abstr., 12 (1986) 765. 3 Baxter, D.B. and Byrne, J.H., Modulation of membrane currents and excitability by serotonin and cAMP in pleural sensory neurons in Aplysia, Soc. Neurosci. Abstr., 13 (1987) 1440. 3aBelardetti, F., Schacher, S., Kandel, E.R. and Siegelbaum, S.A., The growth cones of Aplysia sensory neurons. Modulation by serotonin of action potential duration and single potassium channel currents, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 7094. 4 Boyle, M.B., Klein, M., Smith, S.J. and Kandel, E.R., Serotonin increases intracellular Ca ~+ transients in voltage-clamped sensory neurons of Aplysia californica. Proc. Natl. Acad. Sci. USA, 81 (1984) 7642-7646. 5 Byrne, J.H., Castellucci, V. and Kandel, E.R., Receptive fields and response properties of mechanoreceptor neurons innervating siphon skin and mantle shelf in Aplysia, J. Neurophysiol., 37 (1974) 1041 1064. 6 Castellucci, V., Pinsker, H., Kupfermann, I. and Kandel, E.R., Neuronal mechanisms of habituation and dishabituation of the gill-withdrawal reflex in Aplysia, Science, 167 (1970) 1745-1748. 7 Castellucci, V.F. and Kandel, E.R., A quantal analysis of the synaptic depression underlying habituation of the gill-withdrawal reflex in Aplysia, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 5004-5008. 8 Cleary, L.J. and Byrne, J.H., Light and electron microscopic examination of sensory neurons and motor neurons mediating the tail withdrawal reflex in Aplysia, Soc. Neurosci. Abstr., 10 (1984) 916. 9 Cleary, L.J. and Byrne, J.H., Interneurons contributing to the mediation and modulation of the tail withdrawal reflex in Aplysia, Soc. Neurosci. Abstr., I I (1985) 642. 10 Dale, N., Kandel, E.R. and Schacher, S., Serotonin produces long-term changes in the excitability of Aplysia sensory neurons in culture that depend on new protein synthesis, J. Neurosci., 7 (1987) 2232 2238.
240 I 1 Eskin, A., Byrne, J.H. and Garcia, K.S., Identification of proteins that mediate sensitization in Aplysia, Soc. Neurosci. Abstr., 14 (1988) 838. 12 Eskin, A., Garcia, K.S. and Byrne, J.H., Information storage in the nervous system in Aplysia: specific proteins affected by serotonin and cAMP, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 2458 2462. 13 Gingrich. K.J., Baxter, D.A. and Byrne, J.H., Mathematical models of cellular mechanisms contributing to presynaptic facilitation, Brain Res. Bull., 21 (1988) 513 520. 14 Gingrich, K.J. and Byrnc, J.H., Simulation of synaptic depression, post-tetanic potentiation, and presynaptic facilitation of synaptic potentials from sensory neurons mediating gill-withdrawal reflex in ,4plrsiu, J. Neurophysiol., 53 (1985) 652 669. 15 Gingrich, K.J. and Byrne, J.H., Single-cell neuronal model tbr associative learning, J. Neurophysiol., 57(1987) 1705 1715. 16 Hochner, B., Klein, M., Schacher, S. and Kandel, E.R., Action-potential duration and the modulation of transmitter release fiom the sensory neurons of Ap(vsia in presynaptic facilitation and behavioral sensitization, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 8410 8414. 17 Hochner, B., Klein, M., Schacher, S. and Kandel, E.R., Additional component in the cellular mechanism of presynaptic facilitation contributes to behavioral dishabituation in ApO,sia, Proc. Natl. Acad. Sci. USA, 83 (1986) 8794 8798. 18 Kandel, E.R. and Schwartz, J.H., Molecular biology of learning: modulation of transmitter release, Science, 218 (1982) 433 443. 19 Katz, B. and Miledi, R., A study of synaptic transmission in the absence of nerve impulses. J. Physiol. (Lond.), 192 (1967)407 436. 20 Klein, M., Hochner, B. and Kandel, E.R., Facilitatory transmitters and c A M P can modulate accommodation as well as transmitter release in ApO,sia sensory neurons: Evidence for parallel processing in a single cell, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 7994~ 7998. 21 Klein, M. and Kandel, E.R., Presynaptic modulation of voltage-dependent Ca: ~ current: mechanism for behavioral sensitization in Aplvsia cal([brnica. Proc, Natl. Acad. Sci. U.S.A., 75 (1978) 3512 3516. 22 Klein, M. and Kandel. E.R., Mechanism of calcium current modulation underlying presynaptic facilitation and behavioral sensitization in Aplysia, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 6912 6916. 23 Klein, M., Shapiro, E. and Kandel, E.R., Synaptic plasticity and the modulation of the Ca 2 ~ current, J. Exp. Biol., 89 (1980) 117 157. 24 Montarolo. P.G., Goelet. P., Castellucci, V.F., Morgan, J., Kandel, E.R. and Schacher, S., A critical period for macromolecular synthesis in long-term heterosynaptic facilitation in Aplysia, Science, 234 (1986) 1249 1254. 25 Schacher, S., Castellucci, V.F. and Kandel, E.R., c A M P evokes long-term facilitation in Aplysia sensory neurons that requires new protein synthesis, Science, 240 (1988) 1667- [669. 26 Scholz. K.P. and Byrne, J.I-t., Long-term sensitization in Aplysia: biophysical correlates in tail sensory neurons. Science, 235 (1987) 685 687. 27 Scholz, K.P. and Byrne, J.H., lntracellular injection of c A M P induces a long-term reduction of neurolaal K ' currents, Science, 240 (1988) 1664 1666. 28 Shapiro. E.. Castellucci. V.F. and Kandel, E.R., Presynaptic membrane potential affects transmitter release in an identified neuron in Aplvsia by modulating the Ca 2 + and K + currents, Proc. Natl. Acad. Sci. U.S.A., 77 (198(I) 629 633. 29 Takeuchi, A. and Takeuchi, N., Electrical changes in pre- and postsynaptic axons of the giant synapse of Lol~l,,o, J. Gen. Physiol., 45 (1962) 1181 1193. 30 Waiters, E.T., Byrne, J.H., Carew, T.J. and Kandel, E.R., Mechanoafferent neurons innervating the tail of Aplvsia: I. Response properties and synaptic connections, J. Neurophysiol., 50 (1983) 1522 1542. 31 Waiters, E.T., Byrne, J.H., Carew, T.J. and Kandel, E.R., Meehanoafferent neurons innervating the tail of Aplysia: lI. Modulation by sensitizing stimulation, J. Neurophysiol., 50 (1983) 1543 1559. 32 Zhang, Z.S., Clcary, L.J., Marshak. D.W. and Byrne, J.H., Serotonergic varicosities make apparent synaptic contacts with pleural sensory neurons of Aplysia, Soc. Neurosci. Abstr., 14 (1988) 841.
235
Elsevier Scientific Publishers Ireland Ltd.
NSL 06332
Serotonin acts in the synaptic region of sensory neurons in Aplysia to enhance transmitter release M. Hammer 2, L.J. C l e a r y 1 a n d J.H. B y r n e 1 ~Department of Neurobiology and Anatomy, University of Texas Medical School, Houston, TX 77225 (U.S.A.) and 21nstitutfiir Neurobiologie, Freie Universitdt Berlin, Berlin (F.R.G.) (Received 3 April 1989; Revised version received 25 May 1989; Accepted 26 May 1989)
Key word~: Facilitation; Synaptic release; Sensitization; Serotonin; Aplysia; Spike broadening
An important mechanism that contributes to sensitization in Aplysia is heterosynaptic facilitation of the synaptic connections between sensory neurons (SNs) and motor neurons (MNs). Heterosynaptic facilitation, in turn, is associated with broadening of the spike in the SN. Spike broadening is readily observed in recordings from somata of SNs, and from growth cones of SNs in culture, but broadening in synaptic terminals has only been inferred. Intracellular recordings were made from somata of SNs and from somata of follower MNs. Additional recordings were made from the axons of SNs as they enter the neuropil in the pedal ganglion. Serotonin (5-HT) broadened action potentials in axons of SNs and enhanced excitatory postsynaptic potentials (EPSPs) in the MNs, even after the axons of SNs were surgically separated from their somata. These results indicate that both heterosynaptic facilitation and spike broadening in the axon are due to the local action of 5-HT and can occur independently of modulation of membrane properties in the soma.
One behavioral modification in Aplysh~ that has been studied in great detail is sensitization, a simple form of non-associative learning. Sensitization appears to be due to the enhanced release of neurotransmitter from sensory neurons (SNs) produced by activation of a network of facilitatory interneurons [18]. Facilitation appears to be due, at least in part, to broadening of spikes in the SNs [2, 14, 16, 17, 21, 22, 31]. Although spike broadening, as a result of either sensitizing stimuli or application of serotonin (5-HT; a transmitter that mimics the effects of sensitizing stimuli), is readily observed in recordings from somata of SNs, and from growth cones of SNs in culture [3a], but broadening in the synaptic terminals has only been inferred. In the pleural ganglion, the somata of SNs that innervate the tail are located 2-3 mm away from their synaptic contacts with motor neurons (MNs) in the pedal ganglion [8]. By recording directly from the axons of SNs near their targets in the pedal ganglion, the kinetics of spikes near synapses can be compared with those far away in the soma. Correspondence." L. Cleary, Dept. of Neurobiology and Anatomy, University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77225, U.S.A 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
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The sensory and motor neurons innervating the tail can be readily identified on the basis of several anatomical and electrophysiological characteristics [30]. The tail SNs are located in a discrete cluster on the ventral surface of the pleural ganglion (Fig. 1). The tail M N s are located along a tract on the dorsomedial surface of the pedal ganglion [30]. Axons of SNs were impaled with an electrode in the neuropil of the pedal ganglion adjacent to the somata of the tail MNs. This region is close to the site of synaptic contacts between tail SNs and MNs as determined histologically [8]. Recordings were also made from the somata of a SN and a follower MN. Axons that run into the neuropil of the pedal ganglion from the pleural-pedal connective can be readily penetrated with fine-tipped microelectrodes (Fig. 2). Axons from SNs were distinguished from those of other neurons on the basis of several electrophysiological properties characteristic of the SN somata [5, 30]. While these criteria are not absolutely definitive, we are confident that they allowed us to distinguish SN axons since only a small percentage of the impaled axons met these criteria. Moreover, on two occasions we were able to definitively identify SN axons by recording simultaneously from the soma of the same cell in the pleural ganglion (e.g. Fig. 2). In general, the kinetics of axon spikes are quite similar to those of soma spikes, although axon spikes have higher amplitudes and in some cases are slightly broader. Since the effects of spike broadening are most important at sites of synaptic release, we wanted to establish that the recording sites in the axon were electrotonically close to the terminals. To do this, we took advantage of the fact that the membrane potential of a presynaptic neuron determines the effectiveness of transmitter release (e.g.
MN
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@ Fig. 1. Diagrammatic representation of the experimental preparation. Electrodes were placed in somata of both sensory neurons (SN) in the pleural ganglion and motor neurons (MN) in the pedal ganglion. A separate electrode was used to impale the axon o f a SN in the pedal ganglion. In two experiments, simultaneous recordings were made fiom the axon and soma of the same SN, as shown, but this was not generally the case. To verify the electrophysiological properties of the axon, the posterior pedal nerve (P9) and the pleural-abdorninal connective (P1-A) were stimulated. In some experiments, the pleural-pedal connective was cut before the axon was impaled (arrows). Also indicated for orientation is the pleural-cerebral connective (P I-C).
237 refs. 19, 28, 29). A simple protocol was used to determine the effect o f m e m b r a n e potential o n t r a n s m i t t e r release. While s i m u l t a n e o u s l y recording EPSPs in the M N , 5 spikes were triggered at intervals o f 60 s by passing a brief (1-3 ms) intracellular depolarizing pulse. O n alternate cycles, the m e m b r a n e potential o f the axon was hyperpolarized by 10 m V for 5 s, at which time the spike was triggered. A single series of 5 spikes was analyzed as 2 series of 3 spikes. Thus, the effect o f h y p e r p o l a r i z a t i o n was assessed twice in each series. Because the a m p l i t u d e o f excitatory postsynaptic potentials (EPSPs) decrements when spikes are triggered at regular intervals [6, 7], the effectiveness o f h y p e r p o l a r i z a t i o n was assessed by c o m p a r i n g it to the a m p l i t u d e of the following EPSP rather t h a n to the preceding one. W h e n the c u r r e n t passing electrode was in the axon, the a m p l i t u d e of the EPSP elicited with the SN at hyperpolarized m e m b r a n e potentials was reduced to 71 __+26% of the first EPSP. The third EPSP recovered to 88 _ 34% of the first EPSP. The difference between the second a n d third EPSPs is statistically significant ( t = 2 . 3 2 , 25 dE
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Fig. 2. Effects of 5-HT on spike width and EPSP amplitude. Simultaneous recordings were made from the axon and soma of the same SN and from a follower MN. Recordings were made before (solid line) and after (dotted line) addition of 5-HT. The baselines of all traces were aligned. 5-HT increased the spike duration in both the soma and the axon. Spike broadening is better illustrated at the faster time base shown in the inset (arrow). Both the amplitude and the rate of rise of the EPSP were increased after addition of 5-HT. Similar results were obtained in 5 other experiments. Fig. 3. Effects of 5-HT on spike width and EPSP amplitude persist after SN axons have been cut. Simultaneous recordings were made from the axon of a SN and from a follower MN after the pleural-pedal connective had been severed. Recordings were made before (solid line) and after (dotted line) addition of 5-HT. 5-HT increased the duration of the axonal spike and the amplitude of the EPSP. Spike broadening is better illustrated at the faster time base shown in the inset (arrow).
238 P < 0.05, paired t-test). Thus, the recording site is close enough to the synapse to reduce transmitter release to a level below that expected from synaptic depression alone. These results confirm those reported previously [28] in other neurons of Aplysia indicating that hyperpolarization of a presynaptic neuron reduces synaptic transmission. When the current passing electrode was located in the soma, however, the amplitude of EPSP was unaffected by membrane potential. The amplitude of the EPSP elicited when the soma was hyperpolarized decreased to an average of 92 + 15% of the first EPSP. The third EPSP, elicited with the soma at the resting membrane potential, decreased further to 8 8 + 13% of the first ( n = 14). The small reduction in EPSP amplitude was probably due to homosynaptic depression. The regional effects of 5-HT, were examined by recording simultaneously from SN axons and somata. Recordings were usually made from different neurons, but in two cases we were able to record from the same neuron. When recording from different cells, spikes were triggered separately in the somata and axons at intervals of 60 s and offset by 2 s. When recording from the same cell, the spike was triggered at 60 s intervals by firing the soma. After 5 min, small concentrated aliquots (20/tl) of 5-HT (Sigma) were added to the static bath for a final concentration of 50-300/tM. Within 5 rain spike broadening was observed. The effects of serotonin on the axon spikes were partially reversed by washing out the 5-HT ( n = 4 of 4), but we could not record from the axons long enough to observe complete reversal. Bath applications of 5-HT enhanced the EPSP in the M N and broadened the spike in both the soma and the axon (Fig. 2). However, the degree of spike broadening is clearly less in the axon than in the soma. Nevertheless, the amount of broadening we observed in the axon is of approximately the same magnitude as that shown in the somata of abdominal SNs [16, 23]. The above experiments demonstrated that spike broadening could be observed in recordings from the axon. Because the somata were electrotonically far, it was unlikely that modulation of membrane properties in the soma contributed to this phenomenon. To test further this hypothesis, we surgically isolated axons from their somata by cutting the pleural-pedal connective (see arrows in Fig. 1). The protocol analyzing the effects of 5-HT was identical to that described above. Serotonin produced spike broadening in the axon and facilitated the EPSP recorded from the M N (Fig. 3). The spike recorded from a soma in the pleural ganglion proximal to the cut was also broadened (data not shown). The degree of broadening in the axon was not distinguishable from that observed in intact cells. Results similar to those shown in Fig. 3 were obtained in three other experiments. Thus, it appears that 5-HT does have direct effects in the synaptic region of SNs. This indicates that the somata of SNs are not necessary for short-term modulation of spike kinetics and synaptic transmission by 5-HT. Moreover, the axons themselves, which generally function as signal-conducting elements, may also have receptors for 5-HT. These results do not rule out the possibility that the action of 5-HT in the synaptic terminal itself is more like that in the soma than that in the axon. 5-HT-sensitive channels may be more concentrated in the terminals than the axon, resulting in more
239
prominent spike broadening in the terminals themselves. Calcium channels which are essential for broadening are also more concentrated in terminals than axons. Alternatively, these results may indicate that the cellular mechanisms active in the soma are less prominent in the terminal and that other mechanisms such as mobilization [13-15, 17] or altered Ca 2+ handling [4] may be critical for the enhancement of synaptic transmission. Although the broadening of spikes in somata is unlikely to affect synaptic transmission at the sensory-to-motor synapse in the pedal ganglion, it may have significant effects on synaptic transmission between SNs and their followers in the pleural ganglion [9]. In addition, responses in the somata may reflect other functions of modulation. For example, a plexus of serotonergic fibers infiltrates the cluster of pleural SNs, making contacts with their somata [32]. These serotonergic inputs could produce generalized changes in excitability [3, 10, 20]. In addition, serotonergic input to somata could activate mechanisms that are localized to the soma such as those regulating protein synthesis [1, 11, 12] that are likely to contribute to the changes underlying long-term sensitization [10, 24-27]. While our results demonstrate a local effect of 5-HT on heterosynaptic facilitation and show that spike broadening occurs near the release sites, the experiments do not address the extent to which the broadening accounts quantitatively for facilitation of the EPSP. Further experiments are necessary to examine the relative contribution of spike broadening and other processes such as mobilization of neurotransmitter [14, 17] to facilitation at the pleural sensory to pedal motor neuron synapse. 1 Barzilai, A., Kennedy, T.E., Kandel, E.R. and Sweatt, J.D., Serotonin causes changes in protein synthesis in pleural sensory neurons ofAplysia, Soc. Neurosci. Abstr., 14 (1988) 909. 2 Baxter, D.B. and Byrne, J.H., Serotonin-modulated membrane currents in Aplysia tail sensory neurons, Soc. Neurosci. Abstr., 12 (1986) 765. 3 Baxter, D.B. and Byrne, J.H., Modulation of membrane currents and excitability by serotonin and cAMP in pleural sensory neurons in Aplysia, Soc. Neurosci. Abstr., 13 (1987) 1440. 3aBelardetti, F., Schacher, S., Kandel, E.R. and Siegelbaum, S.A., The growth cones of Aplysia sensory neurons. Modulation by serotonin of action potential duration and single potassium channel currents, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 7094. 4 Boyle, M.B., Klein, M., Smith, S.J. and Kandel, E.R., Serotonin increases intracellular Ca ~+ transients in voltage-clamped sensory neurons of Aplysia californica. Proc. Natl. Acad. Sci. USA, 81 (1984) 7642-7646. 5 Byrne, J.H., Castellucci, V. and Kandel, E.R., Receptive fields and response properties of mechanoreceptor neurons innervating siphon skin and mantle shelf in Aplysia, J. Neurophysiol., 37 (1974) 1041 1064. 6 Castellucci, V., Pinsker, H., Kupfermann, I. and Kandel, E.R., Neuronal mechanisms of habituation and dishabituation of the gill-withdrawal reflex in Aplysia, Science, 167 (1970) 1745-1748. 7 Castellucci, V.F. and Kandel, E.R., A quantal analysis of the synaptic depression underlying habituation of the gill-withdrawal reflex in Aplysia, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 5004-5008. 8 Cleary, L.J. and Byrne, J.H., Light and electron microscopic examination of sensory neurons and motor neurons mediating the tail withdrawal reflex in Aplysia, Soc. Neurosci. Abstr., 10 (1984) 916. 9 Cleary, L.J. and Byrne, J.H., Interneurons contributing to the mediation and modulation of the tail withdrawal reflex in Aplysia, Soc. Neurosci. Abstr., I I (1985) 642. 10 Dale, N., Kandel, E.R. and Schacher, S., Serotonin produces long-term changes in the excitability of Aplysia sensory neurons in culture that depend on new protein synthesis, J. Neurosci., 7 (1987) 2232 2238.
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