kuron, Vol. 6, 679-690, May, 1991, Copyright 0 1991 by Cell Press Target-Dependent Structural Changes in Sensory Neurons of Aplysia Accompany ong-Term Heterosynaptic Inhibition S. Schacher and P. G. Montarolo* Center for Neurobiology and Behavior and New York State Psychiatric Institute College of Physicians and Surgeons Columbia University New York, New York 10032 Summary FMRFamide evokes both short-term and long-term inhi- bition of synapses between mechanosensory and motor neurons in Aplysia. We report here, using dissociated cell culture and low-light epifluorescence video micros- copy, that depression lasting 24 hr of sensorimotor syn- apses evoked by four brief applications of FMRFamide is accompanied by a significant loss of sensory cell vari- cosities and neurites. These structural changes in the sensory cells require the presence of the target motor cell 17. Because the loss of structures known to contain transmitter release sites correlates significantly with the changes in the amplitude of the excitatory postsynaptic potential in 17, our results suggest that the structural changes evoked by FMRFamide reflect a loss of synaptic contacts. Thus, long-term depression parallels long-term facilitation of the sensorimotor synapse produced by serotonin in that both forms of heterosynaptic plasticity involve target-dependent modulation of the number of presynaptic varicosities. Introduction Long-term changes in synaptic transmission between mechanosensory and motor cells accompany long- term habituation (Castellucci et al., 1978) and sensiti- zation (Frost et al., 1985) of the gill withdrawal reflex in Aplysia. These changes in synaptic efficacy evoked by behavioral training appear to involve alterations in the structure of sensory neurons and their active zones within the neuropil of the abdominal ganglion (Bailey and Chen, 1983,1988a, 1988b). Because these observations were made at only one time point, it was not possible to relate directlythese structural changes to alterations in the physiological properties of the sensory neurons examined or to determine the extra- c”?llular and intracellular events mediating the struc- tural changes. Dissociated cell culture of Aplysia sensory neurons along with identified gill and siphon motor cell L7 or other identified cells (Raypot-t and Schacher, 1986; Clanzman et al., 1989a) provides a model system for studying the structure-function relationships accompa- nying synapse formation or synaptic plasticity (Glanz- man et al., 1989a, 1990). In cell culture, the monosynaptic * Present address: Dipartimento di Anatomia e Fisiologia Umana, Corso Raffaello 30, 10125 Torino, Italy. sensorimotor synapse can be modulated by specific neurotransmitters. Repeated applications of serotonin (5-HT), a neurotransmitter involved with eliciting be- havioral sensitization of the reflex (Mackeyet al., 1989; Glanzman et al., 1989b), evoke facilitation of the syn- apse lasting at least 24 hr (Montarolo et al., 1986; Dale et al., 1988; Schacher et al., 1990). Imaging the struc- ture of sensory neurons with low-light epifluores- cence video microscopy both before and 24 hr after repeated 5-HT applications indicated that the long- term facilitation of the synapse is accompanied by an increase in the number of sensory cell varicosities contacting the major axons of the motor cell L7 (Clanz- man et al., 1990). Electron microscope examination of these new varicosities showed that some of them contained activezones (Glanzman et al., 1990; Schacher et al., 1991). In addition, the structural changes in the sensory cells require the presence of the motor cell. Applications of 5-HT to sensory neurons cultured alone resulted in little or no long-term change in structure. The sensorimotor synapse, both in the intact ner- vous system (Abrams et al., 1984; Belardetti et al., 1987; Mackey et al., 1987) and in cell culture (Piomelli et al., 1987; Montarolo et al., 1988; Dale and Kandel, 1990), can be depressed by the neuropeptide Phe-Met-Arg- Phe-amide (FMRFamide). The short-term inhibitory actions of FMRFamide are primarily on the presynap- tic sensory neuron where it increases a K+ current (Belardetti et al., 1987; Brezina et al., 1987a), decreases a voltage-dependent Ca*+ current and Ca2+ influx dur- ing action potentials (Brezina et al., 1987b; Edmonds et al., 1990; Blumenfeld et al., 1990), and decreases spontaneous transmitter release that is independent of Ca*+ (Dale and Kandel, 1990). Similar actions of FMRFamide have been found mediating presynaptic inhibition of identified Helisoma neurons in culture (Man-Son-Hing et al., 1989). As was observed for 5-HT, FMRFamidealso evokes long-term changes in culture. Repeated applications over a 2 hr period produce de- pression of the synapses when reexamined 24 hr later (Montarolo et al., 1988). Does long-term synapticdepression evoked by FMRF- amide involve structural changes such as a decrease in the number of presynaptic sensory cell varicosities, sites known to contain transmitter release sites (Clanzman et al., 1989a)? If structural changesaccompa,ny long-term synaptic depression, do they require interaction of the sensory cell with a correct target motor cell? We there- fore examined the structure of sensory cell neurites contacting motor cell L7 or an incorrect target cell Lll at two time points with low-light epifluorescence video microscopy following intracellular injections of the fluorescent dye 5(6)-carboxyfluorescein. Our results indicate that FMRFamide-induced long-term depres- sion, but not the short-term, is accompanied by de- creases in sensory cell varicosities and neurites. The extent of the structural changes correlates signifi-