(A) Sample EPSPs recorded from cocultures in the four groups in the experiments summarized in (B).
(B) Mean normalized amplitude of the EPSPs for each group in experiments testing the effect of postsynaptic injection of the cap analog. A one-way ANOVA of the data indicated that the group difference were significant [F_(3,63) = 4.89, p < 0.005]. The 5-HT treatment produced significant LTF of the synapses, as indicated by comparing the Day 2 EPSPs in the 5-HT (132 ± 8%, n = 20) and Control (89 ± 13%, n = 15) groups (p < 0.01). Synapses in which cap analog was injected into the motor neuron prior to 5-HT treatment (5-HT-cap analog group, Day 2 EPSP = 88 ± 6%, n = 18) did not exhibit LTF (p < 0.01 for the comparison with the 5-HT group). Postsynaptic injection of cap analog alone did not significantly alter the strength of the sensorimotor synaptic connections (Day 2 EPSP = 101 ± 13%, n = 14; p > 0.05 for the comparison between the Cap analog and Control groups). * indicates significance of the difference between the 5-HT and Control groups, and + indicates significance of the difference between the 5-HT and 5-HT-cap analog groups. Error bars represent ± SEM.
(C) Sample EPSPs recorded from cocultures in the four groups in the experiments summarized in (D).
(D) Mean normalized amplitude of the EPSPs for each group in experiments that examined the effect of postsynaptic injection of gelonin. The differences among the groups were highly significant [F_(3,95) = 4.59, p < 0.005]. Mean normalized EPSP in the 5-HT group (163 ± 16%, n = 23) was significantly greater than that in the Control group (95 ± 12%, n = 25; p < 0.01). The mean normalized amplitude of the Day 2 EPSP in cocultures that received the postsynaptic gelonin prior to 5-HT treatment (5-HT-gelonin group, 111 ± 11%, n = 25) was significantly less than that in the 5-HT alone group (p < 0.05). Postsynaptic injection of gelonin by itself did not significantly alter the strength of the synapse (mean normalized Day 2 EPSP in the Gelonin group = 123 ± 14%, n = 26; p > 0.05 for the comparison between the with the Control group). * indicates significance of the difference between the 5-HT and Control groups, and + indicates significance of the difference between the 5-HT and 5-HT-Gelonin groups Error bars represent ± SEM.

(A) Phase contrast micrograph of a sensorimotor coculture. The arrow points to the major neurite of the motor neuron. Scale bar, 30µm.
(B) Micrographs of sensorin immunofluorescence in the experimental groups. Each micrograph depicts the region along the main axon of a sensory neuron where contacted the major neurite of the motor neuron. Previous evidence indicates that this region represents the area of maximum synaptic contact between the sensory and motor neurons [40]. In cocultures that did not receive a postsynaptic injection of BAPTA, 5-HT treatment increased sensorin staining compared to untreated Controls. In cocultures treated with 5-HT after a postsynaptic BAPTA injection the immunostaining resembled that in Controls. Scale bar, 10 µM.
(C) Intensity of sensorin immunostaining in the four experimental groups. Staining intensity was determined for each coculture by measuring the mean pixel intensity of the fluorescence in four circular regions centered on the main process of the motor neuron. The mean pixel intensity was then corrected for background fluorescence, and the result was normalized to the mean pixel intensity in Control group (Figure S1). A one-way ANOVA indicated that the group differences were highly significant [F_(3,82) = 6.13, p = 0.0008]. A Post-hoc comparison showed that the normalized sensorin staining was greater in 5-HT cocultures (151 ± 15%, n = 20) than in Controls (100 ± 5%, n = 26; p < 0.01). Importantly, the mean normalized sensorin staining in 5-HT-BAPTA cocultures (97 ± 6%, n = 20) was significantly less than that in the 5-HT group (p < 0.01), and not statistically different from that in Control cocultures (p > 0.05). The postsynaptic BAPTA injection alone had little effect on sensorin staining (mean normalized staining in the BAPTA group = 111 ± 8%, n = 20; p > 0.05 for the comparison with the Controls). *, significance of the difference between 5-HT and Control groups; +, significance of the difference 5-HT and 5-HT-BAPTA groups. Error bars represent ± SEM.

(A) Model for short-term synaptic facilitation. The serotonergic facilitatory interneuron, activated in the intact animal by noxious stimulation, releases 5-HT onto the sensory neuron. After binding to its G protein-coupled receptor, 5-HT causes rapid-onset, short-lasting (5–10 min) facilitation of the synapses via processes that involve presynaptic PKA and PKC [41, 42].
(B) Model for long-term synaptic facilitation. Binding of 5-HT to its receptors on the motor neuron causes a rise of intracellular calcium in motor neuron via activation of IP₃ receptors and RyRs [13]. The rise of intracellular calcium drives local postsynaptic protein synthesis (present results) and enhancement of AMPA receptor function [14, 32]. AMPA receptor function may be enhanced through the synthesis of new AMPA receptors, exocytotic delivery of additional receptors to the postsynaptic membrane, or both [13, 18]. The postsynaptic rise in Ca²⁺ also activates one or more retrograde signals (present results). The retrograde signals, released from motor neuron, cause the rapid secretion and enhanced expression (via PKA and PI3K, respectively) of sensorin (Refs. [8, 9 and present results]). After binding to its autoreceptors, sensorin leads to phosphorylation of MAPK and its subsequent translocation into the nucleus of the sensory neuron. Translocated MAPK phosphorylates transcription factors that regulate the gene expression required for LTF [15, 43]. PKA, which is also translocated to nucleus, also plays a critical role in regulating long-term cellular changes accompanying LTF [44, 45]. In addition to changes in presynaptic transcription, LTF is likely to be accompanied by changes in postsynaptic transcription. Furthermore, it is possible that presynaptic effects of 5-HT also contribute to LTF [37]. The dashed lines in [A] and [B] indicate pathways whose involvement in STF/LTF is uncertain at present.

(A) Experimental arrangement. Scale bar, 20 µm.
(A’) Sample electrophysiological records from a Pretest. A single action potential evoked in a sensory neuron produced a monosynaptic EPSP in the motor neuron. Note that the sensory action potentials are not included in the other pre- and posttest records presented in this and following figures. Scale bars, 20 mV and 200 ms.
(A”) Experimental protocol. Note that the sensory and motor neurons in the cocultures were given 3–4 d to form synaptic connections before the start of the experiment. (See Experimental Procedures in the Supplemental Data [online] for additional information.)
(B) Sample EPSPs for four of the experimental groups: Control, BAPTA, 5-HT, 5-HT-BAPTA. Each pair of traces shows EPSPs recorded from the same sensorimotor synapse before (Pre) and 24 h after treatment (Post). Scale bars for these and EPSPs shown in subsequent figures, 10 mV and 80 ms.
(C) Mean normalized amplitude of the EPSPs in the four experimental groups. A one-way ANOVA indicated that the differences among the groups were highly significant [F_(3,49) = 11.20, p < 0.0001]. There was significant LTF of the synapses in the 5-HT-treated group (Day 2 EPSP = 155 ± 14%, n = 18), as indicated by the comparison with the Control synapses (Day 2 EPSP = 88 ± 10%, n = 14), which did not receive 5-HT (p < 0.001). By contrast, synapses in which BAPTA was injected into the motor neuron prior to 5-HT treatment (5-HT-BAPTA group) did not exhibit LTF (Day 2 EPSP = 69 ± 7%, n = 10; p < 0.001 for the comparison with the 5-HT group). Postsynaptic injection of BAPTA by itself (Day 2 EPSP = 73 ± 9%, n = 11) did not cause significant long-term changes in the strength of the sensorimotor synaptic connections (p > 0.05 for the comparison between the BAPTA alone and Control groups). * indicates significance of the difference between the 5-HT and Control groups, and + indicates significance of the difference between the 5-HT and 5-HT-BAPTA groups. Error bars represent ± SEM.