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. 2001 Jul 15;534(Pt. 2):501-10.
doi: 10.1111/j.1469-7793.2001.00501.x.

Serotonin facilitates AMPA-type responses in isolated siphon motor neurons of Aplysia in culture

Affiliations

Serotonin facilitates AMPA-type responses in isolated siphon motor neurons of Aplysia in culture

R A Chitwood et al. J Physiol. .

Abstract

1. Serotonin (5-HT) facilitates the connections between sensory and motor neurons in Aplysia during behavioural sensitization. The effect of 5-HT on sensorimotor synapses is believed to be primarily presynaptic. Here we tested whether 5-HT can have an exclusively postsynaptic facilitatory effect. 2. Siphon motor neurons were individually dissociated from the abdominal ganglion of Aplysia and placed into cell culture. Brief pulses of glutamate, the putative sensory neuron transmitter, were focally applied (0.1 Hz) to solitary motor neurons in culture, and the glutamate-evoked postsynaptic potentials (Glu-PSPs) were recorded. 3. When 5-HT was perfused over the motor neuron for 10 min, the amplitude of the Glu-PSPs was significantly increased. The 5-HT-induced enhancement of the Glu-PSPs persisted for at least 40 min after washout. 4. Prior injection into the motor neuron of the calcium chelator BAPTA, GDP-beta-S or GTP-gamma-S blocked the 5-HT-induced facilitation of the Glu-PSPs. However, the facilitation was not blocked when APV, an NMDA receptor antagonist, was applied together with the 5-HT. 5. The enhancement of the Glu-PSPs by 5-HT was reversed by the AMPA receptor antagonist DNQX, indicating that 5-HT increased the functional expression of AMPA-type receptors in the motor neuron. 6. The presence of botulinum toxin in the motor neuron blocked the 5-HT-induced enhancement of the Glu-PSPs. As botulinum toxin prevents exocytosis we hypothesize that during sensitization 5-HT causes the insertion of additional AMPA-type receptors into the postsynaptic membrane of sensorimotor synapses via exocytosis. This postsynaptic mechanism may contribute to facilitation of the synapses.

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Figures

Figure 1
Figure 1. Effect of 5-HT on the glutamate-evoked response in solitary motor neurons in culture
A, left panel, micrograph showing the experimental arrangement. The black arrow indicates the cell body of an isolated motor neuron in culture. The tips of the perfusion pipettes can be seen at the upper right and lower left of the micrograph. The patch (puffer) pipette used to apply glutamate is visible to the right, and the recording microelectrode is visible to the left, of the cell body. The main motor neurite can be seen projecting from the cell body at twelve o'clock. Scale bar, 500 μm. Centre panel, higher-power view of the same motor neuron shown in the left panel. Immediately to the right of the initial segment of the main neurite of the motor neuron is the tip of the puffer pipette. Right panel, example of a single application of glutamate, made visible using Fast Green, to the motor neuron. The glutamate stream (white arrow) is rapidly removed by the perfusion outflow. Scale bar, 100 μm. B, electrophysiological characterization of an LFS siphon motor neuron. Constant negative current sufficient to hold the cell at -88 mV was applied. When the negative current was removed a characteristic ‘notch’ (arrow) was observed in the membrane potential upon its return from hyperpolarization. Scale bars, 100 ms and 20 mV. C, 5-HT facilitates the response of the motor neuron to glutamate. Summary data for 5-HT and control experiments. Each symbol represents the mean (normalized) amplitude of 6 Glu-PSPs (Methods). Either 5-HT (□, n = 5) or normal perfusion medium (•, n = 5) was bath-applied for 10 min. (In this and subsequent graphs the filled bar represents the period of 5-HT delivery. The 5-HT was typically applied for 10 min, but the duration of 5-HT application occasionally varied ±30 s.) The mean Glu-PSP during 5-HT treatment was 1.26 ± 0.19, whereas it was 0.73 ± 0.06 in control medium. The enhancement of the glutamate response persisted after washout of 5-HT. The mean amplitude of the Glu-PSP during the 10 min period after the perfusion medium was returned to control medium was 1.33 ± 0.19 for the 5-HT-treatment group and 0.74 ± 0.06 for control group. Insets in this and following figures show the averaged traces for 5 consecutive responses from one experiment recorded at the times indicated by the numbers. Scale bars in this and all subsequent graphs, 4 mV and 200 ms. D, 5-HT-induced facilitation of the glutamate response is long lasting. Summary data from experiments (different from those in C) in which all cultures received 5-HT. The mean normalized amplitude during the 40 min post-drug period was 1.65 ± 0.02 (n = 5).
Figure 2
Figure 2. 5-HT-induced facilitation depends upon intracellular Ca2+ and G-protein activation, but not upon NMDA-type receptors
A, summary data for BAPTA (•) and 5-HT control (□) experiments. The mean amplitude of the Glu-PSP during the 10 min post-5-HT period in the BAPTA experiments (n = 14) was 0.89 ± 0.06, whereas it was 1.47 ± 0.09 in the control experiments (n = 8). B, effect of the G-protein inhibitor GDP-β-S. Summary data for GDP-β-S (•) and 5-HT control (□) experiments. The mean Glu-PSP during the 40 min post-5-HT period was 0.84 ± 0.04 in the GDP-β-S experiments (n = 6) and 1.46 ± 0.12 in the control experiments (n = 5). C, effect of occlusion of G-protein activation. Summary data for GTP-γ-S (•) and 5-HT control (□) experiments. The mean Glu-PSP during the 40 min post-5-HT period was 0.73 ± 0.03 in the GTP-γ-S experiments (n = 4) and 1.61 ± 0.11 in the control experiments (n = 4). D, summary data for APV (•) and 5-HT alone control (□) experiments. APV was applied simultaneously with the 5-HT. The APV + 5-HT enhanced the amplitude of the mean normalized Glu-PSP (1.16 ± 0.03, n = 5) compared with that of the 5-HT alone control group (0.98 ± 0.05, n = 5) during the 10 min period of drug application. There was no significant difference in the mean amplitude of the Glu-PSP in the APV + 5-HT group and the 5-HT alone group (1.36 ± 0.08 vs. 1.51 ± 0.08 during the 40 min post-drug period).
Figure 3
Figure 3. 5-HT-induced facilitation of the glutamate response is mediated by modulation of AMPA-type receptor trafficking
A, effect of DNQX on the baseline Glu-PSP. The mean normalized Glu-PSP was 0.78 ± 0.08 during the 10 min of bath application of DNQX (n = 8). B, effect of DNQX on the 5-HT enhanced Glu-PSP. Application of 5-HT increased the normalized Glu-PSP amplitude (mean Glu-PSP = 1.77 ± 0.15 during the 10 min period after returning the perfusion medium to control medium; n = 6). Bath application of DNQX eliminated the 5-HT-induced increase (mean Glu-PSP during DNQX application following 5-HT treatment = 0.97 ± 0.06). C, effect of botulinum toxin on 5-HT-induced facilitation. Summary data for botulinum toxin (BoTox, •) and 5-HT control (□) experiments. The presence of Botox in the motor neuron impaired facilitation of the Glu-PSP by 5-HT. The normalized Glu-PSP was 1.05 ± 0.7 (n = 6) in the Botox experiments and 1.51 ± 0.17 (n = 5) in the control experiments.
Figure 4
Figure 4. Cellular model for postsynaptic facilitation by 5-HT in Aplysia
Stimuli that induce behavioural sensitization cause 5-HT to be released from the terminals of facilitating interneurons. 5-HT binds to receptors on postsynaptic siphon motor neurons. Activation of G-proteins by 5-HT causes a rise in intracellular Ca2+ in the motor neuron via an as-yet unidentified pathway. The rise in intracellular Ca2+ causes vesicles containing AMPA-type receptors to fuse to the postsynaptic membrane and the receptors to be inserted into the membrane. During sensitization 5-HT also binds to G-protein-coupled receptors in the sensory neuron. There it stimulates several intracellular pathways, leading to enhanced presynaptic release.

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