A novel function for serotonin-mediated short-term facilitation in aplysia: conversion of a transient, cell-wide homosynaptic hebbian plasticity into a persistent, protein synthesis-independent synapse-specific enhancement

Abstract

Studies of sensitization and classical conditioning of the gill-withdrawal reflex in Aplysia have shown that the synaptic connections between identified glutamatergic sensory neurons and motor neurons can be enhanced in one of two ways: by a heterosynaptic (modulatory input-dependent) mechanism that gives rise with repetition to long-term facilitation and by a homosynaptic (activity-dependent) mechanism that gives rise with repetition to a facilitation that is partially blocked by 2-amino-5-phosphonovaleric acid and by injection of 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetate (BAPTA) into the postsynaptic cell and is similar to long-term potentiation in the hippocampus. We here have examined how these two forms of facilitation interact at the level of an individual synaptic connection by using a culture preparation consisting of a single bifurcated sensory neuron that forms independent synaptic contacts with each of two spatially separated motor neurons. We find that the homosynaptic facilitation produced by a train of action potentials is cell wide and is evident at all of the terminals of the sensory neuron. By contrast, the heterosynaptic facilitation mediated by the modulatory transmitter serotonin (5-HT) can operate at the level of a single synapse. Homosynaptic activation gives rise to only a transient facilitation lasting a few hours, even when repeated in a spaced manner. The heterosynaptic facilitation produced by a single pulse of 5-HT, applied to one terminal of the sensory neuron, also lasts only minutes. However, when one or more homosynaptic trains of spike activity are paired with even a single pulse of 5-HT applied to one of the two branches of the sensory neuron, the combined actions lead to a selective enhancement in synaptic strength only at the 5-HT-treated branch that now lasts more than a day, and thus amplifies, by more than 20-fold, the duration of the individually produced homo- and heterosynaptic facilitation. This form of synapse-specific facilitation has unusual long-term properties. It does not require protein synthesis, nor is it accompanied by synaptic growth.

Figure 1

Homosynaptic activation produces a facilitation that is cell wide and transient. (A) A single tetanus (20 Hz for 2 sec) applied to the soma of the sensory neuron produces a cell-wide increase in the amplitude of the evoked EPSP recorded in both postsynaptic motor neurons that peaks at 10 min. This homosynaptic facilitation was transient: the increase in the EPSP amplitude was greatly reduced 1 h after training, returned to baseline by 2 h, and no facilitation was present at 24 h. (B) As was the case with a single tetanus, the increase in the EPSP amplitude at sensory-to motor-neuron connections evoked by 4 trains lasted about 1 h and was no longer present at 2 and 24 h. (C) Application of a single pulse of the modulatory neurotransmitter 5-HT produced only short-term facilitation. The amplitude of the EPSP peaked at 10 min and decayed rapidly, returning to baseline at 1 h. The data in A and the 24-h time point in B were taken from bifurcated cultures. The remaining data points in B and C were taken from regular cocultures. No differences were found in the magnitude of response of each type of culture to homo- or heterosynaptic stimulation.

Figure 2

A single heterosynaptic modulatory input enhances the duration of cell-wide homosynaptic facilitation in a synapse-specific way. (A) The time course of the LTF produced by pairing a single homosynaptic tetanus to the cell body with a single pulse of 5-HT to the synapse is shown. Combined homo- and heterosynaptic activation produces robust synaptic facilitation that peaks at 10 min. This facilitation persists only at the branch treated with 5-HT and remains stable from 12 h after training (**, P < 0.01 change in EPSP amplitude in 5-HT-treated vs. untreated branch) up to at least 24 h (*, P < 0.05 change in EPSP amplitude in 5-HT-treated vs. untreated branch). (B) The combination of a single homosynaptic tetanus with a single heterosynaptic pulse of 5-HT produced a long-term enhancement in the amplitude of the EPSP that lasted at least 24 h. This increase in synaptic strength was restricted in its expression to the 5-HT-treated branch compared with the untreated branch (*, P < 0.05). Increasing the number of tetani from one to four also produced an increase in the magnitude of the LTF expressed at the 5-HT-treated branch compared with the untreated branch (#, P < 0.1). In control experiments, untreated cells showed no change in the EPSP amplitude at 24 h. Shown are representative recordings of EPSPs and histograms of the mean change in EPSP amplitude ± SEM. The histograms for the 4 × 20 Hz + 1 × 5-HT illustrated here are taken from the experiments described below in Fig. 3.

Figure 3

Pairing homosynaptic activation with a single heterosynaptic modulatory input produces a form of LTF that is protein synthesis independent and is not associated with synaptic growth. (A) Bath application of 10 μM anisomycin, a translational inhibitor, for 30 min before, during, and 30 min after branch-specific paired training did not block LTF at 24 h (*, P < 0.05, change in EPSP amplitude in 5-HT-treated vs. untreated branch). To test whether the LTF induced by branch-specific paired homo- and heterosynaptic training was accompanied by synaptic growth, we examined the consequences of combining a single tetanus and a single pulse of 5-HT on the number of fluorescently labeled sensory neuron varicosities contacting each motor neuron. We found that although the paired training produced LTF measured at 24 h at the 5-HT-treated branch, it produced no increase in varicosity number. (B) Pairing a single pulse of 5-HT with 4 tetani also produced facilitation present at 24 h but still induced no synaptic growth.