Trophic factor-induced plasticity of synaptic connections between identified Lymnaea neurons

Abstract

Neurotrophic factors participate in both developmental and adult synaptic plasticity; however, the underlying mechanisms remain unknown. Using soma-soma synapses between the identified Lymnaea neurons, we demonstrate that the brain conditioned medium (CM)-derived trophic factors are required for the formation of excitatory but not the inhibitory synapse. Specifically, identified presynaptic [right pedal dorsal 1 (RPeD1) and visceral dorsal 4 (VD4)] and postsynaptic [visceral dorsal 2/3 (VD2/3) and left pedal dorsal 1 (LPeD1)] neurons were soma-soma paired either in the absence or presence of CM. We show that in defined medium (DM-does not contain extrinsic trophic factors), appropriate excitatory synapses failed to develop between RPeD1 and VD2/3. Instead, inappropriate inhibitory synapses formed between VD2/3 and RPeD1. Similarly, mutual inhibitory synapses developed between VD4 and LPeD1 in DM. These inhibitory synapses were termed novel because they do not exist in the intact brain. To test whether DM-induced, inappropriate inhibitory synapses could be corrected by the addition of CM, cells were first paired in DM for an initial period of 12 hr. DM was then replaced with CM, and simultaneous intracellular recordings were made from paired cells after 6-12 hr of CM substitution. Not only did CM induce the formation of appropriate excitatory synapses between both cell pairs, but it also reduced the incidence of inappropriate inhibitory synapse formation. The CM-induced plasticity of synaptic connections involved new protein synthesis and transcription and was mediated via receptor tyrosine kinases. Taken together, our data provide the first direct insight into the cellular mechanism underlying trophic factor-induced specificity and plasticity of synaptic connections between soma-soma paired Lymnaea neurons.

Figure 1

Inappropriate inhibitory synapses develop between soma–soma paired neurons in DM. After 12–24 hr of cell pairing in DM, simultaneous intracellular recordings revealed that a burst of action potentials in RPeD1 failed to alter the membrane potential of VD2/3 (A), whereas compound action potentials in VD2/3 inhibited the firing of action potentials in RPeD1 (B). Similarly, the spontaneous activity in VD4 was inhibited by an induced burst of action potentials in LPeD1 (C), and vice versa (D).

Figure 2

CM promotes excitatory synapse formation and reduces the incidence of DM-induced inappropriate inhibitory synaptic connections between identified Lymnaea neurons. Identified neurons were soma–soma paired in DM for an initial period of 12 hr, at which point DM was replaced with CM. (A) A photomicrograph of soma–soma paired neurons LPeD1 and VD4 in CM. Note that the paired cells do not extend neurites. Twelve to 24 hr after CM substitution, simultaneous intracellular recordings revealed that the action potentials in RPeD1 produced 1:1 excitatory postsynaptic potentials (EPSPs) in VD2/3 (B), whereas a burst of action potentials in VD2/3 failed to alter the frequency of action potentials in RPeD1 (C). Likewise, an action potential in VD4 produced a 1:1 EPSP in LPeD1 (D). (E) Compound action potentials in LPeD1, however, failed to inhibit the firing of action potentials in VD4.

Figure 3

CM-induced plasticity of synaptic connections between identified Lymnaea neurons. Inappropriate inhibitory synapses developed in DM between RPeD1 and VD2/3, and VD4 and LPeD1 (1). Twelve hours after the initial soma–soma pairing, DM was replaced with CM (2), which in turn promoted the formation of excitatory synapses. When DM was replaced with DM, however, neither did the excitatory synapses develop nor were the inhibitory connections eliminated (3). (Solid bars) Excitatory synapses; (hatched bars) excitatory and inhibitory synapses; (dotted bars) inhibitory synapses. The n values for each experiment are noted above the data bars.

Figure 4

CM-induced plasticity of synaptic connections between RPeD1 and VD2/3 requires de novo protein synthesis and transcription. Twelve hours after the initial soma–soma pairing, DM was replaced with CM containing either a protein synthesis (anisomycin, 12.5 μg/ml) or a transcription (actinomycin D, 1 μm) blocker. (1) Twelve hours after initial soma–soma pairing, DM was replaced with CM. Inhibition of either protein synthesis (2) or transcription (3) blocked CM’s ability to promote excitatory synapse formation. Inappropriate inhibitory synapses did however persist. (Solid bars) Excitatory synapses; (hatched bars) mixed excitatory and inhibitory synapses; (dotted bars) inhibitory synapses. The n values for each experiment are noted above the data bars.

Figure 5

CM-induced plasticity of synaptic connections between identified Lymnaea neurons is mediated via receptor tyrosine kinases. RPeD1 and VD2/3 were soma–soma paired in DM for 12 hr. DM was subsequently replaced with CM containing a receptor tyrosine kinase inhibitor. (1)Twelve hours after initial soma–soma pairing, DM was replaced with CM. The CM-induced synaptic plasticity was either blocked completely or reduced significantly in the presence of CM containing a receptor tyrosine kinase inhibitor [(2) Lavendustin A, 10 μm; (5) K252a, 0.1 μm]. CM containing the inactive forms of receptor tyrosine kinase inhibitors [(3) Lavendustin B, 10 μm] were, however, ineffective in perturbing the CM-induced excitatory synapse formation. Similarly, the addition of DMSO [(4) carrier solution, 1%] failed to affect CM-induced plasticity of synaptic connections between RPeD1 and VD2/3 pairs. The n values for each experiment are noted above the data bars.

Figure 6

A model depicting the CM-induced plasticity of synapse formation between soma–soma paired Lymnaea neurons. In the presence of trophic factors [(+) trophic factors] identified Lymnaea neurons form appropriate excitatory synapses. In the absence of trophic factors [(−) trophic factors], however, inappropriate inhibitory synapses develop in vitro. Because these synapses are novel and inhibitory, we have termed them default synapses. These inappropriate inhibitory synapses are reduced in incidence by the addition of CM-derived trophic molecules (thick black arrow), which also induce appropriate excitatory synapse formation between the paired cells. This trophic factor-induced plasticity requires de novo protein synthesis and is mediated via receptor tyrosine kinases (RTKs).