Synapse formation in the absence of cell bodies requires protein synthesis

Abstract

Protein synthesis at distal synaptic sites is thought to play a critical role in long-term synaptic plasticity at preexisting connections. We tested whether protein synthesis in distal neuritic processes contributes to the formation of new synaptic connections by Aplysia neurons regenerating in cell culture after removing their cell bodies. Removal of either the sensory neuron (SN) or motor cell L7 cell body did not affect the formation of synaptic connections during the next 48--72 hr period. Increases in synaptic efficacy after removal of the SN cell body was accompanied by neurite growth and an increase in the number of SN varicosities contacting L7. The increases in synaptic efficacy and the number of SN varicosities were blocked by anisomycin, a protein synthesis inhibitor. The initial formation of synaptic connections was not affected by the absence of the L7 cell body. In the absence of cell bodies from both presynaptic and postsynaptic cells, synaptic efficacy increased for 48 hr and was blocked reversibly by anisomycin. These results support the idea that distal neuritic processes contain stable mRNAs and the macromolecular machinery for protein synthesis that are required for the formation of new synaptic connections.

Fig. 1.

Increases in synaptic efficacy 24 hr after removal of the SN cell body. A, B, Photomicrographs of an SN–L7 culture before (A) and 24 hr after (B) cutting the SN axon at thearrowhead. A portion of the L7 axon can be seen in thetop right corner. An extracellular electrode place near the new axon stump (arrow in B) is used to evoke EPSPs in L7. Dye-filled electrodes inserted at the SN axon stump are used for imaging SN neurites contacting L7 (Fig. 2). Scale bar, 50 μm. C, Examples of two traces before and after cutting the SN axon while recording intracellularly from L7. An initial high-frequency burst of EPSPs is followed by low-frequency bursts. Note the rapid decline in EPSP amplitude typical of homosynaptic depression. Calibration: 10 mV, 0.5 sec. D, Examples of EPSPs evoked in L7 before (Pre) and after (4 hr and24 hr) control treatment (CONT) and cutting the SN axon and removing the SN cell body (−SN CB). EPSP amplitudes increase for both groups. Calibration: 15 mV, 25 msec. E, Summary of the mean ± SEM of the EPSP amplitudes recorded before (Pre) and at two time points after control (CONT) and experimental (−SN CB) treatment. There was no significant difference in the increases in EPSP amplitude over time (see Results).

Fig. 2.

Protein synthesis-dependent outgrowth and varicosity formation in the absence of the SN cell body.A, D, Nomarski optics photomicrographs of a portion of the major axons of L7 in control (A) and anisomycin-treated (D) cultures that are viewed in the epifluorescent images. SN neurites extend and varicosities form along the major processes of L7. B,C, Epifluorescent montage of SN neurites and varicosities interacting with the same region of L7 processes before (B) and 24 hr after (C) removal of the SN cell body. Dye was injected into the SN cell body inA and into the SN axon stump in B. Note the extension of three neurites and the formation of several new varicosities on the major process of L7. E,F, New growth and varicosity formation are blocked by anisomycin. Fluorescent images of SN neurites interacting with the same region of L7 processes before (E) and 24 hr after (F) removal of the SN cell body. Anisomycin was added to the culture 30–60 min after removing the SN cell body. Short neurites have retracted, and there is a loss of several varicosities. Scale bar, 12.5 μm.

Fig. 3.

Anisomycin reversibly blocks increases in synaptic efficacy in the absence of the SN cell body. A–C, Photomicrographs of the same culture before (A), 24 hr (B), and 48 hr (C) after cutting the SN axon at the arrowhead inA. Anisomycin was added 30–60 min after cutting the SN cell body and washed out 24 hr later. After cutting the SN cell body, EPSPs were evoked in L7 by stimulating the axon stump (arrow in B and C) with an extracellular electrode. Note a portion of the L7 axon at thetop of each figure. Scale bar, 50 μm.D, Examples of EPSPs evoked in the same cultures before and after the various treatments. EPSP amplitudes increased at each time point (Day 3 and Day 4) in both the control cultures (CONT) and cultures without an SN cell body (−SN CB). EPSP amplitudes increased in the remaining group only after washout of anisomycin (ANISO WASH; Day 4). Calibration: 15 mV, 25 msec. E, Summary of the EPSP amplitudes (mean ± SEM) before treatments (Day 2) and after treatments (Day 3) and washout (Day 4). An ANOVA indicated a significant effect of treatment (F = 5.006; p < 0.01). Note that EPSP amplitudes in the absence of SN cell body (−SN CB) increase over time as the control group. Treatment with anisomycin blocks the increase in EPSP amplitude, which recovers with washout (see Results).

Fig. 4.

Synapse formation in the absence of L7 cell body.A, Photomicrograph of an L7 in culture alone for 18 hr before cutting the axon at the arrowhead. The cell body of L7 is located at the bottom left portion of the micrograph. B, Photomicrograph of the same culture 48 hr after cutting the L7 axon and then adding a single SN. EPSPs are evoked by stimulating the SN cell body and recording the response with an intracellular electrode in the new L7 stump (arrow). Scale bar, 50 μm. C, Examples of EPSP amplitudes 2 d after adding an SN to a culture without the L7 cell body (−L7 CB) and to a control culture prepared at the same time (CONT). Calibration: 15 mV, 25 msec.D, Summary of the EPSP amplitudes recorded at 48 hr after adding an SN to L7 with (CONT) or without L7 cell body (−L7 CB). EPSP amplitudes were not significantly different (see Results).

Fig. 5.

Protein synthesis-dependent synapse formation by SN neurites with L7 neurites in the absence of both cell bodies.A, Photomicrograph of SN–L7 culture on day 1 without L7 cell body (−L7 CB) just after recording EPSP illustrated in C (ANISO). The SN was added to the culture after cutting the L7 cell body (Fig. 4). The SN axon was cut at the arrowhead. The culture was then incubated in medium containing anisomycin. B, Photomicrograph of the same culture 48 hr later on day 3. The EPSP (seeDay 2 and Day 3 in C) was produced in L7 by stimulating the SN axon stump (arrow). Scale bar, 50 μm. C, Examples of EPSPs produced in a control and an anisomycin-treated culture at each time point. SNs formed synapses with L7 in the absence of the L7 cell body (Day 1, −L7 CB). EPSP amplitude increased in the absence of both the L7 and SN cell bodies (CONT,Day 2 and Day 3, −L7 & SN CB). The increase in efficacy is reversibly blocked by anisomycin (ANISO, Day 2 and Day 3). Calibration: 10 mV, 25 msec. D, Summary of the changes in EPSP amplitudes (mean ± SEM) in the control (CONT) and anisomycin-treated (ANISO) groups. An overall ANOVA indicated a significant difference between the two groups (F = 7.066;p < 0.005). Individual comparisons indicated a significant change in EPSP amplitude on days 2 and 3 in the control cultures over the EPSP amplitude recorded on day 1 (baseline): anisomycin blocked an increase in EPSP amplitude over baseline, and EPSP amplitude increased significantly compared with baseline after washout of anisomycin (see Results).