Neurite growth patterns leading to functional synapses in an identified embryonic neuron

Abstract

We explored the relationship between neurite outgrowth and the onset of synaptic activity in the central neuropil of the leech embryo in vivo. To follow changes in early morphology and the onset of synaptic activity in the same identified neuron, we obtained whole-cell patch-clamp recordings and fluorescent dye fills from dorsal pressure-sensitive (P) cells, the first neurons that could be reliably identified in the early embryo. We followed the development of the P cell from the first extension of neurites to the elaboration of an adult-like arbor. After the growth of primary neurites, we observed a profuse outgrowth of transient neurites within the neuropil. Retraction of the transient neurites left the primary branches studded with spurs. After a dormant period, stable secondary branches grew apparently from the spurs and became tipped with terminals. At this time, neurites of the Retzius (R) cell, a known presynaptic partner in the adult, were observed to apparently contact the terminals. Although voltage-dependent currents were seen in the P cell at the earliest stage, spontaneous synaptic activity was only observed when terminals had formed. Spontaneous release was observed before evoked release could be detected from the R cell. Our results suggest that transient neurites are formed during an exploratory phase of development, whereas the more precisely timed outgrowth of stable neurites from the spurs signals functional differentiation during synaptogenesis. Because spurs have also been observed in neurons of the mammalian brain, they may constitute a primordial synaptic organizer.

Fig. 1.

Extension of primary neurites. Photomicrographs of E7 dorsal P neurons filled with Sulforhodamine B at the earliest stage examined are shown. A, P cell from a ganglion showing axial growth cones on the anterior neurite with conspicuous lamellipodia. In this and subsequent figures, anterior is to theleft. B, P cell from a ganglion two segments rostral in the same embryo used in A. The posterior neurite has partially formed and can be seen alongside the electrode (right). Filopodia are seen primarily in the ganglionic regions and are relatively absent from the neurite passing through the anterior connective (left). A characteristic inflection is seen near the soma (arrow). Scale bars, 20 μm.

Fig. 2.

Contact between R and P cells and formation of terminals. In each micrograph, the P cells were filled with Rhodamine dextran, and in a, d, andf, the R cells were filled with Lucifer yellow using intracellular microelectrodes. a, Posterior branches of the R cell in an E8 embryo seen to follow the P cell branches to the periphery and posterior connective. The arrow indicates retraction of an anterior, lateral branch. b, Photomicrograph of an adult P neuron showing complex terminals with clusters of swellings. c, Enlargement ofb. d, e, g, Confocal images of R and P cell contact in an E13 embryo. Sites of contact (imaged in 40 × 1 μm steps) were observed on each terminal of the P cell (d; two indicated byarrows), seen on its own (e), and enlarged (g; arrowheads point to terminals). f, Confocal image of R and P cell processes in the adult. Note the many sites of contact. Scale bars, 20 μm.

Fig. 3.

Outgrowth and retraction of transient neurites and formation of spurs. Intracellular fills of P neurons with Lucifer yellow in E9 embryos are shown. A, Photomicrograph showing a transient neurite turning anteriorly at the midline (arrow). B, Confocal image of spurs remaining after the retraction of transient neurites (arrows). The cell was imaged in 20 × 1 μm steps. Note tripolar morphology at this stage. Scale bars, 20 μm.

Fig. 4.

Outgrowth of secondary branches. P cells were filled with Sulforhodamine B using whole-cell electrodes.A, V-shaped secondary branches (arrow) seen extending from a spur on the anterior primary neurite of a E11 P cell. B, Y-shaped secondary branches (arrow) with finer neurites (arrowhead) in a E14 P cell. Scale bars, 20 μm.

Fig. 5.

Development of voltage-sensitive currents. Whole-cell patch-clamp recordings using physiological solution reveal voltage-sensitive currents at the first outgrowth of neurites.A, Sample traces showing the voltage steps protocol (top) from E9 (middle) and E10 (bottom) embryos with voltage-sensitive currents.B, Development of voltage-sensitive currents in a group of sibling embryos; mean ± SD for three embryos for each measure. I_Na was estimated from the peak inward (downward) current, andI_K was estimated from the steady current level at the end of the depolarizing voltage steps.

Fig. 6.

Summary of synaptic activity. Histogram plotting the number of recordings obtained from P cells at different embryonic days is shown. Open bars represent recordings without synaptic activity, and filled bars denote the presence of spontaneous synaptic events. Note that at E11 a fraction of the P cells displayed synaptic activity for the first time.

Fig. 7.

Onset of spontaneous synaptic activity.Top, Recordings with high CsCl pipette solution from a P cell in an E10 embryo showed no spontaneous synaptic activity.Middle, Spontaneous activity was first observed in E11 embryos. Bottom, An expansion from the P cell recording at E11 is shown.

Fig. 8.

Interevent interval distribution. The frequency of events (i.e., the number occurring within each time bin) recorded from a P cell is plotted as a function of the interevent interval. The distribution was fit by an exponential function with a time constant of 250 msec. Events separated by <20 msec were excluded.

Fig. 9.

Effects of Cd on spontaneous events. A 10 sec recording from a P cell shows the spontaneous activity in the absence (top) or presence (bottom) of 100 μm Cd²⁺ added to the bath solution. Cd had no obvious blocking activity on the synaptic events up to 30 min after its application.

Fig. 10.

Amplitude, rise time, and decay time distributions of mIPSCs. Whole P cell measurements from a sample experiment for an E13 embryo with high KCl solution in the recording electrode are shown. The number of events is 350. A, Amplitude distribution of events with a mean of 17.2 pA and a median value of 15.8 pA. Minimum amplitude was 7.5 pA (5 pA detection limit), and maximum amplitude was 62.0 pA. Cumulative frequency is plotted as aline. An outlying group of high amplitude events (>50 pA) was observed in all embryos with mIPSCs. B, Rise time (20–80%) distribution with a mean of 1.9 msec and a median value of 1.8 msec. Cumulative frequency is plotted as a line.C, Scatter plot of rise time (20–80%) versus peak amplitude. Note the lack of a linear relationship.D, Mean time constant of decay (tau decay) of 18.8 msec, with a median value of 16.8 msec. Tau decay was best fit with two exponentials for ∼10% of total events in all embryos.