Regulation of synaptic connectivity: levels of Fasciclin II influence synaptic growth in the Drosophila CNS

Abstract

Much of our understanding of synaptogenesis comes from studies that deal with the development of the neuromuscular junction (NMJ). Although well studied, it is not clear how far the NMJ represents an adequate model for the formation of synapses within the CNS. Here we investigate the role of Fasciclin II (Fas II) in the development of synapses between identified motor neurons and cholinergic interneurons in the CNS of Drosophila. Fas II is a neural cell adhesion molecule homolog that is involved in both target selection and synaptic plasticity at the NMJ in Drosophila. In this study, we show that levels of Fas II are critical determinants of synapse formation and growth in the CNS. The initial establishment of synaptic contacts between these identified neurons is seemingly independent of Fas II. The subsequent proliferation of these synaptic connections that occurs postembryonically is, in contrast, significantly retarded by the absence of Fas II. Although the initial formation of synaptic connectivity between these neurons is seemingly independent of Fas II, we show that their formation is, nevertheless, significantly affected by manipulations that alter the relative balance of Fas II in the presynaptic and postsynaptic neurons. Increasing expression of Fas II in either the presynaptic or postsynaptic neurons, during embryogenesis, is sufficient to disrupt the normal level of synaptic connectivity that occurs between these neurons. This effect of Fas II is isoform specific and, moreover, phenocopies the disruption to synaptic connectivity observed previously after tetanus toxin light chain-dependent blockade of evoked synaptic vesicle release in these neurons.

Fig. 1.

Synaptic drive to the aCC/RP2 motor neurons.A, B, Whole-cell current-clamp recordings from either aCC or RP2 (aCC shown) in wild-type young first instar larvae show rhythmic depolarizations that are sufficient to evoke action potentials, which are more clearly visible in trace B. C, Synaptic depolarizations are first evident in late stage 17 embryos (labeled E) and increase in frequency during larval development (L1, first instar;L2, second instar). Values are mean ± SE;n ≥ 10.

Fig. 2.

A subpopulation of cholinergic interneurons coexpresses Fas II. Confocal sections (2 μm) through the ventral nerve cord (VNC) and brain lobe (Brain) in a late stage 17 embryo. Cholinergic neurons are visualized by anti-GFP (cha B19-GAL4 driving UAS-GFP;green), and Fas II expression is reported by a P element driving a nuclear β-galactosidase(β-gal antibody labeled red). A number of cholinergic cells coexpress Fas II in both the ventral nerve cord and brain lobe (merged image on right,arrowheads).

Fig. 3.

Synaptic proliferation during larval development requires Fas II. A, During the first 28 hr of larval life, the frequency of suprathreshold synaptic inputs recorded in aCC/RP2 increases approximately twofold (control line,fas^e93). In the absence of Fas II (fas^eB112), this developmental increase is significantly reduced (p ≤ 0.01). The increase remains normal, however, in Fas II hypomorphs (lines fas^e86 andfas^e76, respectively). Values are mean ± SE; n ≥ 8. B, Ultrastructural analysis reveals that the number of presynaptic terminals that contact aCC (see Materials and Methods) increases during the same period (fas^e93). In the absence of Fas II (fas^eB112), however, this increase fails to occur. C, D, In 28 hr larvae, the presynaptic terminals (arrows) seen to contact aCC are qualitatively similar, regardless of the presence (C) or absence (D) of Fas II. E, An example of a labeled profile of aCC that is not associated with a presynaptic input.

Fig. 4.

Increased levels of Fas II in either the presynaptic or postsynaptic neurons disrupt synaptogenesis.A shows the frequency of suprathreshold synaptic inputs recorded in aCC/RP2 in L1. control represents the average frequency seen in parental GAL4s (1407, RRK, and B19) and UAS transgenic lines (no individual line was significantly different from any other). Expression of Fas II (PEST+) in all neurons of the CNS (1407-GAL4) does not influence the frequency of synaptic inputs. However, selective expression of Fas II in either aCC/RP2 (RRK-GAL4) or presynaptic cholinergic (B19-GAL4) neurons significantly reduces input frequency (p ≤ 0.001 for both treatments). Simultaneous expression of Fas II in both presynaptic cholinergic neurons and aCC/RP2 (B19/RRK-GAL4s) does not reduce the frequency of synaptic drive. The effect of expressing Fas II in aCC/RP2 is rescued by the presence of GAL80 in these motor neurons (RRK-GAL4/GAL80). For all values, n ≥ 8; mean ± SE. B, Expression of Fas II (PEST+) in aCC/RP2 (RRK-GAL4) does not influence the frequency of suprathreshold synaptic drive recorded in the RP3 motor neuron (which does not express GAL4 in these larvae). However, combined expression in both aCC/RP2 and cholinergic interneurons (B19/RRK-GAL4s) results in a significant decrease (p ≤ 0.05; for explanation, see Results). C, Ultrastructural analysis reveals that expression of Fas II (PEST+) in aCC (RRK-GAL4) significantly reduces the number of presynaptic terminals observed to contact this neuron (p ≤ 0.05; χ² test). Simultaneous expression of Fas II in both aCC and cholinergic neurons (B19/RRK) does not, however, affect the number of presynaptic terminals that contact aCC. D, Expression of Fas II (PEST+) in aCC does not reduce the number of presynaptic terminals that contact RP3. Presynaptic terminals contacting this neuron (which does not express GAL4 in these larvae) are significantly reduced in number after the combined expression of Fas II in both aCC/RP2 and cholinergic neurons (p ≤ 0.05; χ² test).

Fig. 5.

Fas II expression in aCC/RP2 reduces the strength of synaptic inputs. Ai, Amplitude distribution of evoked synaptic currents recorded in voltage clamp (V_h −60 mV) from aCC/RP2 in control L1 (GAL4 and UAS parental lines). Current amplitude is normally (Gaussian) distributed with a peak amplitude of −102 ± 1.5 pA (n = 542). Aii, The amplitude distribution of synaptic currents recorded in aCC/RP2 is significantly reduced after expression of Fas II (PEST+) in just these neurons (p < 0.001). Under these conditions, the distribution is centered around a mean amplitude of −48 ± 1.3 pA (n = 487). B, Spontaneous currents (those which persist in the presence of TTX) show no significant difference in amplitude distribution attributable to expression of Fas II in aCC/RP2. In control L1 (Bi; GAL4 and UAS parental lines), currents are normally distributed around a mean of −5.2 ± 0.3 pA (n = 398), whereas after expression of Fas II in aCC/RP2 (Bii), the distribution mean is −6.3 ± 0.2 pA (n = 190). Individual currents were obtained from at least six neurons for each analysis.

Fig. 6.

Fas II is required for TeTxLC-induced reduction in synaptic inputs. Expression of TeTxLC in aCC/RP2 (RRK-GAL4) is sufficient to reduce the frequency of suprathreshold synaptic inputs to these neurons (Baines et al., 1999). However, in the absence of Fas II (fasII^eB112), the TeTxLC-induced reduction of input is diminished. For all values,n ≥ 7; mean ± SE. Similarity ofletters denotes statistical significance atp ≤ 0.05. Control larvae contained either the GAL4 or UAS transgenes (but not both) and normal Fas II levels.