The Journal

Morphological Plasticity Altered Excitability Vivian

Budnik,

Yi Zhong,

and

Chun-Fang

of Neuroscience,

of Motor Axons in Drosophila

November

1990,

fO(11):

3754-3766

Mutants with

Wu

Department of Biology, University of Iowa, Iowa City, Iowa 52242

An anatomical and electrophysiological study of Drosophila mutants has been made to determine the effect of altered electrical activity on the development and maintenance of larval neuromuscular junctions. We examined motor axon terminals of (1) hyperexcitable mutants Shaker (Sh), ether a go-go (eag), Hyperkinefic (Hk), and Dupbcafion of para+ (Dp para+); and (2) mutants with reduced excitability, no action potential (nap’=) and para/yfic (para”‘). Nerve terminals innervating larval body-wall muscles were visualized by using anti-HRP immunocytochemistry, which specifically stains neurons in insect species. In wild-type larvae, motor axon terminals were distributed in a sterotypic fashion. However, in combinations of eag and Sh alleles, the basic pattern of innervation was altered. There was an increase in both the number of higher-order axonal branches over the muscles and the number of varicosities on the neurites. A similar phenomenon was found in the double mutant Hk eag and, to a lesser extent, in Dp para+ and Dp para+ Sh mutants. It is known that at permissive temperature the nap, but not para”‘, mutation decreases excitability of larval motor axons and suppresses the behavioral phenotypes of Sh, eag, and Hk. In the mutant nap’= (reared at permissive temperature), a slight decrease in the extent of branching was observed. Yet, when combined with eag Sh, nap” completely reversed the morphological abnormality in eag Sh mutants. No such reversion was observed in param eag Sh mutants. The endogenous patterns of electrical activity at the neuromuscular junction were analyzed by extracellular recordings in a semi-intact larval preparation. Recordings from wild-type body-wall muscles revealed rhythmic bursts of spikes. In eag Sh mutants, this rhythmic activity was accompanied by or superimposed on periods of strong tonic activity. This abnormal pattern of activity could be partially suppressed by napl in combination with eag Sh.

The invertebrate CNS has long been viewed as having a rigid stereotypic organization that precludes much plasticity (see Murphey, 1986). In recent years, however, studiesof long-term physiological and anatomical changesin Aplysia during behavReceived Apr. 9, 1990; revised July 30, 1990; accepted Aug. 1, 1990. We are grateful to Susan Mikkelsen for her aid in the study of intersegmental differences of motor innervation. We also thank Drs. B. Ganetzky and M. Stem for providing us with the Dp para’ fly stocks and Drs. Michel Gho, Michael Gorczyca, and Mitsuyoshi Saito for helpful comments on the manuscript. Correspondence should be addressed to Vivian Budnik, Department ofZoology, Merrill Science Center, University of Massachusetts, Amherst, MA 01003. Copyright 0 1990 Society for Neuroscience 0270-6474/90/l 13754-15$03.00/O

ioral conditioning (Kandel et al., 1986; Bailey and Chen, 1988a,b), as well as studies of regeneration(Murphey and Lemere, 1984; Shepherd and Murphey, 1986; Loer et al., 1987; Loer and Kristan, 1989) have changedthis view. Instead, it has becomeapparent that, even though the behavioral repertoire of invertebrates is less complex than that of vertebrates, developmental and physiological plasticity is a common feature (reviewed in Lnenicka and Murphey, 1989;seealsoTechnau, 1984; Meinertzhagen, 1989). The mechanismsunderlying neuronal plasticity remain unknown in most cases.However, a factor that has been widely implicated in plastic processesis electrical activity (Hubel et al., 1977;Harris, 1981;Meyer, 1982; Sanesand Constantine-Paton, 1983;Lnenickaand Murphey, 1989;Schmidt and Tieman, 1989). In the optic tectum of vertebrates, for example, it has been shown that correlated activity in retinal ganglion cells determines the fine distribution of retinal terminals in the tectum. Any perturbation of this pattern of activity leadsto defects in the final pattern of connections and therefore in the size and/ or shapeof the receptive field of these neurons in the tectum (Harris, 1980; Meyer, 1982; Schmidt and Edwards, 1983). In the vertebrate neuromuscularjunction, it has also been shown that disruptions in the level of activity of motor axons lead to changesin morphology of axon terminals (Duchen and Strich, 1968; Brown and Ironton, 1977; Holland and Brown, 1980). Very little is known about how activity levels affect the development of neuronal connectivity in invertebrates (reviewed in Lnenicka and Murphey, 1989). Lnenicka et al. (1986) showed that chronic stimulation of relatively silent phasicmotoneurons in the crayfish changed the ultrastructure of its synaptic terminals. This change was reflected in an increasednumber of varicosities in the nerve projecting to the muscle, larger terminals, and greater synaptic contact area. This altered phenotype resembledthe properties of tonic motor axon terminals in arthropods (Atwood and Johnston, 1968;Atwood and Jahromi, 1978;Hill and Govind, 1981; Titmus, 1981). On the other hand, Cohan and Kater (1986) have shown that electrical activity suppresses neurite elongation and growth conemotility in some cultured Helisoma neurons. A genetic approach to the study of neuronal plasticity can be made in Drosophilaby usinga number of neural mutations that either eliminate nerve activity or increaseexcitability (Burg and Wu, 1986, 1989). We have initiated an anatomical and physiological study of neuromuscularjunctions in the hyperexcitable mutants-Shaker (Sh), ether a go-go (eag), and Hyperkinetic (Hk)-and in mutants with reduced excitability-no action potential (nap”) and paralytic (para”‘).

The Journal

Sh, eag, and Hk were isolated by their phenotype of anesthesia-induced leg shaking (Kaplan and Trout, 1969). The Sh gene has been well characterized at the molecular level and found to code for the Z, potassium-channelprotein (Bauman et al., 1987; Kamb et al., 1987; Papazian et al., 1987; Tempel et al., 1987). Sh mutant allelesincreasetransmitter releaseat the larval neuromuscularjunction (Jan et al., 1977; Ganetzky and Wu, 1982a)and decreaseor eliminate the transient K+-current Z, from pupal and larval muscles(Salkoff and Wyman, 1981; Wu and Ha&and, 1985). Mutants at the eag locus display spontaneousaction potentials in larval motor axons (Ganetzky and Wu, 1983).Voltage-clamp analysisoflarval musclecurrents revealsthat the delayed rectifier ZKand, in somealleles,I,, are affected (Wu et al., 1983;Y. Zhong and C.-F. Wu, unpublished observations). The double mutant eag Sh showssynergisticinteractions: both the releaseof neurotransmitter at the larval neuromuscularjunction and the degreeof adult leg shakingare greatly enhanced(Ganetzky and Wu, 1983). Hk mutants show abnormal neuronal activity in adult ventral ganglia (Ikeda and Kaplan, 1970) and stimulus-induced repetitive activity at the larval neuromuscular junction (Stem and Ganetzky, 1989). However, as for eag, the molecular nature of this mutation is still unknown. The mutants para (Suzuki et al., 1971; Siddiqi and Benzer, 1976) and nap (Wu et al., 1978) were isolated on the basisof their temperature-dependentparalytic phenotype. Recent molecular studies indicate that the para gene most probably correspondsto the structural sodium channel gene, and that nap appearsto be a regulatory gene, controlling the expression of para (Keman et al., 1989; Loughney et al., 1989).At permissive temperature, the mutant allele parazs’ appears physiologically normal, whereasnap’” mutants, becauseof a reduction in Na+ channel number (Kauvar, 1982; Jackson et al., 1984) already showalterations in axonal conduction even at room temperature (Wu and Ganetzky, 1980). At room temperature, nap can suppressthe shaking phenotype of eag Sh, as well as somephysiological defects of eag and Sh (reviewed in Ganetzky and Wu, 1985). Many of the above physiological studies have been carried out in the body-wall musclesof Drosophila larvae. In these muscles,4 classesof potassiumcurrents and a calcium current have been well characterized (Wu and Haugland, 1985; Gho and Mallart, 1986;Singh and Wu, 1989).Someof thesecurrents have also been describedat the single-channellevel (Zagotta et al., 1988; Komatsu et al., 1990; Gorczyca and Wu, 1991). Several mutations that disrupt musclecurrents, or alter neuromuscular transmission,have been characterized in the larval preparation (for review, seeGanetzky and Wu, 1986). In addition, anatomical aspectsof the development and distribution of axon terminals in thesemuscleshave beendescribed(Johansenet al., 1989a,b).In this study, we usedthe larval neuromuscularpreparation to examine the possibleeffect of altered activity on the morphology of motor axon terminals. We show that increasing excitability by simultaneousmutations in the eag and Sh genes or in the eag and Hk genes,or by augmenting the number of para+ genes,leadsto an increasein the ramification of branches on the muscles.In addition, there is a greaternumber of putative synaptic sites.These effects seenin eag Sh double mutants can be reversed by the mutation nap”. Although the mechanisms underlying the morphological changesare not known, theseresults provide an independent line of evidence for the involvement of nerve activity in shaping neuronal arborizations in

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invertebrates. In addition, they also provide background for possiblereinterpretation of the physiological defects observed on the neuromuscular junctions of these mutants (Jan et al., 1977; Ganetzky and Wu, 1982a,b;Stem and Ganetzky, 1989). Some of the results have appearedin abstract form (Budnik et al., 1989b).

Materials

and Methods

Fly stocks. All fly stockswererearedat roomtemperature (22-25°C)in standardDrosophila food. As a wild type, the strainCanton-Special

(CS)wasused.Chromosome markersare describedin Lindsleyand Grell (I 968).The mutationsusedarelistedbelow. eag’: Recessive ethyl methanesulfonate(EMS)-inducedmutationlo-

cated at map position l-48 (Kaplan and Trout, 1969). This mutation removes about 50% of I, (Wu et al., 1983;Y. Zhongand C.-F. Wu, unpublished observations). eag4Y This eag allele preferentially affects I, rather than ZKin larval muscles (Ganetzky and Wu, 1983; Y. Zhong and C.-F. Wu, unpublished observations). Hk’: Recessive EMS-induced mutation located at map position l-30 (Kaplan and Trout, 1969). This mutant shows reduced Z, (Wu and Ganetzky, 1988) and, at high-frequency stimulation, displays prolonged excitatory iunctional potentials (EJPs) at the larval neuromuscular iunction (Stern and Ganetzky, 1989). nati*: Isolated bv EMS mutaaenesis (Wu et al.. 1978). Flies bearing this mutation become paralyzedat 37°C due to action potentials block‘: ade. Maps to position 2-56. paral*‘: First described by Suzuki et al. (197 1). These mutants become paralyzed at 29°C. At this temperature, nerve conduction is completely blocked. Physiologically normal at permissive temperature (Wu and Ganetzky, 1980). ShKS’j3: Described by Jan et al. (1977). Shows prolonged EJPs at the larval neuromuscular junction and completely removes Z, in muscle. (For further physiological characterization, see Salkoff and Wyman, 198 1: Wu and Haualand. 1985: Haushland and Wu. 1990.) Sh;KofzO:Reduces-but does not eliminate Z,, in larval muscle. It also has abnormal neuromuscular transmission similar to ShKsf33(Ganetzky and Wu, 1983; Wu and Haugland, 1985; Timpe and Jan, 1987). Maps to position l-57.4. Dp(l:4) r+p. Translocation of the X-chromosome region 14A to 16A2 to the fourth chromosome. This duplication contains the entire para+ gene and also covers l(1) lnzj and Of(l) Pi7 (see below). In this paper, this duplication will be referred to as Dp para’. l(1) lDz3. This is a recessive lethal mutation that lies close to, but is pip from, para (Ganetzky, 1984), and that is covered by Dp(I:4) + “Of(l) rDf7.This is a deletion from 14F6 to 15A6 of the X chromosome that also lies close to Dara. This deletion is also covered bv. D&1:4) _i I r+P _ (Stem et al., 1990). The strains containing Dp(l:4) r+p, l(l) lDz3, and Of(l) P7 were provided to us by Drs. M. Stem and B. Ganetzky. Immunocytochemistry. Late third-instar larvae, staged by the morphology of the spiracles, were used for this study. Larval body-wall muscles were dissected in Ca+-free saline (128 mM NaCl, 2 mM KCl, 4 mM MgCl,, 5 mM HEPES, 35.5 mM sucrose, 5 mM EGTA). Samples were fixed either in nonalcoholic Bouin’s solution [25 ml formalin (from a 37.7% stock solution, Fisher), 5 ml glacial acetic acid, 75 ml saturated picric acid solution] or in paraformaldehyde fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2) for l-2 hr. We found that immunoreactivity was substantially higher and more reliable in Bouin’s fixed tissue. After fixation, samples were washed 2-5 times (15 min each) in 0.1 M phosphate buffer, (pH 7.2) containing 0.2 or 0.3% Triton X- 100 (PBT). Then they were incubated overnight in 1:200 anti-HRP (Sigma) diluted in PBT. After 3 washes with PBT, samples were incubated for 4 hr in 1:20 goat anti-rabbit HRP-conjugated IgG (Cappel), washed 2 times in PBT, 1 time in 0.2 M Tris buffer (pH, 7.6) and reacted using diaminobenzidine (DAB, 5 mg/ml) and 0.03% H,O,. Electron microscopy. Samples were dissected in Ca*+-free saline, as above, and fixed in modified Trump’s fixative [ 1% glutaraldehyde, 4OVo paraformaldehyde, 0.1 M cacodylate buffer, pH 7.0 (CB)] for 2 hr. Samples were then washed 3 times with CB and postfixed in 2% 0~0, for

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electrode (no suction applied). Signals were bandpass filtered at 100 Hz and 10 KHz and taped on a VCR recording system in conjunction with a pulse-code modulation unit (Neuro Data Instrument Corporation). All recordings were carried out in Drosophilanormal saline (same as Ca2+-free saline but with 1.8 mM CaCl, and no EGTA) at 19-20°C.

Results

Figure 1. Toluidine-blue-stained preparation of body wall muscles of third-&tar Drosophilalarva. Arrowheadsindicate (from left to right) longitudinal muscles 7, 6, 13, and 12 of the third abdominal segment. AI-A7 indicate the abdominal segments l-7. Note the CNS in the anterior (top) region, Scale bar = 200 pm. 30 min. After postfixation, samples were washed 3 times with CB and 3 times with distilled water and stained en blocin 2% uranyl acetate for 1 hr. Samples were then dehydrated in an acetone series and embedded in Spurr’s resin. Transverse thin sections were cut from muscles 6, 7, 12, and 13 (identified in thick sections; nomenclature for muscle identification is according to Crossley, 1978), using a diamond knife. Grids were poststained in 4% uranyl acetate (15-20 min) and 1% lead citrate (5-8 min) and visualized using a Zeiss EM 10 microscope. Toluidine-blue staining. To visualize the distribution of muscle nuclei, toluidine blue (TB) was used. Briefly, whole larvae were heat fixed for about 3 set in 60°C water. They were then dissected, stained for a few seconds in TB (6Oh borax, II boric acid, 1% toluidine blue), washed with water, and immediately photographed. Electrophysiology. Extracellular recordings from body-wall muscles 6, 7, 12, and 13 were made by a differential amplifier (DAM-S, WPI) and a capillary glass suction electrode with a tip diameter of 10-20 pm. The positive and negative leads were chlorided Ag wires. Electrical activity was recorded upon touching the muscle fibers with the glass

Distribution of anti-HRP immunoreactive terminals in wild type The body-wall musclesof larvae are composedof identifiable musclefibers arrangedin a segmentallyrepeatedpattern in the thoracic and abdominal segments(Fig. 1). For a description of this pattern and its development, seeCampos-Ortegaand Hartenstein (1985) and Johansenet al. (1989b). Immunocytochemical and physiological studieshave demonstrated that most of the innervation is glutamatergic (Jan and Jan, 1976; Johansen et al., 1989a). However, a subpopulation of muscle terminals also showsproctolin or octopamine immunoreactivity (Anderson et al., 1988; Halpem et al., 1988). Most electrophysiological studies in Drosophila larvae have beenperformed using a particular subsetof the body-wall muscles, the abdominal ventral longitudinal muscles6, 7, 12, and 13 (nomenclature of Crossley, 1978). In this paper, we have centered on these musclesto study the morphology of motor terminals in mutants with altered excitability. In order to visualize motor axon terminals, anti-HRP immunocytochemistry wasused.In insects,the anti-HRP antibody binds to antigens exclusively located in neuronal membranes (Jan and Jan, 1982). The pattern of distribution of these terminals on each identified muscle fiber was characteristic. The description below and resultsof experimentsin this paper were basedon analysesof muscles6, 7, 12, and 13 at the third abdominal segment(Fig. 1). This description is in generalagreement with the description of glutamatelike immunoreactivity at thesemusclesmade by Johansenet al. (1989a, b). Muscles 6 and 7. Anti-HRP immunoreactivity in thesemuscles was located in sparsely ramified branches in the lateral aspectofthese muscles(Figs.24 3A). A branch that liesbetween muscles6 and 7 gives rise to this innervation. Throughout this projection, aswell asin the projections describedbelow in muscles 12 and 13, enlargementsof the neurites, or varicosities, are observed (Fig. 2). In muscles6 and 7, most varicosities measure about 3-5 pm in diameter. However, someshort ramifications show small varicosities in the range of ~2 pm. Muscles 12 and 13. At these more lateral muscles,a nerve branch contactseachfiber at its internal (visceral) faceand gives off sparselyramified branchesthat run anteriorly and posteriorly throughout the length of the muscle (Figs. 2B, 34. Near the initial contact of the nerve with the muscle(proximal terminals), most neurites show large varicosities (3-5 pm). However, all distal neurites show varicosities of the small size (12 pm; Fig. 2B). Although most branchesemergingfrom the initial branch point were restricted to the domain of the particular muscle fiber, in many occasions,the distal portions of the branches extended for a few tens of microns into the adjacent segment, or into a neighboring muscle in the same segment (see, for example, Fig. 4A). This occurred more frequently in muscles of more anterior segments(30% of musclefibers in segment2, 2 1% of musclefibers in segment3, 5% of muscle fibers in segment 6). Motor axon terminals distribute in an anteroposteriorgradient. Even though the generalpattern of distribution of motor

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Figure 2. Innervationof muscles6 and 7 (A) and 12 and i3 (B) asrevealedby anti-HRPimmunccytochemistry, visualizedunderNomarski optics.Note the presence of prominentvaricositiesalongneurites.Arrowheads point to the large-typevaricosities,whereasarrows point to the

small-typevaricosities.SN, segmental nerve.Scalebar, 12pm.

terminals is similar in all abdominal segments,it was noticed that anterior segmentsshoweda higher degreeof neurite ramification. The neuritesof posterior segmentsdisplayed very little or no ramification, and the number ofvaricosities wasalsolower

(Fig. 4). This was particularly clear in muscles12 and 13 from segment6, whereon many occasionsoneof the distalprojections was missing. There was also a continuous change in the size and width of muscle fibers: posterior musclesbecome longer

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iI

i

wild typcii

\12 -.

3. Camera lucida drawings of anti-HRP staining of axon terminals on muscles 6, 7, 12, and 13 of wild-type (A), eug’” Sh’Ko’zo double mutants (II), and eag’” SkKo’zO nap mutants (C). Notice that, in eag’ pM SkKO’*O, the extent of ramification of neurites in muscles 12 and 13 is increased. This alteration is suppressed by nap. Scale bar, 50 pm.

Figure

and thinner (seeFigs. 1, 4A). Even after data were normalized using the surface area of the muscle fiber as drawn in camera lucida tracings(Fig. 4A), the anteroposterior differencepersisted (Fig. 4B, C’). Several studieshave suggestedthat varicosities are regionsof synaptic contacts (see,for example, Lnenicka et al., 1986). In order to addressthis possibility, we studied transverse sections of thesemusclesat the electron microscopicallevel. One-micronthick sectionswere used to determine the presenceof a varicosity. Then, the tissue was thin sectioned and stained with uranyl acetate and lead citrate. We analyzed 2 wild-type samples. We found that varicosities coincided with muscle sites showing synaptic terminals (Fig. 5; for descriptions of the ultrastructure of neuromuscularjunctions in other insect species, seeOsborne, 1975). These terminals were located in the peripheral sarcoplasmas invaginations into the muscle surface. Most terminals were characterized by the presenceof 30-nmdiameter clear vesicles,aswell asby a variable number of synaptic specializations in the presynaptic membrance (Fig. 54). Thesevesiclesweremore concentratedin the region surrounding the synaptic specialization and seemedto be connected by a fibrous material. Terminals were separatedfrom the postsynaptic membranceby a cleft of about 15-20 nm. At the postsynaptic region, and surrounding the whole terminal, a well-developed subsynaptic reticulum was observed (Fig. 5A).

Hyperexcitable double mutants eag Sh show an increased number of branchesand presumptivesynaptic sites In order to investigate the effect of mutations that increased excitability, we studied double-mutant combinations of 2 different alleles of eag and Sh. We found that, in both eaepM innervation wasaltered (Fig. 3). The changescould be described asa substantial ramification of neurites throughout the muscle fiber. Analysis of these ramifications revealed that the number of higher order brancheswas particularly increased(Figs. 6, 7). In the wild type the number of primary brancheswas7.2 -t 0.2 (41%) the number of secondarybrancheswas 9.3 + 1.1 (54%) and the number of tertiary branches was 0.9 + 0.3 (5%). No quaternary brancheswere observed in all 9 samplesanalyzed. In eag’ ShKsJ3j the number of primary and secondarybranches was 8.1 f 0.6 (3 1%) and 11.8 + 1.1 (46%), respectively, and did not differ significantly from the wild type. In contrast, the number of tertiary (4.7 rf: 0.5, 18%)and quaternary (1.3 + 0.4, 5%) brancheswassignificantly increased(p < 0.05). In addition, the number and, in some cases,the density, of varicosities in the neurites was also increased(Fig. 8). The increasein the number of branchesand varicosities was most noticeablein neurites containing predominantly the smallsized varicosities. Therefore, the effect was particularly clear in muscles12 and 13 (Figs. 6, 8A). However, the samepattern of

The Journal

alterations, though less dramatic, was observed in muscles 6 and 7 of eug’ ShKs’j-’ mutants. In these muscles (6, 7) only the number of varicosities was analyzed, because the degree of ramification of neurites was difficult to quantify (Fig. 8B). No changes in the segmental or muscle specificity of the innervation were found in this and all other mutants analyzed in this study. Only a slight but statistically significant decrease in the extent of branching was observed in temperature-sensitive napIs larvae, when reared at permissive temperature (Figs. 7C, 9). However, nap” in combination with eacpM ShrKofzOcompletely reversed the eag Sh mutant phenotype in the body-wall motor axons (Figs. 3C, 7B, 8). In contrast, in eagdPMpatars’ SkKo”O mutants reared at permissive temperature, the pattern of arborization of motor axons remained similar to that observed in eag Sh (Fig. 8). Only a slight but statistically insignificant increase in the number of branches and varicosities was observed in the single mutants eag and Sh (Fig. 9). In vertebrate skeletal muscles, it has been noticed that, during synapse formation, myonuclei accumulate under the nerve terminal, in positions that coincided with clusters of ACh receptors (Nakai, 1968; Kelly and Zacks, 1969). Induction of ACh-receptor cluster formation elicited nuclei movements towards the newly formed cluster (Englander and Rubin, 1987). In Drosophila body-wall muscles, glutamate receptors seem to localize in regions of the muscle that are apposed to neurites (Johansen et al., 1989a.). Therefore, it was important to ascertain if the changes in distribution of varicosities in hyperexcitable mutants were correlated with changes of nuclear distribution. As in most muscles, the body-wall muscles ofDrosophila larvae are polynucleated. These nuclei are distributed superficially in the internally facing (visceral) sarcolemma in a fashion characteristic for each muscle type. In muscles 7 and 13 of the third segment, for example, they distribute in a single row along the anteroposterior axis of the fiber (Fig. 10). In muscle 6, in contrast, nuclei are distributed in 2 alternating rows. In muscle 12, most nuclei were concentrated in the anterior half of the muscle fiber, often surrounding the region of contact of the nerve with the muscle fiber (Fig. 10). The distribution of nuclei in each segment was not identical. For example, in more posterior segments, the double row apparent in muscle 6 was transformed to a single row of nuclei. No change in the distribution or number of these nuclei was detected in any of the excitability mutants analyzed in this study. Other mutants with increased excitability show alterations similar to eag Sh Similar to the 2 eug Sh mutants, the appearance of nerve terminals in Hk’ eag’ larvae was also altered. Again, the alteration was readily apparent in counts of the number of branches and varicosities, which showed an increase similar to that observed in eag Sh mutants (Fig. 11). Because the molecular identity of the eag and Hk gene products is unknown, we wanted to address the question of whether increasing excitability by using other better-defined mutations would also produce the same changes in the morphology of motor axons. Stem et al. (1990) have demonstrated that increasing the number of normal para genes by introducing duplications of the gene (Dp para+) has 2 prominent effects. It suppresses the temperature-sensitive paralysis of napts, presumably by restoring the Na+ channel density, and it enhances the leg-shaking phenotype of Sh Ks*33,resulting in a behavioral phenotype similar to eag’ ShKs”‘.

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Figure 4. Anteroposterior gradient in extent of branching and number of varicosities of muscles 6, 7, 12, and 13. A, Camera lucida drawings of muscles 6,7, 12, and 13 of an anterior segment (A2) and of a posterior segment (A6). Scale bar, 50 pm. B and C, Number of branches and varicosities in muscles 6, 7, 12, and 13 in A2 and A6. B, Number of varicosities per unit area x lo3 in muscles 6 and 7 and 12 and 13. C, Number of branches per unit area x 1O4in muscles 12 and 13. Data are expressed as mean + SEM; n = 10. Note the decrease in both the number of varicosities and branches in the posterior segment. We examined nerve terminals of male larvae that contained at least 1 extra copy of the normal para+ gene [P; Dp(l:4) r+p]. In addition, we analyzed a strain containing the mutation ShKs1j3 in addition to the Dp para+ [Of(l) P7 ShKsljJ; Dp(l:4) r+p]. We found that, in both strains, the number of branches and varicosities was significantly increased, but to a lesser extent than that of eag Sh or Hk eag mutants (Fig. 11). Closer analysis of the data revealed a high variability not observed in any of the other strains analyzed. There appeared to be a group of samples with a phenotype similar to wild type, and a group of samples similar to eug Sh or Hk eag (Fig. 11). This could be the result of either variable penetrance or a different number ofpara+ gene copies (see Discussion). Endogenous pattern of activity in the body-wall muscles Many studies of eag, Sh, and Hk, as well as of their doublemutant combinations, have shown electrophysiological char-

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Figure 5. Electronmicrographs of varicosities.A, A lower-magnification view showingan axon terminal(A) containing30-nmclearvesicles(v) reticulum.B, Higher-magnification view anda regionof presumptivesynapticcontact(arrowhead). 44,muscle;mi, mitochondria;R, subsynaptic (arrowhead). Scale of a synapticregionshowingclearsynapticvesiclesaswell asan electron-dense body typical of insectneuromuscular synapses bar: 0.6 pm,A; 0.1 pm, B.

acteristics consistent with hyperexcitable behavioral phenotypes. However, most, if not all, of these studies have been conducted under conditions where the input from the CNS had been severed. In addition, many of the hyperexcitable phenotypes have been studied at low calcium concentrations (Ganetzky and Wu, 1983), at which reduced muscle contractions presentlesstechnical problemsfor intracellular EJP recordings. As a result of the striking changesin the morphology of axon terminals observedin thesemutants, we were interested in verifying nerve hyperexcitability in thesemutants at normal calcium concentration and with normal CNS inputs. We recorded from the musclesin a semi-intact preparation with the nerve connection between the CNS and the muscleleft intact. Under theseconditions, thesesupercontracting musclescontract considerably, making intracellular recordingstechnically very difficult. However, we found that the CNS-generatedpattern as reflectedby the muscleactivity could be recorded extracellularly for 5-20 min. Figure 12 showsthe typical pattern of activity observed in the wild type, which was composedof a rhythmic pattern of burstingactivity. Eachburst consistedof a train of nerve-evoked spikesin the muscles.These spikescould correspondto action potentials at the muscleand/or to EJPs (Yamaoka and Ikeda 1988; M. Gho, personalcommunication). We believe that these spikeswere nerve evoked and not spontaneousmyogenic activity becausethey were completely blocked by TTX. Inward currents in these musclesare carried by Ca*+ (Gho and Mallart, 1986; Singh and Wu, 1989), but we observed no such spikesin wild-type and mutant larvae at normal Ca*+ concentrations in the absenceof CNS input. Periodsof bursting activity, usually

lasting on the order of minutes, were followed by periods of quiescence.Great variability was found from preparation to preparation regarding duration of bursts, frequency of spikes within a burst, duration of periods of bursting activity, and duration of periodsof silence.Therefore, we did not attempt to quantify further these parameters of the pattern. Figure 13A shows the bursting pattern observed in 3 different wild-type preparations. A common feature of most wild-type patterns recorded wastheir predominantly bursting nature. In eag’” ShKoi20mutants, bursting activity wasalsoobserved in the majority of the musclefibers. However, in contrast to the wild type, periods of strong tonic activity, not organized into bursts, were also observed. This tonic activity was either superimposedon the bursting activity or observed alone. Figure 13B shows representative examples of patterns observed in 3 different eaePM StiKolzOpreparations. In order to characterize the distribution of patterns in these mutants, muscleactivity observedin a period of 5-20 min was classifiedas follows. Bursting (B): at least 3 bursts of spikesin approximately a 3-min period; all spikesgrouped into bursts. Bursting with some tonic component (B/T): spikes mostly grouped in bursts, but a low level of tonic activity (spikesnot grouped into bursts) also observed. Mixed tonic and bursting activity (T+B): Strong tonic nonbursting activity accompaniedby periods of bursting activity, or the tonic activity seemsto be superimposedon bursting activity. Tonic(T): Activity is purely tonic with no clear signsof bursting organization.

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Figure 6. HRP immunoreactivity of neurites containing small-type varicosities (arrowheads) in CS (A) and eug’ LGFJ3 (B) in distal axonal region of muscle 12. Notice the increased ramification of neurites in eug’ Shmi3’. Scale bar, 12 pm.

As shownin Figure 14, in both eaepMShKo120 and Hk’ eag’, there is an increasein the percentageof fibers that show some level of activity not grouped into bursts (categoriesB/T and T+B). This is in contrast with wild-type muscle fibers where activity is mostly organized into bursts (category B). These results show that, in addition to the increasedneurotransmitter releasethat is observed in these hyperexcitable mutants (Ganetzky and Wu, 1983), there are also alterations in the pattern of activity of the motoneurons. However, we found that in

ShKs”3up para+ mutants, though there wasa dramatic increase in the amplitude of EJPs (Stern et al., 1990), the pattern of activity at the muscleswasvery similar to wild type (Fig. 140). In the morphological analysis of nerve terminals, we found that nap could suppressthe increasednumber of branchesand varicosities of eatpM ShlKoIzo.In addition, nap hasbeenfound to suppresssomebehavioral and physiologicalabnormalitiesof eag and Sh (Ganetzky and Wu, 1982a,b, 1983). Therefore, it was relevant to investigate whether the abnormalities in the

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C eag Sh nap 0

Figure 7. Number

of primary,

sec-

ondary, tertiary, and quaternary branchesin muscles12and 13in wild type(n = 9) andeug”” SkKoJzO (n = 9; A), eug’” StiKoJ2* nap (n = 8; B), and napl (n = 7; c). Data areexpressed as mean+ SEM. Note that the increase in branchingobservedin eag’” Sh’Ko’*O is due to an increaseof higher-order branches. Alsonoticethat higher-order branches arereducedin nap mutants.

branch

A 400.

40

l

30

20

cl 8 5 s .z z

10

branch

order

b f z

0

order

branch

order

pattern of activity of motor axons could alsobe rescuedby nap”. As shown in Figures 13C and 14, the hyperexcitable pattern of activity observedin eagpM Sh”0120wasonly partially suppressed by nap’“. While there was an increasein the number of fibers with purely bursting activity, and a decreasein the number of fibers showing strong tonic activity (T+B), the distribution of activity types was not completely like the wild type. In most musclefibers, bursting activity wasreadily apparent. However, in many fibers, a low level of abnormal tonic activity was also observed (Fig. 14). In the singlemutants nap” and eafpM (n = 12 and 11 fibers, respectively; data not shown), the distribution of activity types wasalmost asthe wild type, though in eag’pM,there wasa slight increasein the number of muscle fibers that displayed a low level of tonic activity in addition to the bursting activity (B/T type).

Discussion In this article, we report the effects of mutations that alter neuronal excitability on the morphology of motor axon innervation. 4001

Figure 8. Graphicalrepresentation of numberof varicositiesandtotal

numberof branches in muscles12and 13(A) and in muscles6 and 7 (B). Note the increasein the numberof varicositiesin 2 differenteug S/zdoublemutants.napt, unlikeparuts’, in combinationwith eug 9, completelyreversedthisalteredphenotype.Dataareexpressed asmean isindicatednearthe error bars. Means f SEM. The number of samples werecomparedto wild type usingthe Student’st test.*, p < 0.05; **, p < 0.005;

***,

p < 0.0005.

!I 1 a8

a 5 5

D

Figure 9. Numberof branches andvaricositiesin singlemutants.Data is indicatednear expressed asmean+ SEM. The number of samples the error bars. *, p < 0.05.

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FigureI I. Numberof branches andvaricositiesof muscles 12and 13 in CS,Dp para+,Shhs’rjDppara+,Hk eag’and Hk. Each point in this graph corresponds to counts in an individual sample. Notice the variability of Dp para+and Dp para+ShKsIJ3 data. **, p < 0.005;***, p c

0.0005.

Figure 10. Distribution of nuclei (arrowheads) in muscles 6, 7, 12, and 13of the abdominalsegments A2 andA3, asrevealedby toluidine-blue staining.Scalebar, 80 brn. We used the body-wall musclesof Drosophila larvae because their physiology, anatomy, and development have been well characterized (Ganetzky and Wu, 1986;Johansenet al., 1989b). Anti-HRP staining of axon terminals revealed that they displayed varicosities that could be classifiedinto 2 populations according to their size (small type and large type; seealso Johansenet al., 1989a).Ultramicroscopical analysisrevealed that varicosities coincided with synaptic sites.However, becausewe did not serially section the tissue, we do not know whether varicosities are exclusive sitesof synapsesor whether there are synapsesat other neuritic sites, as well., In crustaceanneuromuscularjunctions, it has been shown that, while varicosities are the region where most ultrastructural synaptic features are found, these are also seenat extravaricosity sites(Lnenicka et al., 1986). Studies are in progressto determine if the sameis the casein Drosophila. We found that, even though the distribution of axon terminals seemedto be characteristic for eachidentifiable musclefiber in different segments,there were clear differencesamongbody segments. The degreeof branching aswell as the number of varicositiesdecreasedin more posterior segments.These intersegmentaldifferencescould be the result of a developmentalgradient at a particular period of synaptogenesis.Alternatively, it could be the result of functional differences,for example, an anteroposterior gradient in the levels of activity of neuromuscular junctions, which in turn could result in variations in the size of the projection and presumably in the number of synaptic sites.

We centered our study exclusively on the third abdominal segment. An interesting observation is that each segmentis mainly innervated by the correspondingsegmentalnerve, but that terminal neuritescan crossthe segmentalboundariesand innervate the distal portions of musclesof adjacentsegments.This implies that there is not strict segmentalspecificity for the body-wall muscleinnervation. Influence of activity on motoneuron terminal arbors Comparisonsof wild-type axon terminals with terminals of several hyperexcitable mutants revealed a significant increasein

Figure12. Extracellular recordings of CNS-evoked responses in larval body-wall muscle 6. Similar activity was observed in muscles 7, 12. and 13. In this preparation, connections between muscles and CNS were left intact (see Fig. 11.Recordinas were carried out at 19-20°C in normal saline. Veitical bar,’150 PCV, h&zontal bar, 5 set in A, 1 set in B, 50 msec in C.

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phenotype presumably by increasing the number of sodium channels(Stem et al., 1990). Even though the number of varicosities and branchesin both cs Dp para+ and Sh Dp para+ sampleswassignificantly higher than the wild type, we observedgreat variability in the morphological data. There appeared to be some samplesvery similar to the wild type, and others with a phenotype similar to eagSh or Hk , ,h eag. This variability is possibly related to the uncertainty in the number of duplicated para+ genespresent in the two strains used [l(l) P; Dp(1,4) r+p or Of(l) rDz3;Dp(1,4) r+p]. Their genetic compositions assurethat at least 1 extra copy of para’ (in addition to the one normally presenton the X chromosome) will be present in males(Stern et al., 1990). This is so because this duplication covers l(1) 1D23 and Of(l) rDi7that are otherwise B lethal. However, there could be either 1 extra copy of the para+ eag Sh genein heterozygotes or 2 extra copiesin flies homozygousfor Dp(1,4) r+p. It is not possibleto distinguish larvae of these 2 genotypes. The fact that hyperexcitability derived from different molecular defects in eag Sh, Hk eag, Dp para+, and Sh Dp para+ producessimilar morphological alterations indicates a possible activity-dependent mechanism that could regulate the growth or maintenance of the motor terminals. Similar observations have been reported in the neuromuscular synapsesof crustaceans(Inenicka et al., 1986) where increasingthe level of activity by chronic stimulation of a motor axon also produced an increasein the number of varicosities at the terminals. C Further evidence consistent with our hypothesis of activitydependentmorphological changeis that the reducedexcitability causedby nap” reversed the effect of eag Sh on motor axon terminals. This mutant has been shown to reverse hyperexcitable phenotypes of eag, Sh, and Dp para+ physiologically and behaviorally (Ganetzky and Wu, 1985; Stem et al., 1990),presumably by decreasingthe expressionof the para+ gene,thereby decreasingthe number of sodiumchannels(Kauvar, 1982;Jackson et al., 1984). It is interesting to note that the naptsmutation alone showsa decreasein the degreeof branching in comparison to the wild type. This could indicate that reduced excitability 20s generateseffects opposite to those causedby hyperexcitability in motor axon terminal morphology. In contrast to nap”, para’“’ Figure 13. Patternsof activity in wild type (A), eag’” Sh’K0’20 (B), did not reverse the eag Sh phenotype. This is not surprising andeag’” SkK0’20 nap*” (C’).Eachtrace in A, II, and C is a recording of a differentlarval preparationrepresenting a typicalpatternobserved. becauseno physiologicaldefectshave beenfound inparatslwhen Noticethat in wild type, CNS-drivenspikesaregroupedin bursts.In reared and testedat room temperature (Wu and Ganetzky, 1980; contrast,in eag*” S&Ko’20, tonic activity is dominant,thoughit is Kauvar, 1982; Jackson et al., 1984). accompanied by periodsof burstingactivity. Most eag”” S&Ko’zo nap An intriguing observation is that the changesobservedin the preparations showa burstingactivity similarto wild type.However,a largeproportionof musclefibersin thisgenotypestill showalow degree number of higher-order branches and varicosities were most dramatically expressedin neurites containing the smaller type of abnormaltonic activity. of varicosities. At present, it is not known whether there is a functional difference between the larger- and smaller-type varthe extent of branching, in particular, higher-order branches, icosities. Studies are in progressto determine if the 2 types can and an increasein the number of varicosities. This was most be differentiated ultrastructurally. A speculation that can be dramatic in the double mutants eag Sh and Hk eag, and to a made is that neurites containing the smallertype of varicosities lesserextent, in Dp para+ and Sh Dp para+. None of the single are plastic portions of motor terminals. Theseregionscould be mutants eag,Sh, or Hk showedstatistically significant difference the sitesat which the size of the projection and the number of from the wild type. It is interesting to note that the mutants eag synapsesare regulated,for example, during the dramatic growth Sh, Hk eag, Dp para’, and Sh Dp para+ have many common that the musclesundergo throughout the larval stages. physiological and behavioral abnormalities related to hyperOur results are in contrast with what was observed by Burg excitability (Ganetzky and Wu, 1983; Stem and Ganetzky, 1989; and Wu (1986, 1989) in sensory neurons of adult Drosophila. Stem et al., 1990). However, the mechanism by which they Genetic mosaicswere generatedin which a singlesensory-bristle increaseexcitability is different. For example, eag Sh mutants complex was genotypically eag Sh or parals’ nap”. However, confer hyperexcitability by decreasingpotassium currents (Z, analysis of the morphology of the sensoryneuron dendritic arand ZK),whereasDp para+ mutants produce an hyperexcitable borization revealed no obvious abnormality with either mutant. A

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A Hk eag

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T

B

Several alternative explanations for these results can be made. First, it is possible that activity-dependent changes of neural development are restricted to a selected population of neurons. In fact, sensory receptors are epithelial in origin and, thus, may differ from motoneurons derived from central neuroblasts. A second possibility is that, in order to produce changes in neuronal morphology and synaptogenesis, it is necessary for the mutation to elicit changes in both the presynaptic and postsynaptic sites. In their work, only the sensory neuron and not the postsynaptic target was most likely mutant as indicated by reflex behavior control of neighboring bristles (Burg and Wu, 1986, 1989). A similar phenomenon, an increase in the extent of branching and the number of varicosities, was observed in a serotonin nerve projection to the digestive system of Drosophila larvae (Budnik et al., 1989a). This change was observed in larvae that were genetically deficient in serotonin and dopamine. It would be interesting to investigate if this change was also an activityrelated phenomenon generated by the lack of neurotransmitter action. Unfortunately, nothing is known about the electrophysiology of that enteric serotonin projection. The effect of activity on the innervation of neuromuscular junctions has been studied in vertebrates. It has been found that motoneurons are saved from cell death by pre- or postsynaptic activity block that results in polyinnervation of the muscles (Laing and Prestige, 1978; Pittman and Oppenheim, 1979; Oppenheim, 198 1). Conversely, electrical stimulation of the nerve results in enhanced neuronal cell death (Oppenheim and Nuiiez, 1982). In Drosophila, the fact that intracellular recordings reveal 2 EJP sizes with different stimulation thresholds (Jan and Jan, 1976) suggests the presence of at least 2 motoneurons. However, we found that the increase in branching observed in hyperex-

0

0

Figure 14. Distribution of muscle fibers with bursting (B), tonic (T), or a combination of both patterns of activity (B/T or T+B; see text) in wild type (n = 35 fibers) and CXZ~“~~SkKoJzO(n = 34 fibers; A), Hk’ eag’ (n = 32 fibers; B), eug’” SkKofzo naps (n = 32 fibers; C), and ShKSiJ* Dp para+(n = 30 fibers; D).

citable mutants was in higher-order branchesand not in the number of primary branches or branches emerging from the nerve (Fig. 7). This suggeststhat the increasedsize of the projection is probably not due to changesin the number of motoneurons innervating the muscles. In adult vertebrate skeletal muscle, it has been found that blocking pre- or postsynaptic activity produces sprouting of motor endings(Brown and Ironton, 1977; Holland and Brown, 1980). We found that increasingrather than blocking activity seemsto produce an increasein the size of muscleprojections in Drosophila. Furthermore, the mutation nap, which decreases excitability, slightly reduces the branching of the projection. Similar to our studies, in crustaceanneuromuscularjunctions, an increasein the number of varicosities as a result of chronic stimulation hasbeen reported (Lnenicka et al., 1986).One possibility to explain the observation in vertebrates is that, in those studies,activity was drastically blocked. In this and crustacean studies, only the activity level was modified. Possiblemechanisms A question that remains to be answeredis what aspectof neuronal activity could produce the morphological changesobserved. Many studieshave shown that synergistic interactions betweeneagand Sh or betweenHk and eagincreasethe amount of neurotransmitter releasedmany fold. In addition, it hasbeen shown that these mutants show repetitive spontaneousaction potentials (Ganetzky and Wu, 1983, 1985; Zhong and Wu, unpublished observations). In this report, the endogenous(presumably neurally evoked) activity wasrecorded extracellularly, and we were not able to measureparameterssuchasthe amount of neurotransmitter releasedor the size of the postsynapticpotential. However, we were able to obtain information about the

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CNS-evoked temporal pattern of activity. We found that most wild-type muscle fibers displayed a rhythmic, bursting pattern of activity. In contrast, in most eugSh and Hk eugmusclefibers, this bursting pattern was accompaniedby a strong tonic (nonbursting) activity. Again, as with the morphological changes observed, Hap’” also reversed the alterations in the pattern of activity in eag Sh. However, in contrast to the morphological changes,this reversion was only partial. In eag Sh nap triple mutants, the predominant type of activity was similar to that of the wild type (bursting), but a low level of abnormal tonic activity was also present. These findings suggestthat the level of activity (frequency of firing, size of the response,etc.) is not the only factor to be consideredin activity-dependent changes, but that the temporal parameters need also to be taken into account. Even though the implication is that hyperexcitability generates hyperinnervation, the mechanismsunderlying this phenomenon are unknown. One possibility is that activity is necessaryto stabilize newly generatedsynapsesand net&es. In the presenceof relatively low levels of activity, the probability that a given synapsewould be stabilized is low, and therefore only a fraction of synapsesare formed (Changeaux and Danchin, 1976).Another mechanismis that there are trophic interactions betweennerve and muscle.For example, atrophic factor (which could be the neurotransmitter itself) whosereleaseand/or transport is activity dependent could mediate both the increasein synaptic sitesand the branching and growth of net&es. This suggestionseemsto have somesupport in certain crayfish motoneurons. Lnenicka and Atwood (1989) applied an axonal conduction block to a small portion of a crayfish motor nerve and chronically stimulatedthe unblocked portion of the nerve, either proximal (near the neuronal cell body) or distal (near the neuromuscular synapse)to the block. They found that long-term physiologicalchangesin the motoneuron nerve terminal activity (from phasicto tonic) could be produced only if the activity of the motoneuron cell body wasaltered. Changesof activity only in the axon and synaptic terminals did not produce any longterm change. In some vertebrate preparations, neuronal excitation is known to bring about gene expressionin the nucleus via changesin the levels of second messengers(Morgan and Ccrran, 1989). In vertebrates, it has been proposed that synapsesundergo continuous turnover. In neuromuscularjunctions, presynaptic terminals unapposed to postsynaptic specializations (newly formed terminals), and postsynaptic specializationsunapposed by presynaptic terminals (regressingsynapses)are normally seen (Wemig et al., 1980; Anzil et al., 1984; Wemig and Fisher, 1986), though the molecular mechanismsthat might regulate this dynamic processare still unknown. In the present study, we only looked at neuromuscularjunctions of third-instar larvae. Therefore, we do not know the time courseof the changes observed, for example, whether in the wild type there could be an early period of hyperinnervation followed by pruning, and whether this pruning is altered in hyperexcitable mutants. However, Johansenet al. (1989b) have looked at the embryonic development of the innervation of the body-wall muscles.At leastup to first instar, they did not seeany profuse ramification of neurites like the onesobserved in this report. In this regard, it is interestingto note that we found an anteroposteriorgradient in the degreeof branching of nerve terminals. A parallel gradient in the levels of activity at certain developmental stagescould explain these segmentaldifferences. This hypothesis could be

tested in our systemby, for example, eliminating nerve activity in temperature-sensitive mutants for a relatively short period and examining the changesproduced in the projections. Basedon our results, it can be suggestedthat the number of synaptic sites and the degree of branching of a given neuron could be the result of the degree of electrical activity of that neuron. Such a mechanism could provide the basis for both developmental and physiologicalplasticity. In Drosophila, a collection of mutations that affect different cellular growth and regulation mechanismsis available for further exploration of this activity-dependent phenomenon. References AndersonMS, HalpemME, Keshishian H (1988) Identificationof the neuropeptidetransmitterproctolin in Drosophila larvae: characterization of fiber-specificneuromuscular endings.J Neurosci8:242255. Anzil AP, BieserA, WemigA (1984) Light andelectronmicroscopic identificationof nerveterminalsproutingand retractionin normal adult frog muscle.J Physiol(Lond) 350:393-399. AtwoodHL, JahromiSS (1978) Fast-axonsynapses in crablegmuscle. J Neurobiol9: l-l 5. Atwood HL, JohnstonHS (1968) Neuromuscular synapses of a crab motor axon. J ExpZoo1167:457-470. BaileyCH, ChenM (1988a)Long-termmemoryin Aplysiu modulates the total numberof varicositiesof singleidentifiedsensoryneurons. Proc Nat1Acad SciUSA 8512373-2377. Bailey CH, ChenM (1988b) Long-termsensitizationin Aplysiu increases the numberof presynapticcontactsonto the identifiedgill motor neuronL7. ProcNat1AcadSci USA 85:9356-9359. BaumannA, Krah-Jentgens I, Muller F, Muller-HoltkampF, SiegelR, KecskemethyN, CasalJ, FerrusJ, Pongs0 (1987) Molecularorganizationof the maternaleffect regionof the Shaker complexof Drosophila: characterization of an IA channeltranscriptwith homologyto vertebrateNa+channel.EMBO J 6:3419-3429. Brown MC, Ironton R (1977) Motoneuronesproutinginducedby prlongedtetrodotoxinblock of nerve action potential.Nature265: 459-461. Budnik V, Wu C-F, and White K (1989a) Altered branchingof serotonin-containingneuronsin Drosophila mutantsunableto synthesizeserotoninanddopamine.J Neurosci912866-2877. Budnik V, ZhongY, Wu C-F (1989b) Projectionof motor axonsin hyperexcitableDrosophila mutants.Sot NeurosciAbstr 15:570. BurgMG, Wu C-F (1986) Differentiationand centralprojectionsof peripheralsensorycellswith action-potential blockin Drosophila mosaics.J Neurosci612968-2976. BurgMG, Wu C-F (1989) Centralprojectionsof peripheralmechanosensorycellswith increased excitability in Drosophila mosaics. Dev Biol 131:505-514. Campos-Ortega JA, HartensteinV (1985) The embryonicdevelopmentof Drosophila melanogaster. New York: Springer. Changeaux J-P,DanchinA (1976) Selectivestabilizationofdeveloping synapses asa mechanism for the specificationof neuronalnetworks. Nature264:705-712. CohanCS, Kater SB (1986) Suppression of neuriteelongationand growthconemotility by electricalactivity. Science232:1638-1640. CrossleyCA (1978) The morphologyand developmentof the Drosophila muscularsystem.In: Thegenetics andbiologyof Drosophila, Vol 2b (AshbumerM, Wright TRF, eds),pp 449-559.New York: Academic. DuchenLW, Strich SJ (1968) The effectsof botulinumtoxin on the patternofinnervationofskeletalmusclein themouse.QJ ExpPhysiol 53:84-89. EnglanderLL, RubinLL (1987) Acetylcholinereceptorclusteringand nuclearmovementin musclefibersin culture.J CellBiol 104:87-95. GanetzkyB (1984) Geneticstudiesof membrane excitability in Drosophila: lethal interactionbetweentwo temperature-sensitive paralytic mutations.Genetics108:897-911. effects GanetzkyB,Wu C-F (1982a)Drosophila mutantswith opposing on nerve excitability: geneticand spatialinteractionsin repetitive firing. J Neurophysiol47:501-514. GanetzkyB, Wu C-F (1982b) Indirect suppression involving behav-

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