Single neuron activity in the Drosophila larval CNS detected with calcium indicators

Journal of Neuroscience Methods 127 (2003) 167 /178 www.elsevier.com/locate/jneumeth

Single neuron activity in the Drosophila larval CNS detected with calcium indicators G.T. Macleod a,*, M.L. Suster b, M.P. Charlton a, H.L. Atwood a a

Department of Physiology, University of Toronto, Medical Sciences Building Rm. 3308, 1 King’s College Circle, Toronto, Ont., Canada M5S 1A8 b Department of Zoology, University of Toronto, Mississauga, Ont., Canada L5L 1C6 Received 19 August 2002; received in revised form 20 February 2003; accepted 29 April 2003

Abstract Although the Drosophila larva has been extensively used for genetic studies of synaptic transmission and locomotion, neurophysiological studies have lagged because it is difficult to investigate circuitry and synaptic function in the larval central nervous system (CNS). Here we introduce an optical technique to monitor neuronal activity in the intact Drosophila larval CNS. We loaded neurons retrogradely through cut axons with dextran-conjugated calcium indicators. Fluorescence responses to changes in the concentration of intracellular calcium are sufficiently fast and large to monitor electrical activity in single neurons. Responses to action potentials were detected in motor neuron cell bodies, axons, neurites, dendrites and sensory neuron afferents identified by genetically targeted green fluorescent protein expression. Our findings provide an experimental procedure for testing synaptic function and connectivity within the intact larval CNS. # 2003 Elsevier B.V. All rights reserved. Keywords: Calcium imaging; Synaptic activity; CNS activity; Identified neurons

1. Introduction Drosophila is an attractive experimental model for neuroscientists because research on gene function and regulation has put it at the forefront of genetics. However, due to the small size and compact structure of the larval Drosophila central nervous system (CNS), its circuitry has resisted a detailed description at the physiological level. In adult flies, the anatomy and physiology of some circuits have been elucidated with electrophysiological techniques (see review in Trimarchi et al., 1999; Nelson and Wyman, 1990) but relatively little has been done in larvae (Rohrbough et al., 2003). In contrast to the larval neuromuscular junction, which is widely used for analysis of the effects of genetic manipulations on synaptic transmission (for review, see Featherstone and Broadie, 2000), the synaptic physiol-

* Corresponding author. Tel.:
/1-512-232-7075; fax:
/1-512-4714651. E-mail address: [email protected] (G.T. Macleod). 0165-0270/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-0270(03)00127-4

ogy within the larval CNS is largely unknown. Genetic research on central synaptic transmission and connectivity would be greatly aided by development of more procedures for monitoring physiological events within the Drosophila CNS. The neurons of the Drosophila CNS are difficult subjects for electrophysiology because they are contained within a tough ensheathing capsule (the ganglionic capsule) in a fluid of unknown composition, regulated by the sheath. Disruption of the sheath in order to introduce electrodes for single-cell recording may alter the extracellular medium in contact with the neurons of the CNS, and thus lead to effects on electrogenesis and synaptic transmission that cannot readily be assessed. In some insects (e.g. locusts), intracellular microelectrodes have been pushed through the sheath to record from somata of individual neurons (e.g. Hoyle and Burrows, 1973). Some studies of adult Drosophila motor neurons were made using highresistance intracellular electrodes (Ikeda and Koenig, 1988; Trimarchi and Schneiderman, 1994) but the success rate for such experiments is low (Broadie,

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2000). In Drosophila larvae, this approach has proven to be relatively difficult because of the small size of the CNS neurons and the difficulty of penetrating the sheath. Electrophysiology on neurons in the Drosophila embryo (Baines and Bate, 1998; Baines et al., 2002) and larva (Baines et al., 2002; Rohrbough and Broadie, 2002) CNS has required the use of proteases and mechanical force to breach the capsule to provide access for patch electrodes. In view of these difficulties, optical methods offer an excellent alternative for monitoring events within the Drosophila CNS. These methods do not breach the ganglionic sheath and could provide simultaneous measurements from many locations. One way to monitor neuronal activity in the CNS is to express, conditionally, protein-based optical indicators. This would permit selected cells to express indicators that signal changes in electrical or chemical activity. For instance, expression of apoaequorin in Kenyon cells in the mushroom bodies of the adult Drosophila CNS allowed detection of synchronized electrical activity in groups of these cells (Rosay et al., 2001). The calcium indicator protein, cameleon, can also be selectively expressed in specific cell types (Kerr et al., 2000), but this construct has insufficient responsiveness to Ca2
to allow the detection of single action potentials (APs) in individual cells. Wang et al. (2003) showed Ca2
responses in single-cell bodies expressing G-CaMP but these are likely due to many APs. The ability to detect single APs or single responses in single cells or dendrites remains to be demonstrated. An alternative procedure is to introduce ion-sensitive indicators directly into the CNS to monitor changes in the activity of selected neurons. For instance, O’Donovan et al. (1993) developed a technique to label anterogradely chick spinal motorneurons (MNs) with dextran-conjugated fluorescent calcium indicators. This technique also succeeded in other systems (zebrafish: O’Malley et al., 1996; Takahashi et al., 2002; honeybee: Sachse and Galizia, 2002; mouse: Bonnot et al., 2002). Recently, we found that dextran-conjugated calcium indicators could detect signals caused by single APs in the presynaptic terminals of larval Drosophila neuromuscular junction (Macleod et al., 2002). We have now explored the utility of this approach in the larval Drosophila CNS. Here we demonstrate a means of monitoring activity in the exposed but intact larval CNS. Dextran-conjugated fluorescent calcium indicators applied to severed nerves radiating from the ventral ganglion (VG) were rapidly transported into the CNS. In response to single-pulse stimulation, neural elements within the VG, including individual cellular processes, display rapid fluorescence changes with a high signal-tonoise ratio. Since several neural elements exhibit both spontaneous and evoked calcium signals that are thought to reflect single APs or synaptic events, our

technique provides a non-invasive means to monitor central activity and connectivity at high temporal resolution.

2. Materials and methods 2.1. Preparation and solutions Larvae were dissected (Jan and Jan, 1976) in either Schneider’s insect medium (Schneider’s) (Sigma, St. Louis, MO), or HL6 physiological solution (Table 1), and pinned to the Sylgard (Dow Corning, Midland, MI) coated bottom of a 0.5-ml perfusion bath. We used an improved physiological solution (HL6; Macleod et al., 2002) that contains many of the compounds in larval haemolymph (Pierce et al., 1999). This solution confers greater longevity to cells in the periphery compared to earlier physiological solutions (Macleod et al., 2002). Activity of the CNS was observed for up to 8 h in this solution. Schneider’s solution, which is often used during dissection, renders the preparation quiescent and stable, but is not suitable for optophysiology due to its autofluorescence, nor is it suitable for electrophysiology due to its high L-glutamic acid content (4.7 mM). HL6 does not contain L-glutamic acid and is not autofluorescent. Table 1 Composition of HL6 physiological solution (mM) CaCl2 MgCl2 KCl NaCl NaHCO3 Isethionic acid (Na
) BES Trehalose×/2H2O L-Alanine L-Arginine×/HCl Glycine L-Histidine L-Methionine L-Proline L-Serine L-Threonine L-Tyrosine L-Valine Troloxa TPENa

0.5 15.0 24.8 23.7 10.0 20.0 5.0 80.0 5.7 2.0 14.5 11.0 1.7 13.0 2.3 2.5 1.4 1.0 1.0 0.0001

pH adjusted to 7.2 with 1 M NaOH:
/1 ml/l. BES-(N ,N -bis[2hydroxyethyl]-2-aminoethanesulfonic acid), trolox (Aldrich; Milw., WI, USA), TPEN-(tetrakis-2(pyridylmethyl)ethylenediamine) (Molecular Probes). All components except BES and TPEN are from Sigma, St. Louis, MO. a Components used for optophysiological studies. Modified after Macleod et al. (2002).

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2.2. Fly stocks Flies were raised on cornmeal agar with dry yeast at 219/1 8C. Experiments were performed on Drosophila Canton-S wandering 3rd instar larvae. GFP expression (UAS-GFP, UAS-EGFP or UAS-mCD8-GFP) was selectively driven in MNs or sensory neurons using the following GAL4 lines: OK6-GAL4 (all MNs; Aberle et al., 2002), RRK-GAL4 (aCC/RP2 MNs, pCC inter neuron; Baines et al., 2002; kindly provided by J.B. Jaynes; for a description of aCC and RP2 MNs, see Goodman and Doe, 1993), 4C-GAL4 (alary muscle MN and lateral bipolar dendrite), md-GAL4 109(2) 80 (multidendritic neurons; Gao et al., 1999), and chaGAL4 (cholinergic neurons; Kitamoto, 2001). UASEGFP, UAS-GFP and UAS-mCD8-GFP all encode variants of GFP and these flies are from the Bloomington Stock Center, IN.

2.3. Loading CNS neurons with fluorescent dyes and indicators

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The most effective loading occurred when fluorescent compounds were applied as soon as possible after the nerve was cut. This was anticipated, because the permeability of cut axon ends to large molecules decreases as ends seal (Eddleman et al., 2000) in a Ca2
-dependent manner (Bittner and Fishman, 2000). Sealing of cut axon ends might be minimized by reducing [Ca2
] in the solution bathing the nerve but we did not do this because we wanted these small axons to take up some indicator and then seal quickly. We hoped that rapid sealing would maximize the longevity of axons. Therefore, Schneider’s or HL6 was used in the loading pipette ([Ca2
]o /5.4 mM and 2 mM, respectively). The loading concentrations and exposure times were determined empirically and may not be optimal (Macleod et al., 2002). If too much Ca2
indicator enters cells, the change in fluorescence with stimulation can be reduced. DMSO, used in other loading protocols (Karunanithi et al., 1997; Umbach et al., 1998) to solubilize ester-linked indictors, was not required in the present technique. 2.4. Optophysiology

The technique used to load fluorescent compounds into centrally located neurons was similar to that described by Macleod et al. (2002) to load dextranconjugated calcium indicators into MN terminals in the periphery. We cut either a hemi-segmental nerve (SN) or a transverse nerve (TN) branch with sharp microscissors and drew the cut end, along with a small amount of Schneider’s or HL6 solution, into the tip of a glass pipette that had been heat-polished to form a snug fit with the nerve (inner diameter of
/12 mm for SN;
/3/6 mm for TN). A fine plastic tube, pushed down the barrel of the pipette to within 100 m of the nerve, contained a 5 mM solution of the selected fluorescent compound in distilled water, and was used to deliver enough solution to give a final concentration of 0.5 mM in the pipette. After the cut end of the nerve had thus been exposed to the fluorescent compoundcontaining solution for 10 /20 min, the solution was extracted from the tip of the pipette. The pipette, still in place, was then filled with fresh Schneider’s or HL6 solution. Fluorescent compounds could be seen in the MN cell bodies within 30 min of application to the cut nerve. The compound concentration (roughly proportional to fluorescence intensity) in cell bodies stabilized approximately 1 h after the cessation of exposure. All fluorescent compounds including rhodamine-B dextran 10 kDa and Alexa Fluor 568 hydrazide sodium salt were supplied by Molecular Probes, Eugene, OR. The batchspecific Ca2
binding affinities (KD) of Ca2
indicators determined in vitro by the manufacturer were: Oregon Green BAPTA-1 dextran 10 kDa (188 nM), Furadextran 10 kDa (140 nM), Fluo-4 dextran 10 kDa (4.1 M) and rhod-2 salt (570 nM).

Monochromatic calcium indicators were imaged with a BioRad 600 confocal scan-head (BioRad, Mississauga, Ont.) on a Nikon upright microscope (Optiphot-2) with a 40/ Olympus water-immersion objective (0.7 NA). The scan-head was controlled using BioRad MRC-600 software. Neurons loaded with Oregon Green-1 or Fluo4 were scanned through a BHS filter set (exciter filter488 nm DF10; emission filter-OG 515 nm LP; dichroic reflector-510 nm LP). Neurons loaded with rhod-2 were scanned through a GHS filter set (exciter filter-514 nm DF10; emission filter-OG 550 nm LP; dichroic reflector540 nm LP). When imaging rhod-2 salt against a GFP background, a dual filter set (A1/A2; A1: exciter filter514 nm DF10, no emission filter, dichroic reflector-527 nm LP; A2: dichroic reflector-565 nm LP, barrier filter (green)-540 nm DF30, barrier filter (red)-600 nm LP) was used. With the photo-multiplier gain maximized and the pinhole opened to its maximum aperture, an acceptable level of fluorescence could be attained even when the output of the argon ion laser (operating at low power) was attenuated to 0.5% by neutral density filters. A green light-emitting diode, placed within the scanhead, was lit briefly (2 or 4 ms) to mark a single stimulus pulse or the beginning of a train of stimuli. Line scan rates for X /Y and Y -time scans were 2 and 4 ms per line, respectively. Pixel values of scanned images were saved in PIC files with 8-bit depth. A look-up table was used to represent pixel values in an 8-bit range (Figs. 3 and 4). Images of fluorescence are shown without the background subtracted. Fluorescence (F ) is reported with background subtracted (arbitrary units: au) (Figs. 3 and 4). ImageJ software (http://rsb.info.nih.gov/ij/) was

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used to convert PIC image files to TIFF format and to measure the average pixel values within selected regions of each image, or within lines of a line-scan image. A region, not containing any calcium indicator fluorescence, was selected beside each fluorescent cell body or process and the average pixel intensity was measured in this region to give a background value. This value was subtracted numerically from the average pixel value of a region or line containing the fluorescent element of interest to generate the value F . DF /F is defined as the change in F during stimulation, relative to F prior to nerve stimulation. N indicates the number of larvae. We detected fluorescence from Fura-dextran-loaded neurons with an intensified CCD camera (PTI, model IC-100; Princeton, NJ) connected to a Lightning 2000 frame grabber controlled by Axon Imaging Workbench 2.2 (AIW 2.2) (both from Axon Instruments, Union City, CA). Emitted light was collected by an Olympus water-immersion objective (40 /, 0.7 NA) through a 5309/35 nm band-pass filter both mounted on an upright Nikon (Optiphot-2) microscope. The loaded neurons were excited with light from a mercury arc lamp

alternately through 3509/5 and 3859/5 nm band-pass filters (Omega Optical, Brattleboro, VT).

2.5. Static imaging Images for Figs. 1A and B and 2C were collected using a CCD camera (Sony, model XC-75, CMA-D2; Empix Imaging, Mississauga, Ont.) mounted on an upright Nikon (Optiphot-2) microscope with Nikon water-immersion objective (20 /, 0.4 NA). Acquisition was through a Data Translation (Marlboro, MA) DT3155 frame grabber supported by AIW 2.2 software. Images for Figs. 1C and F were acquired using a Zeiss laser confocal microscope (LSM 510) with a 40 / Zeiss water-immersion objective (0.75 NA), and 488 and 543 nm lasers. Images for Figs. 1D and E and 2A, B, D and E were acquired using a Leica laser confocal microscope (TCS SL) with a 40/ Leica water-immersion objective (0.8 NA), and 488 and 543 nm lasers. Overlaid images were constructed using Adobe Photoshop 5.5.

Fig. 1. Elements of the SN filled with dextran-conjugated compounds and identified with genetically targeted GFP markers. (A) The severed ends of two contralateral SNs, from the VG, each drawn into a pipette (transmitted light image). A dotted line in each panel indicates the midline of the VG. (B) The same field of view as in (A) observed using epi-fluorescence optics showing fluorescence from the dyes in the lumen of the pipettes. Rhodamine-B dextran 10 kDa (rhod-B) in the left pipette and Oregon Green BAPTA-1 dextran 10 kDa (OGB-1) in the right pipette have traveled up their respective SNs. Each arrow indicates a bolus of dye on either side of the VG immediately inside the ganglionic sheath but not confined in axons. Scale bar 100 mm for both A and B. (C) The VG in (A) and (B) viewed using confocal microscopy, 4 h after the cut ends of the SNs had been exposed to dye, showing the location of a group of MN cell bodies close to the dorsal midline, and their intraganglionic neuropilar processes. Solid white lines indicate the approximate margins of the VG. Neurons filled with rhod-B are shown in red while those filled with OGB-1 are shown in green (z-series projection, 5/1.5 mm steps). Scale bar 20 mm. (D(a)) A confocal microscope image of GFP in the MN cell bodies located along the dorsal midline of the VG in an MN-GFP larva, at the level of the third abdominal segment. (D(b)) The same field of view as in (D(a)), scanned simultaneously, showing cell bodies loaded with rhod-B through a single SN (seen at the left side of the image). (D(c)) (D(a)) and (D(b)) overlain to show dye colocalization in yellow, demonstrating that all rhod-B-filled cell bodies are MNs. An asterisk marks the most posterior MN. Scale bar 20 mm. (E(a)) An image of GFP in RP2 MN cell bodies along the dorsal midline, corresponding to abdominal segments 3 and 4, in an RRK-GFP larva. (E(b)) The same field of view as in (E(a)), scanned simultaneously, showing cell bodies loaded with rhod-B as in (D(b)). (E(c)) (E(a)) and (E(b)) overlain to show dye colocalization, identifying the most posterior MN in the group (marked with an asterisk) as RP2. Scale bars 10 mm in both (D) and (E). (F) A magnified view of the central portion of panel (C) showing a number of neural elements crossing the dorsal midline (z-series projection, 11 /1.5 mm steps) and the identification of two MN cell bodies (aCC and RP2), the suggested identity of MN cell bodies in the more anterior cluster (RP1, RP3 and RP4), the identity of a dendrite from the aCC cell body on the right (arrowhead), and an axon(s) from a multidendritic neuron(s) (MDN) (see Section 3). Scale bar 10 mm. Anterior is at top in all panels. Fig. 2. Elements of the transverse nerve (TN) filled with dextran-conjugated compounds and identified with genetically targeted GFP markers. (A) The VG viewed using confocal microscopy (z-series projection, 20 /3 mm steps), 3 h after the cut end of a TN issuing to the left side of segment 3 had been cut and exposed to rhod-B dextran. A dotted line indicates the midline of the VG. Two cell bodies are visible on the ipsilateral side (left) while a single cell body is seen on the contralateral side (right). All cell bodies are ventrally located, but the cell bodies marked with arrowheads are slightly more posterior and dorsal than the single cell body marked with an arrow. A single large dorsal process (asterisk), projecting from the site of TN entry to the VG is associated with each cell body. Scale bar 10 mm. (B(a)) A confocal microscope image of GFP in a ventrally located cell body in a 4C-GFP larva. (B(b)) The same field of view as in (B(a)) (boxed area in (A)) scanned simultaneously with (B(a)) (z-series projection, 5 /3 mm steps), showing two cell bodies loaded with rhod-B through the TN. (B(c)) (B(a)) and (B(b)) overlain showing GFP co-localization (yellow) with the more posterior cell body (arrowhead). (C) A tracheal dendrite (TD) neuron in the periphery observed using epi-fluorescence optics, associated with the TN (top of image), injected with Alexa Fluor 568 salt using a sharp microelectrode. Scale bar 20 mm. (D(a)) A confocal microscope image of GFP in a TD neuron in an md-GFP larva. (D(b)) The same field of view as in (D(a)), scanned simultaneously, showing MN(s) of the TN, loaded with rhod-B. (D(c)) (D(a)) and (D(b)) overlain to show the close association of the varicose MN(s) and processes of the TD neuron. (E(a)) A confocal microscope image of GFP in a lateral bipolar dendrite (LBD) neuron in a 4C-GFP larva. (E(b)) The same field of view as in (E(a)), scanned simultaneously, showing MN(s) of the TN, loaded with rhod-B, running along the posterior side of muscle 8 as in (D(b)) (muscle at top but not visible). (E(c)) (E(a)) and (E(b)) overlain to show the close association of the MN(s) and the LBD neuron cell body and dendrites. Scale bars 10 mm in both (D) and (E). Images in (A) to (E) are orientated with the anterior at the top. Images in (C) to (E) are orientated with the midline to the left. (F) Preliminary interpretive diagram (not to scale) of the neurons and their approximate pathways in the TN (abdominal segments 2 /4), based on a series of dye loadings of cut TN endings, both toward the VG and toward the periphery, from the sites marked by circled numbers 1 /4. The dye loaded preparation shown in (A) is representative of those obtained by loading the TN dye load from site 2 in (F).

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3. Results Several nerves arise from the larval VG and brain hemispheres, including the eye and antennal nerves, maxillary nerves, segmental and transverse nerves (Bodenstein, 1965), all of which are long enough (minimum length of 100 mm) to load calcium indicators. Here we concentrate on data obtained after loading calcium indicators into the segmental and transverse nerves

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which innervate body wall muscles likely involved in locomotion. 3.1. Ventral ganglion neurons loaded via a segmental nerve We loaded dextran-conjugated fluorescent compounds into motor neurons (MNs) and sensory cell axons in the VG of larvae by exposing cut ends of SNs

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to the compounds. This resulted in retrograde loading of the MNs and anterograde loading of sensory axons. We loaded several fluorescent compounds each conjugated to 10 kDa dextran including rhodamine-B, Oregon Green BAPTA-1, Fura, and Fluo-4. Both sides of the same segment could be loaded simultaneously by drawing up the two SNs into different pipettes for exposure to different dyes (Figs. 1A and B). In panels A /C and F of Fig. 1, rhodamine-B dextran 10 kDa (rhod-B) was loaded from the left side, while Oregon Green BAPTA-1 dextran 10 kDa (OGB-1) was loaded from the right side. Drosophila larvae have a stereotypical arrangement of body wall muscles in each abdominal segment. This is reflected in the arrangement of MN cell bodies in the VG; the same dorsal group of cells is found in each segment. This pattern was consistent in all abdominal segments (2 /6) loaded. Approximately 50 other cell bodies were visible in more ventral focal planes, consistent with the approximate expected number of larval MNs (i.e. 30 per hemi-segment; Hoang and Chiba, 2001).

3.2. Identification of neural elements in the VG loaded via a segmental nerve To help identify cells that were retrogradely filled with fluorescent compounds, we selectively expressed EGFP in MNs with the OK6-GAL4 driver. This revealed MN cell bodies on either side of the VG dorsal midline (Fig. 1D(a)). The MN cell bodies of a single abdominal segment are immediately contiguous with those of the next segment */no segmental boundary is apparent. The use of MN-EGFP expression confirmed that all cell bodies in the dorsal group that were retrogradely filled with rhod-B were MNs (Fig. 1D(b) and (c)). Analogous to the stereotypic arrangement of MN cell bodies in the embryonic CNS (Broadus et al., 1995; Baines et al., 1999), the two most posteriorly located cell bodies in the larval dorsal VG cluster are likely to be those of MNs aCC and RP2. An asterisk marks the most posterior MN in Fig. 1D(c). We established that the two most posterior cell bodies were indeed aCC and RP2 (Fig. 1E(c)) by retrogradely filling single SNs in RRK-GAL4/ UAS-GFP larvae (RRK-GFP larvae: expressing GFP in the aCC, RP2 MNs and interneuron pCC; Fig. 1E(a)) with rhod-B (Fig. 1E(b)). We confirmed the identity of these MN cell bodies on the basis of their terminals in the body wall. In RRK-GFP larvae, GFP was only present, as expected, in MN terminals on muscles 1 and 2. Given the good correspondence in the location of the embryonic and larval aCC/RP2 MNs within the VG, we deduce that the more anteriorly located cell bodies in the dorsal VG cell group (Fig. 1F) are likely to be those of RP1, RP3 and RP4 (Sink and Whitington, 1991; Landgraf et al., 1997; Schrader and Merritt, 2000).

An extensive network of dendritic processes can be seen in the neuropile between the edge of the VG and the MN cell bodies (Figs. 1C and F). A dendrite issues from aCC and projects across the midline (arrowhead in Fig. 1F). We observed an apparently similar dendritic projection extending from beneath RP2 (MDN, Fig. 1F), but this projection is unlikely to be that of RP2, which does not have such dendrites in the embryo (Baines et al., 1999). In addition, this neural process is not labeled in OK6-GAL4/UAS-EGFP larvae (MNGFP larvae in which GFP is expressed in all MNs). We therefore deduce that the GFP-labeled process is likely to be an afferent from a sensory cell. Indeed, in chaGAL4/UAS-GFP larvae (cholinergic-GFP larvae), in which all cholinergic neurons express GFP (including most sensory neurons), such processes are labeled in each segment. This process was also labeled in larvae that express GFP in the multidendritic (MD) neurons (md-GAL4/UAS-GFP; MD-GFP larvae), a subset of the peripheral sensory neurons. Therefore, we presume that this process is the central dendritic projection of an MD neuron whose soma is located in the periphery. 3.3. Ventral ganglion neurons loaded via the transverse nerve Paired transverse nerves (TN) issue from the dorsal midline of the VG, either separately (abdominal segments 1/4) or fused together (segments 5 /9), and innervate a deep body wall muscle (muscle 25) and the alary muscle (Gorczyca et al., 1994) which inserts into pericardial tissues (Curtis et al., 1999). Cell bodies in the VG were retrogradely filled by exposing the cut end of the TN to fluorescent compounds. In the preparation shown in Fig. 2A, the cut end of the TN on the left side of segment 3 was exposed to rhod-B. Two cell bodies were loaded on the ipsilateral side (boxed and shown in Fig. 2B), while a single cell body was loaded on the contralateral side. A dorsal and laterally projecting element (asterisks in Fig. 2A) was filled simultaneously with each cell body. All cell bodies are located ventrally in the VG but the anterior cell body on the ipsilateral side (arrow in Fig. 2A), obscured by processes, is located more ventrally than the two brightly loaded cell bodies (arrowheads). If the TN branch to the right side of segment 3 is similarly loaded (data not shown), the pattern symmetrically complements that already shown, with another cell body being filled more ventrally on the right side, and two of the cells already loaded (arrowheads), filling with dye. This pattern was seen with 26 TN dye fills. When both the SN and TN in the same segment were filled with dye, we observed that the TN enters the VG between MN cell bodies aCC and the cluster of RP1, RP3 and RP4, before branching to more ventral levels (data not shown).

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3.4. Identification of neural elements associated with the transverse nerve When the VG cell bodies in MN-GFP larvae (expressing GFP in all MNs) were retrogradely filled with rhodB via the TN, we identified all rhod-B labeled cell bodies as MNs (N /3, data not shown). In 4C-GAL4/UASCD8-GFP larvae (4C-GFP larvae), GFP is expressed in many cell bodies throughout the CNS and in a number of peripherally located sensory cell bodies in the more anterior abdominal segments (Suster et al., 2003). We noticed that in the CNS, 4C-GFP labels four cell bodies on either side of the VG very prominently. In these larvae, GFP is also expressed in the TN and the MN terminals that innervate the alary muscle in abdominal segments 1/4, but not in the MN terminals of muscle 25. By retrogradely filling the VG cell bodies in these larvae with rhod-B via the TN (Fig. 2B(b)), we could identify the more posterior cells (arrowheads in Figs. 2A and B(c)) as the MNs which innervate the alary muscle. By a process of elimination, the cell body more anterior (arrow in Fig. 2A) is the MN that innervates muscle 25. Two sensory cells are located on the TN on the posterior edge of muscle 8; one is the lateral bipolar dendrite (LBD) neuron and the other is the tracheal dendrite (TD) neuron (Gorczyca et al., 1994). These large and distinct sensory cell bodies are located one above the other, with the TD neuron more posterior. Either cell body can be reliably impaled with a sharp microelectrode for injection (Fig. 2C), stimulation or recording. The TD neuron in Fig. 2C has been injected with Alexa Fluor 568 salt. In md-GAL4/UAS-GFP larvae, GFP is expressed in the TD neuron (Fig. 2D(a)) and forward-filling of the TN with rhod-B (Fig. 2D(b)) demonstrates close apposition between the MN(s) and the processes of this sensory cell (Fig. 2D(c)). Dendrites of the TD neuron extend in either direction along the TN, but the projection back to the CNS is through the SN rather than the TN. In 4C-GFP larvae, GFP is expressed in the LBD neuron (Fig. 2E(a)) and forwardfilling of the TN with rhod B (Fig. 2E(b)) demonstrates apposition between the MN(s) and the sensory cell (Fig. 2E(c)). We confirm that dendrites of the LBD neuron extend out to the MN terminals on the alary muscle (Gorczyca et al., 1994; data not shown) and along the TN past the branch point to muscle 25, but do not project all the way back to the CNS (embryo: Merritt and Whitington, 1995; first instar larva: Gorczyca et al., 1994). The diagram in Fig. 2F is consistent with our observations from many dye fills, both toward the VG (retrograde, N ]/3) and away from the VG (anterograde, N ]/3), from each of the sites marked by circled numbers 1, 2, 3 and 4 in Fig. 2F. The observation that neither the TD nor LBD neuron cell bodies loaded with dye when the TN was cut at site ‘‘3’’ and

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filled with rhod-B towards the periphery confirmed that neither sensory cell projected back to the VG through the TN. 3.5. Calcium signals in motor neuron cell bodies, neurites, dendrites and sensory afferents We next attempted to obtain physiological evidence of neural activity in the larval CNS by the use of calcium indicators retrogradely loaded into CNS neurons. The high-affinity (188 nM) calcium indicator OGB-1 was loaded into MN cell bodies in
/80% of attempts (25 out of 31) through the SN, and in
/80% of the attempts through the TN trunk (site 1: 3/3) or a TN branch (site 2: 7/9). Stimulation of the TN caused OGB-1 fluorescence in the alary muscle-MN cell body to increase slowly (Figs. 3A and B). Stimulation pulses were 1.5 /3 V in amplitude and 0.3 ms in duration. The peak DF /F obtained during trains of pulses was approximately a linear function of frequency from 5 to 20 Hz (Figs. 3D and E). The same cell bodies showed spontaneous changes in their resting fluorescence (Fig. 3C), lasting from tens of seconds to minutes. Stimulation at any frequency could trigger a lasting rise in resting fluorescence; the decay phases showed considerable variance (Fig. 3D). Some of the variation in the decay phase may result from the superposition of stimulated responses on changes caused by spontaneous activity but this was not investigated in detail. The more dorsal elements (asterisks in Fig. 2A) commonly displayed spontaneous changes of a more rapid time course than the ventral cell bodies. A change in fluorescence in the alary muscleMN cell body could be detected in response to a single stimulus pulse (5.99/1.9%, DF /F ; S.D., N /3) (data not shown). Fluorescence responses to single pulses could most easily be detected in those MN cell bodies loaded with a low concentration of indicator. These responses are comparable in amplitude to responses representing single APs (7 /10%) measured in zebrafish MN cell bodies filled with Calcium Green dextran 10 kDa (Fetcho et al., 1998). However, in Drosophila larval CNS, we cannot be certain that the response to a single pulse represents only a single AP. When OGB-1 was loaded into the dorsal cluster of MN cell bodies in the VG through the SN (Fig. 4A), cell body fluorescence did not increase in response to stimulation of the ipsilateral SN. However, in lightly loaded preparations, MN cell body fluorescence fluctuated spontaneously as seen in the alary muscle-MN cell body and the rate of these fluctuations could be increased by stimulation of the contralateral SN. On the other hand, cellular processes loaded with calcium indicator were responsive to stimulation of the ipsilateral SN. These events were recorded by confocal line scanning through a fixed point on the selected element

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Fig. 3. Fluorescence changes of dextran-conjugated indicator in cell bodies of the transverse nerve (TN) within the VG. (A) A diagram (not to scale) showing the approximate relative locations of the cell bodies of the TN MNs within the VG. The TN has been cut and loaded with OGB-1 at a site before it bifurcates into separate hemi-segment TNs (site 1 in Fig. 2F). All four cell bodies in the VG have loaded but only one (green: an alary muscle MN) was examined here (B /E) during stimulation of the TN through the loading pipette. (B) Four sequentially scanned images of an alary muscle MN cell body (anterior at right, midline at top). Each image is scanned left to right in 512 ms, while the distance between consecutive images represents an acquisition delay of 506 ms. A single white scan line in the second image marks the start of a 5-s train of electrical pulses (2 V, 0.3 ms) delivered to the nerve at 20 Hz. Arrow marks indicate the time at which each pulse was delivered. The look-up table used for pixel intensity (0 /255) is shown to the right. Scale bar 10 mm. (C) Time course of the average pixel fluorescence intensity, minus background, of the cell body (area of fluorescence measurement indicated by a circle in the fourth image in (B)) during a single 5-s train of pulses. The open symbols correspond to the four images in (B). The red line indicates spontaneously high cell body fluorescence in the absence of prior applied stimulation. (D) Cell body DF /F in response to 5-s stimulation trains of different frequencies (5, 10, 20 Hz), where each data series is the average of three individual image series (as shown in (C)), each separated from the other by a minimum of 2 min and aligned with other image series of the same stimulation frequency using the frame containing a white scan line (see image 2 in (B)) ([Ca2
]o 0.5 mM). The stimulated responses are superimposed on spontaneous changes. (E) A plot of the average maximum DF /F (avg. of two points at 5.90 and 7.08 s) achieved during the stimulus trains of different frequencies in (D).

at a rate of 4 ms per line. A cellular process projecting contralaterally from beneath RP2, identified earlier as an MD neuron afferent (MDN, Fig. 1F), displayed a large (130%) and rapid (10 /90% rise-time B/4 ms) response to single isolated stimulus pulses (Fig. 4B). This element displayed a long refractory period and could not be excited within several seconds of the preceding pulse or spontaneous event. This element also displayed spontaneous changes in fluorescence indistinguishable in amplitude and time course from those evoked with a single stimulus pulse. In MN terminals, the change in fluorescence in response to a single stimulus pulse reaches a maximum within 4 ms of the stimulus pulse (Macleod et al., 2002). Responses in cellular processes in the VG, reported here, have a similar rise time, but the latency following a stimulus pulse is often as much as 12 ms. The reason for the longer delay in the CNS is not clear. It is possible that many of the fluorescence events do not result directly from stimulation of peripheral axons connected to the CNS element, but instead arise from synaptic

input of other neurons*/part of the delay may result from synaptic delay and signal integration. In the mid-levels of the VG (different focal plane from those in Fig. 1) within the neuropile, a longitudinally oriented structure loaded though the SN appears similar in size, orientation and location to the central projections of peripherally located lateral chordotonal sensory cells (lch5) described by Schrader and Merritt (2000). This structure is also similar to those seen in cholinergicGFP larvae that express GFP in the chordotonal organs This structure displays a frequency-dependent increase in OGB-1 fluorescence in response to SN stimulation frequencies between 5 and 20 Hz (data not shown) and rapid spontaneous activity in the absence of stimulation (Fig. 4C). The amplitude of single events in both elements loaded through the SN (DF /F /100%; Figs. 4B and C) is large relative to that observed in MN terminals in the periphery where the fluorescence response to single stimulus pulses is usually B/50% (Figs. 3 and 4; Macleod et al., 2002). The time constant of decay (t)

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Fig. 4. Fluorescence changes of dextran-conjugated indicator in neural elements of the SN within the VG. (A) A diagram (not to scale) showing the approximate relative locations of the most dorsal MN cell bodies within the VG, loaded through the SN. The SN has been cut and loaded with OGB1 midway between the VG and the hemi-segment it travels to (segment 3). All MN cell bodies and sensory axons from the SN have loaded but only the MD neuron axon (blue) and the chordotonal axon(s) (red) have been examined in (B) and (C), respectively. The thin vertical boxes indicate the approximate location and direction of line scanning of each element, i.e. all line scanning is along the antero-posterior axis. (B) The MD neuron process running across the dorsal midline, from beneath the cell body of RP2, gives a fluorescence response to a single stimulus pulse to the SN through the pipette. (B(a)) A serial line scan transversing the MD neuron axon while three pulses were delivered (2 V, 0.3 ms) at 2 Hz to the SN. Scale bars: vertical */2 mm; horizontal */200 ms. The same look-up table is used for pixel intensity as in Fig. 3B. (B(b)) A plot of fluorescence for each scan line in (B(a)) shows a response to only the first stimulus of the three-pulse train. (C) Chordotonal axons (identified by their similarity to those described by Schrader and Merritt (2000)) running longitudinally in a cluster close to the midline on the ipsilateral side, display rapid spontaneous changes in fluorescence. (C(a)) A serial line scan obliquely through a short section (4 mm) of the sensory axon(s) during spontaneous activity. Scale bars: vertical */2 mm; horizontal */200 ms. (C(b)) A plot of fluorescence for each line in (C(a)). No data averaging or smoothing in panel (B) or (C). Anterior at top.

is on the order of several hundred ms and is slower than that reported in the periphery either for single pulses or for trains (t
/60 ms; Macleod et al., 2002). Changes in fluorescence, whether stimulated or spontaneous, could be detected in all VGs examined. The elements continued to respond to stimulation and often manifested spontaneous activity for as long as they were observed; in one case, activity continued for almost 8 h after loading began (avg. 3059/87 min, S.D., N /5). 3.6. Loading of the calcium indicators Fluo-4, rhod-2 and Fura The low-affinity (4.1 mM) calcium indicator dextranconjugated Fluo-4 10 kDa (Kreitzer et al., 2000) was also loaded into the CNS. This indicator gave a small fluorescence response with low-frequency stimulation but the response was easily quantified at high frequencies (20 /50 Hz). Unfortunately, it has negligible fluorescence at resting intracellular calcium levels making pre-stimulation focusing difficult. The salt form of Alexa Fluor 594 has been simultaneously loaded with Fluo-4 to assist with focusing and calibration of signals

(Sabatini et al., 2002). Here we were able to load Alexa Fluor 568 salt into the VG cell bodies (data not shown) indicating that this approach is feasible. However, we would recommend that Alexa Fluor 568 or 594 conjugated to 10 kDa dextran be used rather than the salt form that may cross gap junctions or become compartmentalized. GFP expression is commonly driven in identified neurons using the GAL4/UAS system and it would be desirable to find a calcium indicator whose spectra can be discriminated from GFP for use in these neurons. rhod-2 is potentially useful as its excitation and emission spectra can be discriminated from GFP. We were able to load rhod-2 salt (a dextran conjugate is not available) into cell bodies in the VG via either the SN or TN. Like Fluo-4, rhod-2 has negligible fluorescence at resting intracellular calcium levels but the GFP in the same neurons can be used to assist pre-stimulation focusing. Although stimulation-evoked changes in rhod-2 could be detected against a GFP background, these changes were also small at low frequencies of stimulation. An unanticipated finding was that rhod-2 crossed into MN cell bodies in contiguous segments, and rhod-2 fluores-

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cence changes were observed from the MN cell bodies of a number of segments in response to single electrotonic shocks (0.8 mA, 0.3 ms) delivered close to the surface of the VG with a suction pipette. Fura-dextran 10 kDa was loaded into the VG through the SN. Emission /510 nm was easily detected from the dorsal group of MN cell bodies with both 350 and 380 nm excitation wavelengths, and was not obscured by autofluorescence. However, Fura-dextran displayed little change in the 510 nm emission ratio with 350/380 nm excitation at low stimulation frequencies. There are significant methodological hurdles in calibrating Fura fluorescence against an absolute intracellular Ca2
concentration ([Ca2
]i) in the cell bodies of the VG. The cells we have observed cannot be driven to high enough [Ca2
]i to saturate the indicator, possibly due to a combination of countermanding inputs and weak antidromic AP propagation (Ogawa et al., 2002).

saturated at the level at which the events peak (DF /F 5/ 200%) and is thus not reporting all APs contributing to the event. We have observed that OGB-1 sustains increases of /300% in MN terminals (Macleod et al., 2002). It is also possible that some of these signals could be due to Ca2
entry through ligand-gated channels during a synaptic event. The ability to detect single events, both evoked and spontaneous, in individual neural elements including cell bodies and dendrites provides valuable information about neuronal activity and connectivity. Furthermore, the ability to detect events without signal averaging allows for both spontaneous APs (Fig. 4C), and evoked APs (Fig. 4B) with unknown latencies, to be monitored. This is an advantage over the use of genetically encoded indicators. Although dextran-conjugated indicators have been used to monitor CNS neural activity in a number of other animal preparations (see Section 1), this report is the first to document their utility in any Drosophila CNS preparation.

4. Discussion 4.1. Neuronal activity in the CNS The data shown here demonstrate that a simple technique can be used to detect intrinsic and stimulated activity in a wide variety of identified cells in the CNS of Drosophila larvae. This study exploits the fact that sufficient Ca2
crosses the membranes of cell bodies, dendrites, axons and neurites during APs to be easily detected as changes in the fluorescence of Ca2
indicators. The rapid events that occur in processes within the VG, either in response to stimulation of the loading nerve, or spontaneously, have a rise time (10 /90%) of less than a line scan interval (4 ms). We interpret these events to be the product of single APs. For two APs to occur within the line scan interval requires a firing frequency of ]/250 Hz. Although higher firing frequencies (as high as 650 Hz) have been reported for the campaniform sensilla in the locust (Burrows and Pflu¨ger, 1988), other characteristics of these data lead us to dismiss tentatively the possibility of these events being the product of multiple APs. The similarity in size of spontaneous events and those evoked with a single stimulus pulse is consistent with both types of event being the product of a single AP. There was no indication of an underlying arithmetic factor in the amplitudes of either type of event to suggest that they derive from multiple APs. For instance, we did not notice signal amplitudes that were multiples of a smaller signal; such summated signals would be expected if a single stimulus triggered several APs. In addition, the peak of the response gives no indication of a plateau, no matter how brief, that would have indicated a number of APs saturating the calcium indicator. It is unlikely that the indicator OGB-1 is fully

4.2. Considerations for the retrograde-filling technique Tight junctions maintain a ‘‘blood/nerve barrier’’ at the site where motor axons issue from the nerve sheath (Auld et al., 1995); therefore, cutting the hemi-segment nerve should breach the blood /nerve barrier. However, although the interaxonal space of the hemi-segment nerve is exposed to fluorescent compounds, they do not diffuse into the interneuronal space of the VG. There appears to be a barrier to movement of large exogenous molecules (10 kDa), within the perineural space, at the margin of the VG as marked by the arrows in Fig. 1B. The absence of freely diffusing fluorescent compounds within the VG provides good contrast for the axonally loaded neuronal elements. The 10 kDa dextran-conjugated dyes did not move from cell bodies in one segment to cell bodies in an adjacent segment. This is expected because gap junctions in Drosophila exclude 10 kDa rhodamine dextran but may pass molecules as large as 3 kDa fluorescein dextran (Phelan et al., 1996). Consoulas et al. (2002) describe dye coupling between an MN cell body and an interneuron in the Drosophila larval VG and we noticed that rhod-2 tripotassium salt (869 MW) moved between cell bodies in contiguous segments */presumably via gap junctions. This ability of rhod-2 salt to cross into other cells offers the possibility of imaging activity in completely intact circuits and in neurons such as interneurons that have no processes external to the VG. However, it may be difficult to identify from which cells the signals originate owing to passage of rhod-2 between cells. Here the application of confocal optical sectioning might allow cell identification.

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4.3. Future prospects While there are many model organisms suitable for cellular studies of neural circuit function (Marder and Bucher, 2001), few organisms are as readily amenable to genetics as Drosophila . The prospect of gaining access to a simple neural circuit amenable to genetic manipulation is an attractive one, particularly if the technique described here can be used to assay the activity of multiple cells within a circuit. The classic sensory feedback reflex arc may be a basic unit of behavior; this would be an appropriate simple circuit with which to begin genetic analysis. Here we could examine the activity of identified MNs in response to defined sensory input from the periphery. Stimulation of individual sensory neurons (e.g. LBD or TD) might provide specific ‘‘feedback’’ to MNs in the CNS. This approach could provide a means of monitoring the function of basic sensory feedback loops in the semiintact larva. The ability to detect Ca2
activity in the terminals of sensory neurons and Ca2
oscillations in MNs (that presumably reflect the activity of the central pattern generator) may allow identification of some of the CNS elements that drive the activity of MNs associated with a variety of motor patterns. Multiphoton microscopy may prove useful to monitor the activity of fine cellular processes (i.e. dendrites) in the live CNS thereby providing data for studies on biophysical mechanisms (e.g. Sabatini and Svoboda, 2000) and synaptic plasticity (e.g. Engert and Bonhoeffer, 1999). In the future, this technique may yield valuable data for studies into the molecular mechanisms underlying spontaneous and evoked activity of MNs in the larval CNS. Targeted expression of neural toxins (Sweeney et al., 1995) or non-inactivating K
channels (White et al., 2001) could provide the means to selectively silence subsets of sensory or motor neurons. Single-gene mutations could be used to eliminate specific sub-cellular or molecular components in MNs, interneurons or sensory neurons. More generally, a basic sensory feedback loop in the Drosophila larva, the activity of which can be measured in the CNS, and which is accessible to genetic manipulation, could provide a unique opportunity to test in vivo, the role of candidate genes associated with sensory-motor disorders of the human nervous system (Reiter et al., 2001).

Acknowledgements This study was supported by grants from the Canadian Institutes for Health Research (CIHR) to G.T.M., M.P.C. and H.L.A., and from the Natural Sciences and Engineering Research Council of Canada (NSERC) to H.L.A. M.L.S. thanks Prof. M.B. Sokolowski (Depart-

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ment of Zoology, University of Toronto, Mississauga) for his support.

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