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First published online January 12, 2004
Journal of Experimental Biology 207, 633-644 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00784

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Modular organization of the silkmoth antennal lobe macroglomerular complex revealed by voltage-sensitive dye imaging

Hiroyuki Ai^* and Ryohei Kanzaki

Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

View larger version (72K): [in a new window]   Fig. 1. Sensory fibers stained by back-filling with cobalt-lysine from the cut end of the medial nerve (MN) and the lateral nerve (LN). (A) Axons running in the MN have their source in the medial flagella (arrowheads in i), and axons running in the LN have their source in the lateral flagella (arrowheads in ii). (B) Typical bipolar cells were stained on the surface of the medial flagella (arrowhead in i) and the lateral flagella (arrowhead in ii). Scale bar, 1 mm (A), 100 µm (B).

View larger version (42K): [in a new window]   Fig. 2. Antennal lobe (AL) of the silkmoth. (A) Confocal image of the AL. In this focal plane, two macroglomerular complex (MGC) compartments – cumulus and toroid – and 12 ordinary glomeruli are visible in the AL. AN, antennal nerve; C, cumulus; Gs, ordinary glomeruli; T, toroid; D, dorsal; L, lateral. Scale bar, 100 µm. (B,C) 3-D reconstruction images of the AL. These images are from two different directions (anterior and posterior). In the anterior image, the toroid and the cumulus are visible. However, the horseshoe (H) is not visible because this compartment resides near the posterior surface. With our optical recording technique, the anterior side of the AL is always oriented toward the MOS-type image sensors.

View larger version (92K): [in a new window]   Fig. 3. Projection pattern of the sensory fibers in the medial nerve (MN). (A) The axons were stained by forward-filling from the cut end of the MN with tetramethylrhodamine–dextran. The preparation was counterstained with Lucifer Yellow to visualize the boundary of the tissue. Serial confocal optical slices were acquired and projected to a frontal plane. In the image, the tetramethylrhodamine–dextran-stained fibers were superimposed on the counterstained image of the brain. Axons originating from the MN run through the medial half of the antennal nerve (AN) and project into the antennal lobe (AL). (B) Projected AL image of optical sections at 50±20 µm in depth. The image is of the same preparation as that in A, with a high magnification. The macroglomerular complex (MGC) was always stained more densely than the ordinary glomeruli. The axons projecting to the MGC are biased towards the medial MGC; however, those projecting to the ordinary glomeruli distribute homologously to each ordinary glomerulus. (C) The projected image of the thick axons projecting to the antennal mechanosensory and motor center (AMMC; 105±20 µm in depth). Most of the thin axons running from the MN project to the AL anteriorly, and most of the thick axons project to the AMMC, passing through the posterior AL. (D–F) Confocal images of an anterior plane (D; 28 µm in depth), a medial plane (E; 50 µm in depth) and a posterior plane (F; 73 µm in depth) of the MGC. The MGC is composed of two compartments: toroid, which is a donut-shaped structure, and cumulus, which is a disk-shaped structure situated more proximally to the AN input. In both the anterior and the medial planes, the stained axons are biased towards the medial MGC: medial toroid and medial cumulus. In the posterior plane, the stained axons are distributed homologously. OL, optic lobe; cT, central toroid; mT, medial toroid; lT, lateral toroid; mC, medial cumulus; lC, lateral cumulus. Scale bar, 200 µm (A), 100 µm (B,C), 50 µm (D–F).

View larger version (94K): [in a new window]   Fig. 4. Projection pattern of the sensory fibers in the lateral nerve (LN). (A) Axons originating from the LN run through the lateral half of the antennal nerve (AN) and project to the antennal lobe (AL). (B) Projected AL image of optical sections at 50±20 µm in depth. The image is of the same preparation as that in A, with a high magnification. The axons projecting to the macroglomerular complex (MGC) are biased towards the lateral MGC. (C) The projected image of the thick axons projecting to the antennal mechanosensory and motor center (AMMC; 100±25 µm in depth). (D–F) Confocal images of an anterior plane (D; 34 µm in depth), a medial plane (E; 50 µm in depth) and a posterior plane (F; 65 µm in depth) of the MGC. In all optical planes, the stained axons are biased towards the lateral MGC; lT and lC. For abbreviations, see Fig. 3. Scale bar, 200 µm (A), 100 µm (B,C), 50 µm (D–F).

View larger version (38K): [in a new window]   Fig. 5. Optical responses in the antennal lobe (AL) elicited by electrical stimulation of the medial nerve (MN) and the lateral nerve (LN). (A) Real image of the moth brain (boxed area of inset). The boxed area in the schematic drawing corresponds to the MOS-type image sensor used by optical recording. Scale bar, 500 µm. OL, optic lobe; PC, protocerebrum. (B) Optical responses in the AL. All optical images were superimposed on the bright real image of the AL. Consistent response patterns in the macroglomerular complex (MGC) were evoked by the electrical stimulation of the medial nerve (MN) or the lateral nerve (LN). The pattern was initially a depolarization of the antennal nerve (AN; 3.6–7.2 ms after the onset of the stimulation) and, subsequently, a depolarization of the MGC (4.8–9.6 ms). At 7.2 ms from the stimulus of the MN, the area strongly (>0.4% of the background fluorescence) responding to stimulation of the MN was restricted to the medial half of the MGC. By contrast, the area strongly (>0.4% of the background fluorescence) responding to stimulation of the LN was restricted to the lateral half of the MGC. (C) Time course of the optical signals in the AL evoked by stimulation of the MN (upper panel) and the LN (lower panel). The optical signal was calculated by averaging signals recorded in areas that had a response of >0.3% (–F/F) at 7.2 ms from the stimulus onset and filtered at 246 Hz (Fc). The response, evoked in the MGC, had a peak at 7.2 ms after the stimulus onset. The response had another slow component (arrowheads) after the first peak of the depolarization. The annodal break was visible just after the stimuli (S).

View larger version (22K): [in a new window]   Fig. 6. Time course of the effect of Ca2+-free saline (A) or 10–4 mol l–1 bicuculline (B) on the optical signals in the antennal lobe (AL) evoked by stimulation of the medial nerve (MN). The optical signals were compared in five square areas (mT, cT, lT, mC and lC). The optical signals were filtered at 246 Hz (=Fc). Scale bar in real image, 100 µm. (A) Slow components (arrowheads) were blocked under Ca2+-free conditions (green lines). Postsynaptic activities (red lines) were calculated by subtracting the response under Ca2+-free saline conditions (green lines) from the response under normal saline conditions (black lines). The excitatory postsynaptic responses (blue lines) were calculated by subtracting each normalized GABAergic optical response from the postsynaptic optical response (red lines). The peak amplitudes of the excitatory postsynaptic responses in the optical signals in the mT, cT and mC were significantly larger than those of the lT and the lC, respectively (see text for details). (B) Bicuculline had no effect on the first peak of the optical signals but increased the second peak of the optical signals (arrowheads on green lines). The possible time course of the GABAergic inhibitory postsynaptic response (orange lines) was calculated by subtracting the responses under bicuculline conditions (green lines) from those under normal conditions (black lines). The maximum amplitudes of the GABAergic inhibitory postsynaptic responses in these MGC sub-regions were not significantly different from each other (double arrowheads).

View larger version (28K): [in a new window]   Fig. 7. Comparison of the postsynaptic activity, evoked by stimulation of the medial nerve (MN) and the lateral nerve (LN), among these macroglomerular complex (MGC) sub-regions. The maximum amplitudes of the three responses, the postsynaptic responses, the GABAergic inhibitory postsynaptic responses and the excitatory postsynaptic responses were compared among different MGC sub-regions. (A) When the MN was stimulated, the maximum amplitudes of the postsynaptic responses in the medial toroid (mT) and central toroid (cT) were significantly larger than those of the lateral toroid (lT). In the cumulus, the maximum amplitude of the postsynaptic response in the medial cumulus (mC) was significantly larger than that of the lateral cumulus (lC). Asterisks show that the maximum amplitudes of these responses are significantly different (P<0.05, one-way repeated-measures ANOVA, N=8). The peak amplitudes of the GABAergic inhibitory responses were not significantly different among these MGC sub-regions. (B) When the LN was stimulated, postsynaptic responses in the lT and the lC were detected, but not in other MGC sub-regions. In the toroid, the maximum amplitude of the excitatory postsynaptic response of the lT was significantly larger than that of the cT (P<0.05, one-way repeated-measures ANOVA, N=7). In the cumulus, the maximum amplitude of the postsynaptic response of the lC was significantly larger than that of the mC (P<0.05, one-way repeated-measures ANOVA, N=7). The maximum amplitudes of the GABAergic responses were not significantly different among these MGC sub-regions except mT. N.D., not detected.

View larger version (20K): [in a new window]   Fig. 8. Time course of the effects of Ca2+-free saline (A) or 10–4 mmol l–1 bicuculline (B) on the optical signals in the antennal lobe (AL) evoked by stimulation of the lateral nerve (LN). The optical signals were compared in five square areas (mT, cT, lT, mC and lC). The optical signals were filtered at 246 Hz (=Fc). Scale bar in real image, 100 µm. (A) The optical signals under normal saline conditions (black line) were superimposed onto those under Ca2+-free saline conditions (green lines). Postsynaptic activity (red lines), blocked by Ca2+-free saline, was evoked in both the lT and the lC (arrowheads). The peak amplitudes of the excitatory postsynaptic responses (blue lines) in the optical signals in the lT and the lC were significantly larger than those of the other macroglomerular complex (MGC) sub-regions (see text for details). (B) Optical signals blocked by bicuculline were detected in all MGC sub-regions except for the mT (double arrowheads on orange lines). The maximum amplitudes of the GABAergic inhibitory postsynaptic responses in these MGC sub-regions were not significantly different from each other.

View larger version (30K): [in a new window]   Fig. 9. The GABAergic inhibitory postsynaptic response (arrowhead) always preceded the postsynaptic responses separated under Ca2+-free conditions (double arrowhead). The sensory responses remained under Ca2+-free conditions, postsynaptic responses separated under Ca2+-free conditions and GABAergic inhibitory postsynaptic responses blocked by bicuculline in each MGC sub-region were superimposed. The delay time of the GABAergic inhibitory postsynaptic response from the first peak (0.2±0.2 ms) was shorter than the delay time of the postsynaptic response blocked by Ca2+-free saline (1.5±0.5 ms) (P<0.05; paired t-test, N=7).

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