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First published online June 7, 2004
Journal of Experimental Biology 207, 2389-2400 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01036

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Development and steroid regulation of RFamide immunoreactivity in antennal-lobe neurons of the sphinx moth Manduca sexta

Joachim Schachtner^1,*, Björn Trosowski¹, Wolfgang D'Hanis¹, Stephan Stubner² and Uwe Homberg¹

¹ Department of Biology, Animal Physiology, Philipps-University, 35032 Marburg, Germany
² Department of Biogeochemistry, MPI for Terrestrial Microbiology, 35043 Marburg, Germany

View larger version (114K): [in a new window]   Fig. 1. Normarski images of 40 µm sections showing cell types I–V of the AL labeled with the RFamide antiserum. (A) Type I cells in the lateral cell group (LC) of a stage P15 AL. Staining within the glomeruli (GL) primarily results from type I neurons. No immunostaining is visible in the median cell group (MC). (B,C) Consecutive horizontal sections through a P13 AL. (B) In addition to glomerular staining, type I cell bodies give rise to stained fibers in the root of the outer antenno-cerebral tract (arrows). (C) Section ventral to B. The large cell body of the type II cell (arrow) has a posterior-medial position within the LC. Inset shows the primary neurite (arrows) leaving the AL without sending processes to the AL neuropil. (Di–iii) Type II–V neurons in a stage P1 pupa. Di and Dii are both superimposed images of two consecutive sections. Diii combines Di and Dii (total of four consecutive sections) with parts of the anterior laying cell groups omitted to better see the connections between cell bodies and neurites. The neurite (double arrow in Di) of the type II cell (large arrow in Dii, iii) leaves the AL towards the tritocerebrum (TC) while the neurite of the type V neuron (double arrowhead in Di) enters the AL. The neurite (arrowheads in Di) of the type IV neuron gives rise to processes that cover most of the developing AL area (solid outline in Di). The cell bodies of type II, III (arrowheads in Dii, iii), and IV neurons (small arrows in Dii, iii) are located anteriorly to the AL neuropil. Small arrow in Di, Dii points toward the connection site of the type IV neuron with its cell body. The type IV cell body lies in a cell group (broken outline in Dii), which is different from that of type II and III neurons (solid outline in Dii). A, anterior; D, dorsal; L, lateral; CN, coarse neuropil; MGC, male specific macroglomerular complex; TC, tritocerebrum. Scale bars, 100 µm (A–C); 50 µm (inset in B, Di–iii).

View larger version (59K): [in a new window]   Fig. 2. Confocal images of 40 µm sections showing double immunostaining in the lateral cell group (LC) of P10 antennal lobes with antisera against RFamide (green, Cy2; Ai, Bi) and GABA (red, Cy3; Aiii, Biii). (Aii, Bii) Overlays of the respective immunostainings in Ai/Aiii and Bi/Biii. (A) RFamide-ir cell bodies in the LC, of which two show double labeling with the GABA antiserum (large arrows in Aii). The arrowhead (Ai) points to the large type II cell body, which like the root of the outer antenno-cerebral tract (small arrows) shows only RFamide and no GABA immunostaining. (B) In another section all type I cells colocalize RFamide and GABA immunoreactivity. Scale bars, 40 µm.

View larger version (109K): [in a new window]   Fig. 3. Confocal images of 40 µm sections showing RFamide immunoreactivity (green, Cy2) during AL development. (A) Area of the larval antennal center (LAC) in a wandering larva (W2, 2-day-old wandering stage shortly before entering the prepupal stage). Within the developing lateral cell group (LC, encircled area) type II (arrow) and III (arrowheads) somata are stained. The LAC is labeled (red) by dextran backfill of the antennal nerve. The double arrowhead marks a dextran-labeled axon bundle coming from the larval antenna. (B–F) Double immunolabeling with the RFamide antiserum (green, Cy2) and the synaptotagmin antibody (red, Cy5). (B) Anterior section through the developing AL showing the type V neuron entering the AL from the tritocerebrum (TC). (C) In stage P9 developing glomeruli (GL) can be distinguished by synaptotagmin immunostaining. RFamide immunostaining in basal parts of the developing glomeruli is weak compared to staining of the type V arborizations (arrowheads) and to staining intensity in stages later than P12/13 (see D–E). In the LC the large type II cell (large arrow) with its neurite leaving the AL (smaller arrows) and smaller type I cells are labeled. (D) Frontal section through posterior parts of a stage P16 AL with strongly labeled type I cells, strong labeling in the glomeruli (GL) and intensely labeled fibers in the root of the outer antenno-cerebral tract (arrows). (E) Oblique section showing typical labeling of glomeruli in an adult AL. The type V cell enters the AL neuropil from the TC (arrow). (F) Magnification from E showing the neurite of the type V cell entering the AL neuropil. A–E, frontal sections; orientation bars in C (D, dorsal; M, medial) also apply to A–D; CN, coarse neuropil; Es, Esophagus; MC, median cell group; MGC, macroglomerular complex. Scale bars, 40 µm (A,B); 80 µm (C–E); 20 µm (F).

View larger version (17K): [in a new window]   Fig. 4. Developmental time course of numbers of RFamide-ir cell bodies in the lateral cell group. Acquisition of RFamide immunostaining in AL neurons occurs in two phases. A first increase from stage P5 to P7/8 results in about 25 cells. The second increase in cell number (up to about 60 cells) occurs from P11/12 to P16. Each data point indicates mean ± S.D. The number of antennal lobes is indicated at each stage; stages P0+P1 and P2+P3 were pooled. The three cells at stages P0 to P4 are the type II and III neurons. The dotted line indicates adult eclosion at stage P20. Inset shows hemolymph concentrations of 20E during the pupal–adult transition as reported by Warren and Gilbert (1986). The open and filled large arrows mark the times of 20E injection at P1, P9 (asterisk) and P10/11, respectively, the open and filled circles (asterisk after P9 injection) mark the times when the injected animals were killed and processed for immunocytochemistry (see Fig. 5).

View larger version (14K): [in a new window]   Fig. 5. Numbers of RFamide-ir neurons in the AL of M. sexta after hemolymph injection of 20E at P1 (A), P9 and P10/11 (B) and dissection several days later (see inset in Fig. 4). (A) Animals were injected with 20E at stage P1 and dissected 2 (P3) to 8/9 days (P9/10) later. Hormone-treated animals (open circles) showed a progressive increase in the numbers of RFamide-ir neurons compared to controls (filled circles) injected with saline. (B) Injections of 20E at P9 or P10/11 and dissection 6/7 days later (P9, open triangles) or 3/4–5/6 days later (P10/11, open circles) resulted in cell numbers that were not significantly different from the control animals (filled circles or triangles). Values are means ± S.D. The number of Als is shown for each stage. Inset in B shows wing pigmentation 6 days after injection of saline (control) or 20E at stage P10/11.

View larger version (61K): [in a new window]   Fig. 6. Confocal images of 40 µm sections showing RFamide immunoreactivity (green, Cy2) and synaptotagmin immunoreactivity (red, Cy5) in antennal lobes (ALs) after hemolymph injection of 20E or after injection of the vehicle alone at stage P1 and dissection 3 days later at pupal stage P4. In A and B, additional Normarski images are shown (gray). (A) After injection of the vehicle alone ALs were indistinguishable from ALs of untreated animals at stage P4. In the lateral cell group (broken outline) two cell bodies are labeled. (B) 3 days after 20E injection the number of RFamide-ir cells in the LC (broken outline) increased to levels normally observed about 2 days later (see Fig. 5A). The glomerular pattern reached a stage normally observed at pupal stage P7/8 (Dubuque et al., 2001). Arrows point to developing glomeruli. (C) Posterior section through an AL showing a dense meshwork of strongly RFamide-ir arborizations, which is normally not observed in P4 or any other pupal stage. Frontal sections; scale bars, 80 µm (A,B); 40 µm(C). M, medial; D, dorsal.