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Neurochemical fine tuning of a peripheral tissue: peptidergic and aminergic regulation of fluid secretion by Malpighian tubules in the tobacco hawkmoth M. sexta

N. J. V. Skaer1, D. R. Nässel2, S. H. P. Maddrell1,* and N. J. Tublitz3

1 Department of Zoology, Downing Street, University of Cambridge, Cambridge CB2 3EJ, UK
2 Department of Zoology, Stockholm University, Svante Arrhenius väg 16, SE-106 91 Stockholm, Sweden
3 Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403 USA



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  		Fig. 1. Effects of serotonin and octopamine on fluid secretion by isolated pharate adult M. sexta Malpighian tubules. (A) Effects of serotonin at three different concentrations. (B) Effects of octopamine at three different concentrations. In this and all subsequent figures, fluid secretion data are normalized to the rate immediately prior to test substance application. Values are means ± 1 S.E.M. (N=5). Each control trace in A and B represents data from a single, separate trial.

 


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  		Fig. 2. Effects of 1 mmol l-1 cyclic AMP (cAMP) and 1 mmol l-1 cyclic GMP (cGMP) on fluid secretion by isolated pharate adult M. sexta Malpighian tubules. Cyclic nucleotides were added at the time indicated by the vertical line. Values are means ± 1 S.E.M. (N=5). Control trace represents data from a single trial.

 


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  		Fig. 3. Effects of leucokinin I (LK-I) at three different concentrations on fluid secretion by isolated pharate adult M. sexta Malpighian tubules. Values are means ± 1 S.E.M. (N=5). LK-I was added at the time indicated by the vertical line. Control trace represents data from a single trial.

 


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  		Fig. 4. Effects of individual cardioacceleratory peptides (CAPs) on fluid secretion by isolated pharate adult M. sexta Malpighian tubules. See text for CAP dosages. The CAPs were added at the time indicated by the vertical line. Values are means ± 1 S.E.M. (N=5, except for CAP 1a/1b where N=6). Control trace represents data from a single trial.

 


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  		Fig. 5. Effects of tachykinin-related peptides (TRPs) at four different concentrations on fluid secretion by isolated pharate adult M. sexta Malpighian tubules. (A) Effects of Locusta TK-1, (B) Leucophaea TRP-1 and (C) Leucophaea TRP-4. The TRPs were added at the time indicated by the vertical line. Values are means ± 1 S.E.M. (N=3). In each panel, control traces represent data from a single, separate trial.

 


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  		Fig. 6. Interactions of leucokinin I (LK-I) and cyclic AMP (cAMP) on fluid secretion by isolated pharate adult M. sexta Malpighian tubules. LK-I (60 µmol l-1) or cAMP (1 mmol l-1) were applied at the times indicated by the arrows. Solid line and arrows: cAMP added first followed by LK-I; broken line and open arrows: LK-I added first followed by cAMP. Values are means ± 1 S.E.M. (N=8).

 


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  		Fig. 7. Tracings of LTKLI neurons in the brain of a fifth instar larva of M. sexta. (A) Cell bodies of the brain (filled cell bodies are posterior, unfilled anterior). DN, cell body of the large descending neuron. (B) Tracing of cell bodies and processes of some of the major posterior LTKLI neurons. Note the varicose fibres distributed in the brain neuropil and in the four commissures connecting the hemispheres. The arrow indicates two of the ascending axons derived from the ventral nerve cord, with arborizations in the tritocerebrum and protocerebrum. DN, the large descending neuron with processes in the protocerebrum and axon (also at arrow) to the ventral nerve cord. Scale bar, 100 µm.

 


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  		Fig. 8. Micrographs of LTKLI cells in M. sexta. (A) Whole mount of brain of fifth instar larva. (B) Other focus and higher magnification shows varicose branches of LTKLI neurons in larval brain. Note fibres in commissures. (C) Unfused abdominal ganglion with LTKLI fibres in neuropils and entering from anterior nerve root (arrow). Cell bodies are in other focal plane. (D) Terminal abdominal ganglion with LTKLI cell bodies (arrows) and fibres in neuropil. (E) Surface view of LTKLI endocrine cells of the midgut (e.g. at arrows). The cells appear irregularly distributed partly because the intestine became contracted at fixation. (F,G) Immunoreactive endocrine cells seen in longitudinal view. Gut lumen is at bottom of panels. Scale bars: 100 µm (A); 50 µm (B-D); 100 µm (E); 25 µm (F,G).

 


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  		Fig. 9. Micrographs of cryostat sections of brain from pharate adult M. sexta labeled with antiserum to LomTK. (A) Fibres in the upper division of the fan-shaped body of the central body complex (frontal section). Note also fibres in superior median protocerebrum (above central body). (B) Fibres in lower part of mushroom body calyx (large arrow) seen in frontal section. Also in the upper parts there are thinner LTKLI fibres (small arrows). (C) Antennal lobe with LTKLI fibres in all the glomeruli. Note also cell bodies (arrow) which are part of a cluster of about 30 neurons supplying LTKLI fibres to the glomeruli. (D) Also in the macroglomerular complex of the antennal lobe there are varicose LTKLI fibres. (E-G) Immunoreactive neurons in optic lobes (overview in G). (E) Large cluster of cell bodies at the anterior base of the medulla. (F) Fibres in a thin layer of the medulla. (G) An overview of the medulla (Me), lobula (Lo) and lobula plate (LP) is shown in horizontal section. Note immunoreactive fibres in medulla and lobula plate (arrows). Scale bars, 50 µm (A-E); 100 µm (G).

 


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  		Fig. 10. Tracing of LTKLI cell bodies in ventral nerve cord of fifth instar larva of M. sexta. SOG, suboesophageal ganglion; T3, metathoracic ganglion; A5, fifth unfused abdominal ganglion; TAG, fused terminal abdominal ganglion. The median neuron in T3 is dorsal, other neurons are ventral. Scale bar, 100 µm.

 

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© The Company of Biologists Ltd 2002