Experimentally induced release of a neurohormone influencing hemolymph trehalose level in Calliphora erythrocephala (diptera)

GESERAL

AND

COMPARATIVF:

EiYDO(‘RINOLOGT

Experimentally

12, 449-459

Induced

(1969)

Release

of a Neurohormone

Influencing Hemolymph Trehalose Level Calliphora erythrocephala (Diptera)

in

Evidence is presented that the corpus cardiacum (c. card.) of the blowfl> Calliphora is the source of a neurohormone, which in less than 20 min after stimulation may cause a significant rise in the hemolymph trehalose level in uiuo. Sssay is based on thin-layer chromatography of hemolymph samples from before and after the experiment for each individual fly, and the effect is recorded as the difference in size and drnsity between the two spots, not as absolute trehalose amounts. Electrical stimulation, combined with various surgical experiments, has shown that the c. card. may br activated via the brain to release the hyperglycemic hormone, provided that, the nerve connection betwvcrn the brain and the c. card. (the “~ardinc-rrcurrcnt, nrrvc”) is intact.

In an clcctron microscope study (Norhas shown that stimulation of the brain mann, 1965) of the corpus cardiacum of evokes the release of a cardioaccelerator the blowfly, C’ctllipimx eryth,rocephnla, this from the corpus cardiacum of Periplaneta. organ appeared to offer crrtain advantages Kater’s experiments, which in important for the study of some, perhaps general, respects are very similar to ours (see Disnturosecretory mechanisms. Thus the re- cussion): did not, come to our knowledge lease of neurosecretory granules (by exo- till after t’he completion of the present cyt.osis 1 and its dependence on impulses work. probably conducted by neurosecretory As n-ill be considered later, electron ins.1 cells would seem open to analysis by microscopy of corpora cardiaca from flies so stimulated might be expected to elucombined electrophysiological and cytological techniques. As a first approach it cidate furt,her the mechanism of secretion. was planned to provoke the release of hor- However, since this must still rest on pure monee by electrical stimulation and, sub- morphologic evidence, it appeared necesscquently, to inspect the preparation by sary t’o find a bioassay allowing a strict clrctron microscopy. correlation to be made between cytological That electrical stimulation mav induce ant1 physiological evidence for hormone ;I rclea~ of stainable n.s. matel:ial from liberation. For that purpose secretions with the corpus cardiacum (r. card.) has been a rather short-term action would be prefshow11 for Bluberus crnniifer by Hodgson erablc. The best known active principles of this an<1 Gcldiay (1959). This could be correlatecl with a reduced content of a phar- typr, arc a heart-accelerating substance n~acologically active substance in the (review by Davey, 1964‘1 and a hormone Ol'pill. In Xchisfocerra gregaria Highnam positively influencing the concentration of I 1961, 1962) similarly ohtained a depletion trehalosc in the hemolymph (Steele, 1961, 1963; Bowers and Friedman, 1967). of 11,s:. mattcrial from the c. cartl. by electrical Ftimulation. Recently Kattr (1968) This paper describes an investigation of 449

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the latter effect in Calliphora. The hemolymph trehalose level, and the influence upon this of the different experimental procedures, has been measured by thin-layer chromatography, and electrical stimulation combined with various surgical experiments have provided evidence, firstly that it is possible to provoke the release of a hyperglycemic neurohormone by electrical stimulation of the brain, secondly that this substance is liberated from the c. card., and t,hirdly that, the liberation will take place only if the nerve connection between brain and c. card. is intact’. MATERIATS

AND

METHODS

Virgin, adult Cnllipl~om females, kept at, 25”, were used for all experiments. Within 2 hr after hatching the flies were placrd individually in small cages and frd sugar crystals and water ad libifum. The flies to be used-age 4 to 5 dayswere lightly anaesthetized with ether. Shortly after immobilization the fly was mounted in a dish by means of two strips of plasti&rr. one over the thorax, the other holding down thr head in a forward t,ilted position. The stretc*hc~d neck mcmbranc was partly removed, allowing hemolymph to be withdrawn. At the S:UIIC time the c. card. and adjacent organs, bring drawn out from the thorax, could be observed and, when desired, operated upon (see later). The posterior surface of the head, now nearly horizontal, could be picrcned by the> st imulnting electrodes. Some of the operations took place under Ringer. This Ringer represents a first step in the development of a Calliphora-Ringer. It has the following composition : NaCl, 7.48 g; KH?PO.,, 1.39g; Na2HPOI. l.OOg; CaCl, 6 H,O, 0.095g; MgCL, 6 H,O, 0.030 g; benzyl-pcnicilline, 0.040 g. Water to 1000 ml. pH = 6.8. Semi-isolated hearts may survive and maintain pulsation in this solut,ion for more than 24 hr (Normann, unpublished). Hemolymph Samples. Hemolymph was wit,hdrawn from the neck of the fly by means of a 0.5~pl capillary micropipette. Paraffin-coating of the pipette ensured perfect repeatability in delivering the liquid, and for every fly the same pipette was used for both first and second withdrawal of hemolymph. Withdrawal of these samples, amounting to at most one-tenth of the total hemolymph volun~r, did not affect the flies detectably. (According to later measurements, the total hemolymph volume may vary krt,ween 7 and 15 ~1 in 4-5-day-old stigar-fed flies from the

AXD

DUVE

&rain used for our esprrimc~nts (H. Duve, unpublished)). Electrical Stimulation. Bipolar stimulation electrodes were mado from stain&+steel insect needles, clcctrolytically pointed to a tip diameter of a few microns. The electrodes were insulated with lacquer to the tip. For cvcry two flies stimulated the clcct,rodrs wvcrp rcpointcd and insulated, lmxust~ of erosion produced during stimulation. The> two cl(~ctrotlc~-sl)a~etl 0.8 mm apart-werr mounted in a micropositiomr, allowing them to I)r inxrrted rather precisely on the dorsal surface of the brain through the head capsule. When in tjosition the, elcrtrode tips were situated s?-mlllc~t,ric,:lll?at either aide of the medial muroaccrctory ~~~11s and in the neighbourhood of the lateral ns. cells. Rc~ctangular pulws (F&10 V) with a duration of 2 mscc wcrc dclivcrcd by a “Disa ministim” ~liniul:~tor. Freqtirncy and duration of stimulation will Ix> mcntioncd later. Stimulus voltage and cmrent (0.05-0.1 mA) were monitored by means of a cld beam oscilloscope. The ware-form of stimulus current showed a slight but constant ckviation from the rectangular shape (ser Rowcll, 1963). 2’hiu-Layer Chron&ograph~. The thin-layer t)l:rtes wcr(l prepared with Desaga equipment, tt,qing 30~ Kisclghur G powder per 60 ml water. Aliquots of 0.5~1 standard carbohydrates and hemolymth (0.5 ~1 hcmolymph diluted with 5.0 ~1 10% isopropanol) were applied. Chromat.ography took place in a Dcsnga tank containing 65 ml ethylacc>tate f 35 ml isopropanol and distilled water 2: 1. The solvent was allowed to move 10 cam from the starting point (about 25 min). After chromatography the plates were dried under a stream of void air until all solvent had been removed. The plates wcrc then sprayed with a solution of anisaldchy-de/cone. HSO, (Randerath, 1965) and dcvrloped 15 min at 100”. Smsitivity wan about 0.05 ,ug. Ezlalttation of Chromatograms. Since we have not yet found a quantitative method allowing accurate measurement of the trehalose content of thr 0.5-~1 hrmolymph samples, we had to make determinations on the basis of the devrloped chromatograms, the colour reaction of these being amply sensitivr. However, not having ac-cess to equipment for direct drnsitometric evaluation of the plates. we designed the following photometrical method for USC with photographic nrgativcs. 2111 rhromatograms were photographed on 7 X ‘7-(&m Agfa AGEPE negative plates. The spots of trehalose (and glucose) appeared light on the nrgntivcx and tmnsmission through t,hese spots

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ww found to be linearly correlated with the amount of material (see later in t,his section). Transmission was measured with a modified Zeiss photometer (for microphotography). The photocell was enclosed in a chamber with a window of opal glass rendering the inside light diffuse. The aperture was ovoid to fit the shape of the spots and was 10 mm in length. For measurement the negat,ive was placed in the enlarger and the image focused on the level of t)he which was surrounded by photocell window, white cardboard to make the spots visible. Magnification was adjusted to allow the largest spot of the negative just to fit into the window. For every spot to be measured the photocell was first moved to a region adjacent to the spot. Here the amplifier was adjusted to give zero reading (the background density). Then the photocell was moved to let all the light of the spot into the window, whereafter the reading was made. For all determinations great care was taken to standardize the zero adjustment procedure (compcnsation for variations in background density). The two samples from each fly (before and after experimental treatment) were always spotted next to each othrr and then compared on the basis of the photometer readings. For each fly we obtained a figure wliich for convenience WC shall term the trehalose index, or TI, and which represents the photometer reading of the second

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sample in percentage of the first reading. For example, with a photometer reading for the first spot of 0.25 PA and for the second 0.45 PA, the TI for that fly will bc (45 X 100)/(25) = 180. Provided the size of the spots dots not exceed that of the photocell window, plotting of different trehalosc concentrations results in linear standard curves (cf. Fischer, Parsons, and Morrison, 1948). We therefore feel justified in regarding the trehalosc indices as approximately reflecting the true changes in the hemolymph trchalose content of ilip flies. To compensate for the individual variation of the flirs, both with regard to initial trchalose level and responses to experimental treatment, we decided to make enough experiments to ensure that for each group the significance of the difference between the averages for the experimental flies and the controls would be as high as p < 0.001. Statistical treatment (t test) was carried out according to Croxton (1953). The initial hemolymph trehalose levels recorded appeared rather variable (see Fig. 1); we tentatively ascribe this to diurnal fluctuations modified by the actual physiological state of the animal. However, response to experiments was rrrordcd as a difference in hcmolymph trehalose content in one and the same fly before and after exprrimental treatment. Moreover, for each experimental animal the corresponding control was

FRONT GLUCOSE

FIG. 1. Chromatogram with two C experiments, showing spot,s of trehalose and glucose. The spots were applied from right to the left. The following trehalose indices were recorded: First control (Cl), TI = 100; i.e., no change. First experimental recipient (E,), TI = 130. Cz, TI = 130. Er, TI = 190 (trehalose level nearly doubled). Note the difference of the first spots (initial levels) for example between Ci and CL The standard trehalose spots (and those of glucose) contain 0.5 pg (Si) and 1.0 pg (St). When compared with the spots of the hemolymph samples it should be noted that these were diluted 1:ll before aliquots of 0.5 ~1 were applied on the plate. Spots of glucose were generally present although usually smaller than the trehalose spots. In many cases they decreased or disappeared during the experiment (see Es). An increase might, however, also occur.

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made immediately afterwards. Thus by perimental and control flies alternately sider them comparable as groups with rearing and sampling conditions. EXPERIMENTS

Experiment

AND

A: Electrical

using exwe conregard to

RESULTS

Stimulation

This group consisted of 21 flies stimulated via the brain. After withdrawal of the first hemolymph sample for chromatography the flies had the stimulating elect,rodes implanted as previously described. The stimulator was turned on before the electrodes were introduced through the head capsule, and the tips were advanced until jerks of the legs and wing bases indicated that the surface of the brain had been reached. Stimulation lasted 15-20 min, whereupon the second hemolymph sample was taken. One half of the flies were stimulated at a pulse repetition rate of lO/sec, and thus received 10-12.000 shocks. The rest of the flies received only about 5.000 shocks (5/set). However, no difference in response could bc detect,ed between the two subgroups (see Discussion), and statistically they were treated as one group. As Fig. 2 shows, the mean trehalose index resulting from stimulation amounted to 266 t 99 (SD). Thus on an average the trehalose content of the hemolymph more than doubled in 20 min. Not a single fly failed to show at least some rise in trehalose content, the weakest response being a TI of 130. Controls. For every fly stimulated as described above, a control was made immediately afterwards. (Two controls were lost). Great care has been taken to treat the controls exactly as the stimulated flies. The same micropipette was used for hemolymph collections, and the fly had electrodes implanted in the same way. Again shocks were given during introduction of t.he electrodes, the stimulator being swit’ched off as soon as t’he jerks of the ar,pcndages showed that the tips were in place. The control spent the same time in the fixed position as the stimulated fly, t#he difference in treatment being only that of the number of shocks given Cup to 20 for the cont,rols).

AND

DCVE

O/o 3603403203002802602&O-220-m 200-p IBO160ILO-

I

120IOOA

@

B

C

FIG. 2. Hemolymph trehalose levels after experiments. Group averages and their SD expressed as percent,age of initial level: the “trehalose indices.” Control groups cross-hatched. Group A. Stimulation through bipolar electrodes implanted in brain; II = 21. Controls. Electrodes implanted, a few shocks given at beginning (see text); ,L = 19. Level of significance: p < 0.001. Group B. Stimulation as group A. Recurrent never with nervi rorporis cardiari and aorta cut; IZ = 18. Controls. Stimulation as (1; aorta with nerves cut behind c. card. n. = 17. Level of significance p < 0.001. (iroup C. Effect of injection of hemolymph bathing c. card. from donor fly, st,imlllated as in A; IL = 24. Controls. Injection of hemolymph from same donors, withdrawn before stimldation; n = 23. Level of significance: p < 0.001.

A certain rise in trehalose level might occur in t’he controls (see Fig. 2). In one case the TI was as high as 170. However, the average TI for the controls only amounts to 107 2 32 (SD), and the difference between this figure and t’he mean for the experimental flies (266 I+ 99) is highly significant, (p < 0.001). As will be considered later, the mere handling of the flies inevitably introduces a certain degree of “strrss,” which almost, certainly influences t’hc trehalose 1~~1.

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Meut-Fed Flies. Since sugar and water alone may be characterized as a “maintenance diet,” whereas meat is necessary for the females to carry through the reproductive cycle (Fraenkel, 1940; E. Thomsen, 1952), it seemed of interest to ascertain that the effect of stimulation on trehalose level in meat flips is similar to that found in sugar-fed flies, in spite of differences with regard to mrt,abolic conditions. To t,hat end experiment, “A” was rcpentrtl wit,11 :L group of 4-day-old meat-fed flies. Ten of these were stimulated and ten flica served as controls. After the second hemolymph collection every fly was dissected to ascertain that growth of ovaries was normal. The mean TI for the stimulated flies was 189 +- 63 (SD) and 125 t 45 for the controls. The difference between the stimulat,rd sugar fly and mt>at fly groups and between the two control groups respectively was not significant. For the meat flies, however. the differencebetweenstimlllat,edand control groups was again significant (p < 0.05). For practical reasons WP dGdrd to continue using supar flitIs. Experiment B: Stimulation after of Yerue Connection between oncl Corpus Cardiacusn

Section Bra&

Though the experiments described (“A”) were based on t,he assumption that the c. card. might, be provoked to liberate a hyperglycemic substance after stimulation of the brain, our positive result might nevertheless be interpreted in different ways. Thus t,he substance in question might, be produced in and liberated from the brain itself, this region being directly affected by the stimulation. Alternatively, some organ outside the brain might be affected by electrotonic spread. (At this point it may be mentioned that, while the electrode tips are spaced 0.8 mm apart, the distance between these and the c. card., when the fly is in position for stimulation, is about 2 mm). We consequently decided to examine the effect, of stimulation after transection of t,he nrrvc connection between the brain and the c. card. If this were to abolish the hyperglycemic effect of stimulat,ion, it would appear to rule out the two alternative c~xplanations mentioned, and would also strengthen the hypothesis of impulse conduction and transmission from brain to c. card. (Normann, 1965). Kerve section was carried out as follo\vs.

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The fly was mounted in the dissecting dish as for stimulation. The operation was made under Ringer. After removal of the two neck muscles crossing each other above the c. card., the “cardiac-recurrent nerve” (E. Thornsen, 1954) was cut together with the aorta. This operation was quickly performed, and when transferred to its cage the fly recovered its ability to walk and fly in lo-15 min. Three hours later the first hemolymph withdrawal was made, and the flies were stimulated with 10 shocks/set for 20 min. (All manipulations, including the hemolymph collect’ions, were well tolerated by the flies, which were all alive the day after the experiment). As can be seen from Fig. 2, these flies did not respond t’o electrical stimulation, their TI being 102 + 21 (SD). The difference between this group, which consisted of 18 flies, and the stimulated “A” flies is significant (p < 0.001). Except for the operation they were treated exactly as group A. Thus w,c always made an experiment of the “A” type and ran the hemolymph samples of this on the same chromatography plates as were used for the B experiments to ascertain that the whole set-up functioned in a normal manner. These “controls” const,itute part of the material of group A. Furthermore, since the nerve cut belongs to the 3tomatogastric syst,em, and in fact consists of the rccurrent nerve (Fig. 3) together with the ncrvi corporis cardiaci, whereas the ventral connective between the brain and the thoracic ganglion was unsevered, stimulation could still hc seen to produce appendage movements. We thcrcforc suggest that, the lack of effect of stimulation is due to the intcrruption of the nervous pathway from the brain to the c. card. C’ontrols: doytn

Stiwulntion after Section of the behind the Corp~rs Cnrdiacum

As control for experiment B, we decided to examine the effect of stimulation after section of the aort’a hchind the c. card. This operaCon was carried out, like the first one. These controls t,hrn had an intact connection bctwcm brain and c. card., whereas

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ti.R.

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N.OES.

FIG. 3. The corpus cardiacum (c. card.) and some adjacent organs, seen from the left. The c. card. and the hypocerebral ganglion (G.H.) are located in the triangular space between the oesophagus (OES.), the aorta (A.), and the proventriculus (PROV.). The corpus allat,um (C. All.) is innervated by two nervi corporis allati (N.C.A.), one on either side of the aorta. The c. card. and the hypocerebral ganglion are connected with the brain by the “cardiac-recurrent nerve,” consisting of the nervi corporis cardiaci (not shown), which have joined the recurrent nerve (N.R.). Behind the hypocerebral ganglion two pairs of nerves are found; viz., the nervi oesophagei (N. OES.), which innervate the crop duct, and the aortic nerves (AOR.N.), which run backwards on either side of the aorta. The length of the c. card. proper is 100 ,.L In experiment A the whole complex was intact. For experiment B the nerve connection to brain together with the aorta were cut just anterior to the c. card. The controls had the aorta and the aortic nerves cut behind the hypocerebral ganglion. For experiment C the nerve connections of the donor’s c. card., including the innervation of the oesophagus and the aorta wall, were all cut. The aorta and most of the hypocerebral ganglion were removed. Only the cardiac-recurrent nerve was left intact.

the operation involved the cutting of the aorta and the ‘Laortic nerves,” w,hich emerge from the retrocerebral complex (M. Thomsen, 1969) and which probably correspond to the lateral cardiac nerves described in other insects. The operat’ed controls had 3 hr to recover and were then stimulated. As Fig. 2 shows, these flies did respond t’o stimulation level. The

with The

a rise mean

in TI

hcmolymph was 136

t

trehalose 26 (SD’).

difference bctwcen this and the mean of the first, B group (102 * 21) is significant (p < 0.001). We regard this as supporting importance

our

conclusion of an intact

between

brain and c. card. C. Injection

with regard to the nerve connection

Ezperimen ts

The experiments so far described seem to indicate that stimulation of the brain may cause secretion of a hyperglycemic substance from an organ innervated by the so-called cardiac-recurrent nerve. Secretion must t,hus he due to some part of the

retrocerebral endocrine complex, which, however. in addition to the c. card. consists of the corpus allatum, the aorta wall containing neurosecretory axon endings, andin young adults-remnants of the thoracic gland (Normann, 1965; M. Thomsen, 1969). Direct. evidence that the active principle is liberated from the c. card., as in other insects (SW Discussion), could be obtained through the injection of corpora cardiaca extracts. Unfortunately, all such extracts (regardless of method of preparation) failed t,o give positive responses comparable to that of the A experiment. A further complication was that control injections (for example of Ringer solution) alone might bring about a rise in trehalose level. This problem will be dealt with in the Discussion. Assumirlg that the c. card. must be the source of the hyperglycemic hormone, we decided to make a new approach and above all t’o omit, any use of art.ificial solutions which might interfere with trehalose produrtion. The idea was to separate the c.

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card. from all other organs, leaving only the nerve comrection with the brain intact, and to keep the gland isolated, immersed in a small volume of hemolymph, during stimulation via the brain. This particular sample should be enriched in hyperglycemic hormone compared to the rest of the hemolymph, only if t’hc factor was actually liberated from the c. card. In order to test this, every experiment would demand the use of three flies; namely, one donor fly, one recipient to be injert’ed with the sample bathing t,he donor’s c. card. during stimulat,ion (‘lexperimental recipient”), and one control recipient t’o be injected with “ordinary” hemolymph from the donor. Experimental routine was as follows. The donor /I?/ was etherized and mounted for dissection and st,inmlatcd in the usual way. At first O.fi ~1 hemolymph was withdrawn, and the sample was injected into a recipient from which a hcmolymph sample for chromatography hat1 already been taken. This recipient was the control. Twenty minutes after the injection the second hemolymph collection was made. Meanwhile, t.hc c. card. of the donor was dissected free of the adjacent, organs (Fig. 3). With a fine scalpel it was first separated from the aorta, whereby the ncrvi corporis allati were also cut. Then the posterior nerves were severed (the nervi oesophagei and the aortic nerves). The c. card. and the cardiac-recurrent nerve just in front of it had to be separated from the oesophagus. It was not possible to remove totally the hypoccrchral ganglion, since t’his is partly fused with the c. card. The opcration was difficult, because the use of Ringer had to be avoided. However, the bluishwhit’e colour of the glancl was a great. help. When dissection had been successful, the c. card., including parts of the hypoccrehral ganglion, had hcen severed from all other organs except the hrnin, with which it was still connected hy the cardiac-recurrent nerve. The c. card. could now be sucked into a micropipett’e (the same as previously used for the control sample), mounted in a Leit’z micromaninulator. Care was t.akcn to suck

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the same vol~mc of hemolymph (0.5 and to avoid injuring the nervous PI), connection from the brain, now entering the “chamber” containing the gland surrounded by hen~olyn~l~l~.The donor fly was t’hen stimulated for 30 min (10 shocks per second). The hemolymph that had been bathing the c. card. during stimulation was now injected into the experimental recipient, from which the first hcmolymph sample had already been taken. hfttr nnothcr 20 min the second hemolymph sample was withdrawn. These samples were t,hen chromatographcd together with those of the control recipient, which had received an injection of hcmolymph collected from the same donor before stimulation (Fig. 1). The trehalose indices of flies having had this “physiological extract” injected show a mean of 169 t 47 (SD), the corresponding figure for the controls being 111 t 28 (Fig. 2). Significance is at the level of p < 0.001. We therefore conclude that the hyperglycemic hormone is in fact secreted by the corpus cardiacum. up

DISCUSSION Our experiments support the hypothesis that, the c. card. may bc activated to release a hyperglycemic hormone by electrical stimulation of the brain, and that this response is directly dependent on nervous conduction. Our findings are well in line with those of Kater (1968). Kater has showu that stimulation of the brain of Pcriplrr),etn may evoke the release of a potent cardioaccclerator from the c. card. and that, this response is dependent on nervous connection between the brain and corpora cnrdiaca. In Periplnnetn the different IICI~S to the c. card. are anatomically well separated (in contrast to Cnll&horn) , ant1 hv cutting particular nerves to t,he c. carcl. Katcr was able to show that only the nervus corporis cardiaci I was important for the eliciting of the release of the rardionccclerator by brain stimulation. Elcct8rically induced liberation of n.s. material has earlier heen reported not only in other insects, as mentioned in the In-

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troduction, but also in crabs (Cooke, 1964) and an oyster (Nagabhushanam, 1964). Corresponding results have been obtained with vertebrates, both for the caudal n.s. system of fishes (Fridberg, Iwasaki, Yagi, Bern, and Nishioka, 1966) and for the hypothalamo-ncurohypophyseal system (e.g., Harris, 1947; Douglas and Poisncr. 1964; Jasinski, Gorbman, and Hara, 1966). In different n.s. systems hormone liberation t,hus seems to be under neural control. The responsible mechanism may be of a grneral character and probably should he linked with the increasing body of evidence for the neuronal (impulse-conducting) nature of ns. cells (see survey by Knowles, 1967). Our experiments do not allow us to decide from which part of the c. card. the hyperglycemic factor is being secreted, because the so-called extrinsic and intrinsic portions are not anatomically well separated. Some indirect evidence may, however. be relevant to that problem. In the desert locust, Highnam (1961) has found that’ an “adrenalin-like” substance (not necessarily identical with the hyperglycemic factor in Cnlliphora) is produced by t,he intrinsic cells of the c. card. In other insects, injection of extracts of corpora cardiaca has generally resulted in a marked elevation of hemolymph trehalose (Steele, 1961; McCarthy and Ralph, 1962; Bowers and Friedman, 1963; Ralph and McCarthy, 1964; Friedman, 1967, and Dut’rieu and Gourdoux, 1967). Steele (1961) reported that control inj.ections of brain ext’racts may have a slight hyperglycemic effect’, but. ascribes this to traces of cardiaca tissue remaining attached t,o the brain after dissection. Ralph and McCarthy, on the other hand, who also obtained a definite, although weak, effect. of brain cxt’racts, considered the possibility t’hat the hyperglycemic hormone mav be produced by n.s. cells in the brain to he then stored and released from the c. card. In this connection it should be mentioned that gel filtration analysis of insect hyperglycemic principle by Natalizi and Frontali (1966) has revealed that t.wo different hyperglycemic hormones may hc present in

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the corpora cardiaca. These hormones could be of a different origin. The effect on trehalose level of brain extracts might still be explained otherwise. In int’act animals the system responsible for the mobilization of glycogen and product.ion of trehalose seems t,o be very sensitive. Brain extracts may contain factors which at sufficiently high concentration may have direct or indirect effect on trehaloae production. In view of the findings of Steele (1965)) Murphy and Wyatt ( 1965’)) and Wiens and Gilbert (1967) with regard t.o certain properties of the enzyme system, such as the effect of inorganic ions, it may be suggested that ionic constituents of such preparations may also have an effect. This could explain the rise in trehalose level, which we encountered as a result of injection of Ringer solution. Anyhow, the c. card. is the source of a hormone directly influencing trehalose synthesis (though not necessarily the site of its synthesis) ; this has also been found by in vitro experiments in which the fat body has been incubated with extracts of the gland (Ralph, 1962; Wiens and Gilbert, 1965, 1967; Friedman, 1967). Release of this hormone can be experimentally provoked, and as earlier mentioned it has been t,he main purpose of the present study to obtain physiological evidence for secretory activity in order to study the accompanying cellular phenomena with the electron microscope. The result’s of this examination will he published separately (Normann, 1969). With regard to the physiological significance of the hyperglycemic neurohormone, our results are in accordance with a concept of a short-termed effect; viz., a mechanism for the rapid mobilization of reserve carbohydrates, advantageous for the insect in situations of “strrss.” This term was adopted by Hodgson and Geldiay (1959) to describe certain conditions leading to depletion of c. card. hormone. We use the word “stress” to tlenotc any situation of discomfort or the like, which dcmands an increased energy expenditure (not unlike provocation of adrenalin secretion in vcrtehrates) .

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In pilot experiments we recorded a rise in hemolymph trehalose content immediately after stimulation for only 5 min. Other pilot’ experiments seemed to indicate that stimulation for more than 20 min might, not result in a much higher trehalose index, and we consequently used t,hat period for nearly all experiments. As earlier inentioncd, a stimulation frequency of 5/ set resulted in a TI nearly equal to that brought about by IO shocks/set. Probably hormone output is proportional to the number of shocks administered, and is therefore larger at higher frequency. However, one limiting factor seems to be the phosphorylase activity, which may be kept, at a maximum by a hormone titer produced by less int.ense stimulation than WChave used. The trehalose level at any moment is dependent on additional factors. Thus trehalose it’self act’s on t,rehalose product,ion through at least one negative feedback mechanism (Murphy and Wyatt, 1965; Friedman, 1968). Moreover, the expenditure of trehalose during the experimental period may be of significance although impossible to evaluate. The flies wake from the anaesthcsia and use trehalose when attempting t’o get loose. We always anaesthetized t’he flies lightly in order to minimize t’he effect on &culat,ion. Delayed recovery of heart pulsation may be the cause of the weaker response to stimulation, now and then encountered. It is interesting that, in the control flies (group A) there was a certain rise in trchalose level (mean 7%, see Fig. 2). Probably this is due to stress caused by experimental handling, although a pharmacological effect of etherization on the neuroendocrine system cannot be excluded. The mean trehalose indices of the controls of experiment A and C, both having an intact “stress-reactive” system, should be compared to the experimental group B. Though intensely stimulated the latter appeared practically unaffected as regards the trehalose level. When carrying out experiment B, we were not’ aware of the probable significance of the ionic conditions which became apparent, in connect.ion with the injection

NE~ltOHO1~MOiTE

4*57

experiments. Although the preceding operations were made under Ringer solution, we nevertheless regard the B experiments as satisfactory, because the flies had at least 3 hr to recover before the actual experiment was made. In that period homeostatic conditions must haye been restored, and in fact the trehalose content of the first hemolymph sample was at, the usual level. However, although the operated controls did show a significant rise in trehalose level after stimulation, this increase was rather small compared to the experimental groups A and C. Whether this decreased response could be due to the application of Ringer or to the interference with a more specific physiological mechanism (e.g., an effect on heart activity of cutting the aortic nerves) ha:: not yet been rstablislicd. The somewhat smaller average TI for the C experiment than for group A was only to be expected, because the operation is difficult and may cause some damage to the cardiac-recurrent, nerve and so reduce the responsiveness of the n.s. system. Besides, the chamber containing the c. card. during stimulation must have contact with t#he hemolymph outside at t#he opening through which the nerve ent,ers; the ‘(hermane-enriched” hemolymph inside may therefore hc somewhat diluted. On the other hand it is of interest, that the control recipients show virtually the same TI as the controls of experiment ,4. This means that injection of hcmolymph from another fly does not per se have any recognizable effect’ on t’he reripicnts. .ACKNOWLEDGMENTS We are greatly indebted to Prof. C. Ovrrgaarrl Nielsen, Ph.D., for raluablr discussions and suggestions during the course of this work and the preparation of the mannscript. The work has received support, from the Danish State Research Foundation and by a grant to one of us (T. N.1 “JX@bmand i Odense Johann og Hannca from Weimann. f. Ssedorffs lrgat.” REFERENCES ROWERS,

ization corpus

V. S.. .~ND FRIEDMAN, S. (1963). Mobilof fat, body glycogen by an extract of cnrdiacum. Nnt~re 198, 685.

458

NOIZMANN

1. M. (1964). Electrical activity and rcleas> of neurosecretory material in crab pericardial organs. Comp. Biochem. Physiol. 13, 353-366. CROXTOIY, F. E. (1953). “Elementary Statistics with Applications in Medicine and the Biological Sciences.” Dover, Kew York. D.AWJY, K. G. (1964). The control of visceral muscles in insects. In “Advances in Insect Physiology,” Vol. 2, pp. 219-245. ilcademic Press Xew York. DUTRII~, J., AND GOURDOUX, L. (1967). Le controlc neuroendocrinien de la trehalosemie de Carausius morosus. Compt. Rend. 265 Sbr. D 1067-1070. FIYCIIEII, R. B., PARSONS, D. S., ASD MORILISON, G. A. (1948). Quantitative paper chromatography. Nature 161, 764-765. 1%.4I~;sIwL, G. (1940). Utilization and digestion of carbohydrates by the adult blowfly. J. Erptl. Biol. 17, l&29. FHIDBERG, G., IWASAKI, S., YAGI, Ii., BERN, H. A., WILSON, D. M., AND WISHIOKA, R. S. (1966). Relation of impulse conduction to electrically induced release of neurosecretory material from the urophysis of the teleost fish Tilapia WLOSsambica. J. Exptl. Zool. 161, 137-150. FRIEDMAN, S. (1967). The control of trehalose synthesis in the blowfly Phormia Tegina Meig. J. Insect Physiol. 13, 397-405. FRIEDX~N, S. (1968). Trehalose regulation of glucose&phosphate hydrolysis in blowfly extracts. Science 159, 110-111. HARRIS, G. W. (1947). The innervation and actions of the neurohypophysis : an investigation using the method of remote control stimulation. Phil. Trans. Roy. Sot. London Ser. B 232, 383-442. HIOHNAM, K. D. (1961). Induced changes in the amount of material in the neurosecretory system of the desert locust. Nature 191, 199-200. HIGHSAM, K. D. (1962). Xeurosrrretory control of ovarian development in Schistocerca gregaria. Quart. J. Microscop. Sci. 103, 57-72. HOD~SOX, E. S., AND GELDI~Y, S. (1959). Experimentally induced release of neurosecretory materials from roach corpora cardiaca. Biol. Bull. 117, 275-283. JASINSKI, A., GORBMAN, A., AND HARA, T. J. (1966). Rate of movement and distribution of stainable neurosecretory granules in hypothalamic neurons. Science 154, 776-778. KATER, S. B. (1968). Cardioaccelerator release in Periplaneta americana (L). Science 160, 765 767. KNOWLES, F. G. W. (1965). Neuroendocrine corC:OOKE,

AND

DUVE

relations at the level of ultrastructure. Arch. At&. 111icroscop. Morphol. Exptl. 54, 343-357. I
RELEhSE

erythrocephnla 172. E. neurosecretory cephaln by Erptl. Bid. THOMREN, M. of thP adult histological THOMSEN,

Mrig.

J.

Exptl.

OF

Biol.

HYPERGLYCEMIC

29,

137..

(1954). Studies on the transport of material in Callipk.ora erythromeans of ligaturing experiments. J. 31, 322-330. (1969). The neurosecretory system Calliphora erythrocephala. IV. A study of the corpus cardiacum and

459

NEUROHOHYOiVE

its connections with Zellforsch. 94, 205-219.

the

nervous

system.

Z.

A. W., AKD GILBERT, L. I. (1965). Regulation of cockroach fat-body metabolism by the corpus cardiacum in vitro. Scie?ze 150, 614-616. WIENS, A. W., AND GILBERT, L. I. (1967). The phosphorylase system of the silkmoth, Hyalophora cecropin. Camp. Biochenz. Physiol. 21, 145-159. WIENS,