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The neuropilar neurosecretory reservoir of Locusta migratoria migratorioides R & F
IXNERAL
ASD
<‘OMP.4R.4TIVF:
The
16, 574-585
k:SDOCRI401,0~:Y
Neuropilar
( 1971)
Neurosecretory
migratoria
Reservoir
migratorioides
Received September
of
Locusta
R& F
11, 1970
In Locusta migratoria migmtorioides, neurosecretory fibers from the intracerebral portions of the nervi corporis cardiaci interni (NCCI) leave the major axon tracts and ramify through the adjacent neuropile before reentering the same or the opposite tract. The fibers carry droplets of neurosecretion, and the complex forms a “reservoir” of neurosecretory material within the neuropile. It is concluded that reservoir fibers which cross to the opposite NCCI. and subsequently recross the ,midline at the major decussation of the tracts, must originate in median neurosecretory cells which consequently innervate the ipsilateral corpus cardiacum. This conclusion is strongly supported by the distribution of nemosecretion following unila.teral cautery of the pars intercerebralis. Neurosecretion accumulates in the neuropilar reservoir during starvation, and diminishes during subsequent feeding. The reservoir can accommodate large variations in the amount of material within the neurosecretory system, and can explain the absence of clear-cut cycles in the content of neurosecretion within the perikarya of the median neurosecrctory cells.
Descriptions of insect neurosecretory systems sometimes mention the presence of droplets of neurosecretion lying within the neuropile of the protocerebrum, outside the paired axon tracts from the median neurosecretory cells. In Sy72agr& (Parngris) calida, the droplets are confined within fine fibers which leave the main axon tracts and appear to reenter after a short detour (Thomsen, 1954). The fibers are difficult to see in Oncopelfus fasciatus, although the neurosecretory granules are obvious (*Johansson, 1958). Stainable granules in “intercellular spaces” close to the major axon tracts are present in Melanoplus sanguinipes (Dogra and Ewen, 1970). In Carausius morosus, the amount of neuropilar neurosecretion is such that the region is called a reservoir (Herlant-Meewis and Pacquet, 1956). Large amounts of neurosecretion, apparently lying outside the major neurosecretory tracts, are present in Locwta migratoria cinerascens (Girardie, 1967, Figs. 1-3) and have been briefly mentioned by Girardie and Girardic (1967). 574
During the course of experiments designed to inhibit and accelerate the release of material from the cerebral neurosecretory systems of locusts, it was noticed that the median neurosecretory cells of Locusta migmtoria migratorioides reacted differently from those of Schistocerca gregaria. In the latter, starvation (among other treatments) eventually causes a marked accumulation of neurosecretion within the perikarya of the A-cells of the pars intercerebralis, the amount diminishing rapidly on subsequent feeding (Highnam et al., 1966; Highnam, 1967). In L. m. migratorioides, on the contrary, starvation results in no large accumulat’ion of neurosecretion within the perikarya of the A-cells. The protocerebral neuropile of L. ~1. migratorioides contains very much larger amounts of neurosecretion than that of 8. gregaria. The possibility that this neuropilar material functions as a “reservoir” in L. vz. naigratorioides, modifying the bistology of the protocercbral A-cells during periods of accumulation and release of WI!-
IGEUROPILAR
NEUROSECRETORY
rosecretion, has been investigated. The way in which the intracerebral portions of the nervi corporis cardiaca interni (NCCI) are modified to produce the reservoir-like structure is first described, since to our knowledge no detailed account is yet available. MATERIALS
AND
METHODS
Reariblg. Female L. m. migratorioides were rcarcd from fledging with males in glass-fronted cages of 60-liter capacity, at a temperature of 30 t 2°C. and with a 12-hr photoperiod. The animals were fed fresh letture and bran daily until the beginning of the experiments. Stnwutiw and subsequent fee&g. Forty 6- to ‘I--day-old female locusts were placed in a clean cage, provided with tap water in a petri dish. and starved for 4 days. Dead animals were removed to prevent cannibalism. Five animals were sacrificed at the end of this period. Fresh lettuce and bran were then placed in the cage and samples of 5-6 locusts were taken 5, 15, 60, and 120 min after feeding had begun. Females drawn from the same hatching group were allowed to feed normally, and a sample of 8 individuals sacrificed at the same time as the starred locusts. With two replicates, no obvious differences were found between samples. The observations reported in the Results section comprise 20 continuously fed. 11 starred. and 44 starved/fed individuals. Hwtology. The neuroendocrine systems of the locusts were processed histologically and stained with paraldehyde-fuchsin (Highnam. 1962). Each staining rack contained slides from fed and starred individuals, and from each of the starved/ fed samples. Some neurosecretory systems were stained in siiir with periodic acid/Victoria blue and the brains and corpora cardiaca mounted complete (Dogra and Tandan, 1964). Unilateral cautery of the pars itltercerebralis. In 34 6- to g-day-old locusts. neurosecretory cells on the left side of the pars intercerebralis together with some near the midline from the right side (to ensure complete destruction of all cells on the left side) were destroyed with an electric cautery needle (Highnam, 1962; Hill. 1962) ; 9 locusts were treated similarly, except that the needle was not heated when it touched the pars intercerebralis. The locusts were fed fresh lettuce and bran for 4 days. After this recovery period, the animals were starved for 4 days, 25 operated, and 7 shamoperated animals surviving the treatment. The neuroendocrinr s.vstems were stained with paraldehyde-fuchsin ; each staining rack contained sections from oprrated and sham operated individuals.
575
RESERVOIR
Measurement of cell and wxlenr diameters. Neurosecretory cells from histological sections of the pars intercerebralis of starved and continuously fed locusts were projected onto the scrren of a television monitor viq a transist.orized camera channel, giving a magnification of X2400, The cell and nuclear diameters were measured on the acrren with a pair of dividers and read off on a centimeter rule. At least 40 A-cells were measured in each animal. The average volumes of cells and nuclei were found for each individual, and mean and standard errors were calculated for ~a& s:kmple. Estimatio,l of content of neurosewetion witltiu the system. Using an Integrating Mkrodensitomcter, Type GS2 (Barr & Stroud). ahsorhnncc values were measured at fixed positions along each neurosecretory system. The numbers of rcadings in each individual were as follows: nearosrcretory cells, 6 : dorsal wings of reservoir. 4 : uIq,cTr central reservoir, 3; lower central reserl-oir. 3; crossover point, 3; both exit points, 6; both NCCI, 6: corpora cardiaca. 3. Different frame areas were used depending upon the structur:d limits of the neurosecretory syskm in each SYtion, and the instrument readings were corrected for background and scanning area. lifter each *ession of use, any variation in instrument performance was corrected using a standard Zeks Graufilt’er. absorbance value 0.33. Light source wavelengths were 5400 x for counterstained seetions, and 5600 X for sections stained only with paraldehyde fuchsin. The va!ues for the integrated absorhances are given in units of pL2X lo-‘. RESULTS
The Structure of A’eurosecretory
the
Newopilar
Reservoir
Each major axon tract from the two groups of median neurosecretory cells curves anteriorly through the brain, crossing its fellow about half-way along its intracerebral length, and turns posteriorly to leave the ventral surface of the brain as the NCCI. The neuropilar reservoir is restricted largely to the region dorsal to the decussation of the major axon tracts (Fig. 5A). The reservoir is diffuse, composed of many fibers containing beads or elongated droplets of neurosecretion. Its limits are relatively well defined, and the volume contained within these limits is constant between individuals of different ages and de-
Yelopmental conclition. HOWWY, t11v to tlica rcgioll of (l(~(~us>:ttioll. ‘l‘h(~y ~‘21111~8 amount of neurosecrction carried l)y the tlivitlrd into two yet::: tllcb more cbrxntr:il fibers can vary considerably I F~C below 1. axons sl)iral with a steel) I)itcah (Figs. I :~n(t The most dorsal part of the rc>.qt*rvoir lies 5C 1 ; the l>eriphcral axons n-it11 a very sh:~llateral to the median neurosecretorp ~11~ low f,itrh (Figs. I and 51).E,F I. The tloul&~ and abo\Te their yentral limits. Here the slkal is lost ventral to the tlecusaation of reservoir is in the form of two “wings.” the the t’racts, but realq,ears within the dorsal tips of which are 1)ostcrior to each major lobcls of the coqlora rartliacn. C’uriously, the axons coml)osing tlicb tTv0 neurosecretory tract although the greater major tracts spiral in the same direction: part of each wing lies lateral to its own anticlockwise \&en viewed from the dorsal tract (Fig. 5AI. The two dorsal l\.ings surface of the brain (Fig. 1 i, shown parswing downward and anterior to the axon clearly by the t>eripheral axons. tracts, and meet about loo-140 IL above the ticularly The two groups of median neurosecretory region of decussation. The reservoir now forms an inverted triangle, with its allex cells on either side of the midline must consequently be considered asymmetric in this just below the decussation (‘Fig. 5A 1. respect. Since the axons of the neuropilar In the dorsal third of the reservoir, venreservoir originate and terminate in the tral to the dorsal wings, the clensity of fibers major tracts, this asymmetry results in difis uniform, but below this region the fibers ferences in the patterns of the reservoir are contained in two loosely connected sheets which cross in the midline to give a fibers associated with the two tracts. The shallow ljitch of the peripheral axons figure of eight appearance in horizontal section (Fig. 5H). The lool~ of the “8” enclose enables the reservoir fibers to leaye and less dense regions of fibers. The al)ex of the enter the major tracts more or less horizontally. The fibers constituting the right reservoir also contains few fibers (Fig. 5A). dorsal wing of the reservoir leaye the right, The axons composing each major neurotract’ posteriorly, or encircle the tract besecretory tract follom7 a spiral course dorsal
FIG. I. Diagramof t.hemajor axon tracts and neuropilar reservoir fibers in Locusta migrato~ia migratorioides’ as viewed from the anterior surface of the brain. The central (cj) and peripheral (pf) axons in the tracts spiral in the same direction on both sides. The fibers in the dorsal parts of the reservoir (f.i.) enter and leave t,he same tract; those in the central part of the reservoir (f.ii) leave one tract and cross the midline to enter the ot,her; those in the ventral part’ of the reservoir (f.iii) leave one tract, do not cross the midline, but ent.er the other t,rart vetlt,ral to the major decussation. Compare Fig. .5.
NEUROPILAR
XEUROSECRETORT
fore leaving laterally. The fibers loop extensively in the neuropile before returning to the right tract anteriorly. Fibers constituting the left dorsal wing of the reservoir, on the contrary, leave the left tract anteriorly and after traversing the neuropile, return to the tract posteriorly (Fig. 1) , Where the dorsal wings of the reservoir begin to swing anteriorly to the two major tracts, the departing and returning fibers cross each other at right angles lateral to each tract (Fig. 5B). On the right side the departing fibers leave the posterior face of the tract and reenter anteriorly, whereas on the left side the directions of the fibers are again reversed. The asymmetry of the reservoir caused by the similarly spiraling right and left major tracts is most obvious centrally. It will be remembered that here the reservoir is anterior to the major tracts (Fig. 5A). Manv of the fibers in this part of the reservoir leave one of the major tracts and enter the other: fibers leave the lateral face of the right tract, cross the midline in a smooth curve and enter the lateral face of the left tract (Fig. 5G). But the fibers passing in the opposite direction leave the inner face of the left tract, move anteriorly, and loop tightly to enter the inner face of the right tract (Fig. 5H). The different paths of the fibers moving from right to left, and from left to right, give the reservoir its figure of eight appearance in horizontal section. ,Just above the decussation of the major tracts, reservoir fibers move anteriorly and then downward, remaining on the same side of the midline (Figs. 1 and 5A). They then enter the opposite tract after it has crossed its fellow. In the crossover itself, the right tract moves either anterior or posterior to the left tract: in 100 brains selected at random from other L. m. nrigmforiaides material, the former occurred in 52 specimens and the latter in the remaining 48. Depending upon where thev leave and enter the major tracts, t)he fihers of the neuropilar reservoir can thus be divided into three groups (Fig. 1) : (i) those composing the dorsal and lateral wings of the reservoir which leave and enter the same
RESERVOIR
577
tract (Fig. 5B) ; (ii) those forming the central part of the reservoir which leave one tract, cross the midline, and enter the other tract (Fig. 5G,H) ; and (iii) those near the ventral part of the reservoir which leave one tract, do not cross the midline, but enter the other tract after this has crossed over. It is not known whether the neuropilar reservoir is formed from axons which leave and reenter the major tracts more than once during their passage through the brain, or whether only a single diversion occurs. The dense network of reservoir fibers suggests the former, although it is possible that the fibers branch within the reservoir. It is likely, however, that the type ii fibers, crossing from one tract to the other and then recrossing the midline in company with other axons at the major decuesation, originate in neurosecretory cells which consequently innervate their ipsilateral corpus cardiacurn. Type iii fibers would similarl? innervate the ipsilateral gland. This possibility has been tested experimentally. Ipsilateral Innervation of the Corpora Cardiaca by Median Neurosecretory Cells After cautery of the left side of the pars intercerebralis followed by 4 days starvation, the neurosecretory cells on the right’ side contain dispersed aggregates of ncerosecretion, and their axons together with the dorsal wing of the reservoir on this side are normal. No neurosecretory cells are present, on the left side, and axons and dorsal wing of the reservoir are indicated only by a very few droplets of neurosecrction (Fig. 6A). Ventral to the dorsal wing of the reservoir, fibers carrying neurosecretion pass from the right major tract to the remains of the left (Fig. 6B). Below this region, the left tract now contains considerable amounts of neurosecretion, although always less than the right. The tracts decuseate normally, and the right tract (originally left) carries less neurosecretion than the left (originally right) (Fig. SC). The right XCCI and the right dorsal lobe of the corpora cardiaca also contain significant
57x
HIGHSA\I
amounts of neurosecretiou, although less than the left XCCI and the left dorsal lobe of the corpora cardiaca (Fig. 6D). Kcurosecretion is usually evenly dispersed through the unpaired ventral lobe of the corpora cardiaca, although in some sperimcuu the right side of this lobe contains more neurosecret,ion than the left, suggesting that some axons from the NCCI which traverse the dorsal lobes of the gland once more cross each ot,her in the ventral lobe. In the sham-operated individuals, the distribution of neurosecretion throughout the neurosecretory system, including the reservoir, is similar to that in starved but otherwise normal females. The absence of neurosecretion from the proximal parts of the left axon tract and the left dorsal and lateral wings of the neuropilar re.zervoir after cautery of the
FIG. 2. Diagram of the neurosecretory system of a female after cautery of the left side of the pars intercerebralis and subsequent starvation, as viewed from the anterior surface of the brain. Neurosecretion (shown by stippling) is absent from the proximal parts of the major axon t,ract, and the reservoir on the left side, but, appears within the left tract at the level of t,he cent,ral part of the reservoir, and can then be followed into the right NCCI (m) and right lobe of the corpora cardiaca (WC). Compare Fig. 6.
ASI)
\VEST
left groul) of median nr~rlroscc*rc~tory cell>. together with its reappcarancc in the tract at the lcvc~l of the central part of the rcscrvoir (Fig. 2)) strongly supports the nssumption that type ii reservoir fibers cro.ds from one major tract to the other in normal animals. That fibers bearing neurosecretion can then be traced through the major decussation of the t.racts and into the right NCCI and the right dorsal lobe of the corpora cardiaca can only mean that a proportion of the neurosecretory cells on the right side of the brain innervates its ipsilateral corpus cardiacum. Eflects of Starvation and Subsequent Feeding upon the Neuropilar Neurosecretory Resewoil The amount of neurosecretion in the perikarya of median neurosecretory cells of lo- to 11-day-old females which have been starved for 4 days differs little from that in the cells of fed females of the same age (Fig. 3). In similar females starved for 6 days, the mean volume of the neurosecretory cells together with their mean nuclear volume are both significantly less than those of fed individuals (Table 1). Moreover, all parts of the reservoir together with the axon tracts at the points of exit from the brain and the NCCI of starved individuals contain more neurosecretion than those of the fed animals (Fig. 3). The corpora cardiaca of starved animals, on the contrary, contain less neurosecretion than those of fed individuals (Fig. 3). When previously starved females begin to feed, the perikarya of the neurosecretory cells appear to contain more neurosecretion, although sample variation is such that the amounts are not significantly different from those in starved animals (Fig. 4A). The neurosecretory material is clumped together in the cells of the starved animals, and is dispersed through the perikarya in the feeding individuals, although these differences are not sufficiently clear cut to alter significantly the microdensitometer absorhante values. Five minutes after previously starved females begin to feed, the amounts of neuro-
NEUROPILAR
NEUROSECRETORY
TABLE
379
RESERVOIR
1
COMP.IRISON OF CILLLVL.IR MD NtTCLt:.\ti VOLUMES OF THE A-NWROSECRETORY CELLS IN THE BRMNS OF STARVLD .~ND CONTINUOUSLY FED FEMALES OF Locusta migratoria migratorioides Treatment (number of animals) Starved for 6 days prior Continuously fed (9) Comparison
of means:
cellular
Age at sacrifice (days)
to sacrifice
(9)
volumes,
11-14 11-14
p = 0.01-0.001;
secretion decrease significantly in the dorsal wings and ventral (reservoir II) parts of the reservoir, the points of exit from the brain of the axon tracts, and the NCCI (Figs. 4A,B). Neurosecretion does not decrease in the central parts (reservoir I) of the reservoir, the region of crossover of the major axon tracts, and the corpora cardiaca (Figs. 4A,B).
; Q ii
nuclear
Cellular volume ($) ( * SEM)
Nuclear volume w ( f SEW
924 * 52 1199 5 75 volumes,
5.55 f 39 706 5 40
p = 0.0’2-0.01.
Fifteen minutes after the recommencement of feeding, all parts of the reservoir show a large variability in neurosecretory content, although the dorsal wings, the points of exit from the brain of the major tract’s, and the NCCI, still contain less material than those of starved animals (Figs. 4A,B). The corpoka cardiaca at this time have an average content of neurosecretion
Controlr
(
Fed
m
Starred
4 days
f { CdlS
2
Dorsal
Wings
Reservoir
I
Reservoir
II
Crossover
Exit Point
NCC
I
CClrpOr0 Cordiaco
110 loo. 90 . 80 . 70
.
60 50 40 30 -
-I-
20 10 -
FIG. 3. Microdensitometer absorbance values for different parts of the cerebral neurosecretory systems of continuously fed and 4-day starved locusts. The animals were 10-11 days old at sacrifice. The vertical lines indicate the standard error of the mean for 5 continuously fed animals and 5 starved animals. Note that all parts of the neurosecretory system of the starved locusts contain more neurosecretion than those of the fed, except the perikarya of the neurosecretory cells and the corpora cardiaca.
.\SI)
much greater than that of atarvecl aniinals, although there is a large individual variation (Fig. 4B). One and two hours after prcriously starved animals begin to feed, all parts of the neurosecretory system show great variation in their content of material, although the dorsal wings and ventral part of the reservoir, the exit points from the brain of the major axon tracts, and the NCCI all contain less material, on average, than the corresponding regions in the neurosecretory systems of starved individuals (Figs. 4A,B j . DISCISSION
In many insects, histological changes in the median neuroeecretory cells of the brain
\\‘ES’l
are tisrociatctl with :I \.:~ric~tv of clevc~lol~mental or l~hysiological events;, n-hcbrcw in ot11cra no such changes can bc obsc~rvccl (\l?gglesworth, 1964). In I,. rr/,iy~ntoricr in particular, the absence of cyclical change> in the median neurosecrctory cells forms the basis of an elaborate hypothesis on the mode of action of the thoracotropic hormone (Clarke and Langley, 1963 1. In S. gregarin, starvation is associated with the accumulation of material within the cerebral neurosecretory system, including the perikarya of the median neurosecretory cells; subsequent feeding results in a rapid diminution in the amount of this material (Highnam et al., 1966). In L. m. migratorioides, starvation and subsequent,
FIG. 4A. Microdensitometer absorbance values for parts of the cerebral nemosecretory system of starved locusts, and of locusts at different times after the recommencement of feeding. Vertical lines indicate the standard error of the mean of samples of 5 or 6 individuals. Note the sharp decreases 5 min after the beginning of feeding in the dorsal wings of the reservoir, and the ventral part, of the reservoir (Reservoir II). FIG. 4B. As Fig. 4A, but for the remaining parts of the cerebral neurosecretory system. Note the decreaseafter 5 min at the points of exit of t#he NCCI from the brain, and in the NCCI themselves. Note also the temporary increase in the corpora cardiaca 1-i min after the recommencement of feeding.
NEUROPILAR
NEUROSECRETORY
feeding produce no such dramatic changes in the perikarya of the median neurosecretory cells, and a first conclusion would be that the cells in this species react to the treatments in a manner different from those of S. gregaria. However, the present results show that neurosecretion accumulates within the neuropilar reservoir of L. 1)~. migratorioides during starvation, and subsequent feeding is correlated with changes in the content of material within the reservoir and the remainder of the neurosecretory system. When Calliphora erythrocephala is fed upon sugar water, neurosecretion accumulates within the perikarya of the median neurosecretory cells, disappearing when the flies are subsequently fed upon meat (Thornsen, 1965). In this species, the nuclei of the “full” neurosecretory cells are smaller than those of animals of the same age fed upon meat (Thornsen, 1965). In S. gregaria also, the nuclear volumes of “full” neurosecretory cells are less than those of cells containing “dispersed aggregates” of neurosecretion (Highnam, 1966). Assuming that nuclear volume reflects synthetic activity in the neurosecretory cells, it is likely that the synthesis of neurosecret.ion is ultimately depressed when material accumulates within the neurosecretory cells of both C. erythrocephala and I$. gregaria. In L. m. migratorioides, the nuclear volumes of the median neurosecretorp cells are smaller in starved than in continuously fed females. It is suggested that in this insect also, the synthesis of neurosecretion is depressedwhen release is retarded and material accumulates, but that by the time the neuropilar reservoir becomes filled with neuro
RESERVOIR
581
In L. m. ,migratorioides, the amount of neurosecretion in the proximal and distal parts of the neuropilar reservoir diminishes markedly within 5 min after previously starved females begin to feed. Neurosecretion similarly decreases in the axon tracts and NCCI distal to the reservoir. This suggests that feeding stimulates the proximodistal flow of neurosecretion along the system, resulting in a temporary accumulation of material in the corpora cardiaca by the fifteenth minute after the recommencement of feeding. The apparent lack of changes in the central part of the neuropilar reservoir within 5 min of feeding could be due to the replacement of neurosecretion, in this area of dense fibers, by material flowing down from the dorsal wings. Similarly, at the region of decussation of the major tracts, where the axons follow a more direct course, the apparent lack of change could result from the rapid flow of neurosecretion along the axons. By the end of 60 min after the recommencement of feeding, the neurosecretion which had accumulated in the corpora cardiaca has been discharged, and the content of material in the glands has returned to its original value. In the reservoir, the individual variation in amount of neurosecretion in samples taken at 15, 60, and 120 min after the recommencement of feeding increases markedly. It is likely that this variability is due not only to individual differences in the rate of flow of neurosecretion (perhaps due to variations in feeding rate), but also to increases in the rate of synt,hesis of material by the neurosecretory cells. In S. gregaria, feeding after prior starvation produces an initial increase in the amount of neurosecretion in the major axon tracts just proximal to their points of exit from the brain (Highnam et al., 1966). In both S. gregaria and L. m. migratorioides, therefore, the stimulus of feeding causesan increased axonal flow of neurosecretion. In 8. gregaria, the corpora cardiaca of starved individuals contain large quantities of neurosecretion ; the initial increase in the rate of flow of material causesit to “back up” in the distal parts of the axons. In L. m. migratorioides. on the contrary, the corpora
582
FIG. 5. The structure
HIC;HNA.\I AND IVEST
of the neuropilar neurosecretory reservoir of Locusta migratoria migf’alorioides. (A) Whole mount of the pars intercerebralis, stained in .s& with periodic acid/Victoria blue, viewed from the anterior surface. Note that the dorsolateral parts of the reservoir are associated with their respeo tive major axon tracts, but that the central parts of the reservoir connect the two tracts. The lettering indicates levels of horizontal sections shown in B, G, and H. (B) Horizontal section of the right major axon tract (at) at the level a in Fig. .5A. Note that the incoming and outgoing reservoir fibers cross more or less at right angles (arrowed).
NEUROPILAR
NEUROSECRETORY
cardiaca of starved individuals contain less neurosecretion than those of continuously fed animals, so that the increased flow consequent upon feeding can be accumulated within the glands, at least temporarily. The short delay before material accumulates within the corpora cardiaca reflects
RESERVOIR
583
the time taken for neuroeecretion to pass at least from the NCCI, and probably from more distant sites. The rate of flow of neurosecretion along the axons in L. m. migratorioides is thus of the same order as that in S. gregaria (Highnam, 1966). In L. m. migratorioides, each median
FIG. 6. Horizontal sections through parts of the neurosecretory systems of animals whose left. group of median neurosecretory cells has been cauterized. (A) Section just below level a in Fig. 5A. No fibers or neurosecretion can be seen on the left side, although gaps in the neuropile (arrowed) occur. (B) Se&on at, about level 6 in Fig. 5A. Central reservoir fibers swing across from the right tract (compare Fig. 5G), and fibers carrying neurosecretion can now be seen in the position of the left tract (arrowed). (C) Section just below the major decussation of the axon tracts. Although smaller in amount than in the left (originally right) tract, significant amounts of neurosecretion are present in the right (originally left) tract (arrowed). (D) Section through the dorsal lobes of the corpora cardiaca. The right NCCI (m) and the right dorsal lobe of the corpora cardiaca @cc) contain neurosecretion, although in smaller amount than in the left NCCI (In) and left dorsal lobe (ICC). Stained paraldehyde fuchsin. Scales in p.
(C) Vertical section through the origin of left major tract showing central fibers (arrowed). (D) Vertical section through the periphery of the left major tract showing fibers (arrowed) which seem to run almost horizontally. These are parts of the peripheral axons which spiral with a much shallower pitch than those shown in 5C. (E) Vertical section through the right axon tract showing peripheral fibers (arrowed) running from left to right of the photograph. (F) The same section as 5E, but focused through the thickness of the axon tract, to show the peripheral fibers (arrowed) on t,he opposite side; these now run from right to left of the photograph. (G) Horizontal section of the axon tracts and reservoir at the level b in 5A. Fibers (arrowed) leave the outer face of t,he right tract and enter the outer face of the left tract. (H) Horizontal sect,ion of the axon tracts (at’s) at the level c in 5A. Fibers (arrowed) leave the inner face of the left tract and enter the inner face of the right tract. B - H stained with paraldehyde fuchsin. Scales in p.
group
of
cerehl
neuro~wretory
~~~~11s ill-
nervates its ipsilatersl ai n-cl1 ai its contralateral corpus cardiscum. The l)lienomenon is difficult to demonstrate cxperimentally, because partial cautcry of the cell groups is followed by increased activity in the remaining cells (Highnam and Hill, in preparation), and neurosrcretion within the corpora cardiaca can be made obvious only when its release is retarded, for example by starvation, as in the present experiments, Such double innervation of the corpora cardiaca may have important consequences, as when conclusions about the control of the activity of the corpora allata are based upon experiments involving unilateral cautery of the pars intercerebralis (e.g., Strong, 1965). Moreover, sotne axons may again cross the midline within t,he corpora cardiaca in L. m. migratoriaides, as in Melanoplzts snnguinipes (Dogra and Ewen, 19701. Arborizations of fibers associated with t)he roots of the intracerebral portions of t#he NCCI in Periplaneta americana are considered to be dendritic processes of the neurosecretory cells (Adiyodi and Bern, 19681. These arborizations, containing neurosecretory material, bear a remarkable resemblance t’o the neuropilar reservoir in L. m. migratorioides, and were indeed considered to be part of a small reservoir when the neurosecretory systems of our own stocks of P. americana were examined in 10 p thick sections and in whole mounts. In P. awericana, Rlatta germanica, Carausius morosus, and S. gTegaria, some of bhe “arborizing” fibers pass from one major neurosecretory axon tract to the other very like the type ii fibers in L. II). ntigratorioides. In this latter insect especially, the neuropilar neurosecretory rcservoir cannot be composed primarily of dendrit’ic processes, since both the histological and experimental evidence described here points to the carriage of neurosecretory material by the reservoir fibers to the corpora cardiaca. The asymmetry of the spiraling major axon tracts in L. m. migratorioides is of considerable interest,. In iMeZn?lopZzrs sangwinipes, the “medial” groups of ncurose-
cwtory
~11~
n-it11
th+r
;wOciatc~l
;txoik
are founcl together on one side of the brain or the other in 10$4 of the inclividuals examined (Dogra and Encn, 19701. The asymmetry of the cells in both sljccies perhaps reflects a unilateral origin during embryogenesis: a detailed esamination of the early stages of devclo1n~~et~t~ of the cells will prove worthwhile. ACKSOWLEDGMENTS We are very grateful to Mr. Roger Webb, made the photomicrographs. Part of the work carried out during the tenure of an S.R.C. search Studentship held by M.R.W.
who was Re-
REFERESCES IL G., AND BERN, H. A. (1963). ?u’curonal appearance of neurosecretor~cells in the pars intercerebralis of Periplanet of the neurosecretory system and the retrocerebra1 endocrine glands of the adult migratory 3felanoplu.s .w,tguinipes (IFah) grasshopper. (Ort.hoytcra: .\rrididac). J. .lIornhr~l. 130, 451465. DOGRA, G. S., AND TANDAN. B. E. (1964). Adaptation of certain histological techniques for in situ demonstration of the ncuro-endocrine system of insects and other animals. Q~nrl. /. .Wioosc. Sci. 105, 455466. GIR.~RDIE, A. (1967). Contrirle ncuro-horruornd dr la metamorphose et de la pigmentation rhpz Loc~~~tn migmtori-r cinertrsceks (OrthotJterc~). Bull. Biol. Fr. Belg. 101, 79114. GIRARDIE, A.. AND GIR.~RDIE. J. (1967). hutie hiatologique, histochimique et, ultrastructurale de la pars intercerebralis chez Locust/r rnigrrctorilc L. (Orthopter~). Z. Zellforsch. diic~~s~. Anti. 78, 54-75. HERLANT-MYE;WIS, H.. AND PACSET, I,. (1956). Neurosecretion et mue chez Carazrsius morosus Brdt. Ann. Sci. Nut. (ZOO/). 18, 163-169. HIoHNaM. K. c. (1962) IYrurosecrrtorrvontrol of ovarian dcvelopmcnt, in Schistncerr~r grrgrrricc. Or~nrf. /. Microsc. Sci. 103. 57-72. _ ADIYODI.
NEUROPILAR
NEUROSECRETORY
K. C. (1966). Estimates of neurose-rctory activity during maturation in locusts. Pror. Int. Sump. Insect Endocrinol. BTIIO. V. J. A. Novak. Ed., Prague. HIGHNAM. Ii. C. (1967). Insect hormones. J. Endocrinol. 39, 123-150. HIGHNAM. K. C., HILL. L.. AND MORDUE, W. (1966) Thr endocrirw system and oocvte growth in Schistocerc~~ in relation to starvation and frontal ganglionectom~. J. lrwect Physiol. 12, 977-994. HILL, L, (1962). Xeuroaecretory control of haemolymph protein concentration during ovarian develoljment in the desxt locust. J. Insect Plqsiol. 8, 609-619. Jow.~sssor, A. S. (1958). Relation of nutrition to cndorrinr,-reproduc.tivc functions in the milkwc:~d hu< Oucope1trt.s frrsciat~ca (Dallas) (HrtHIGHNAM.
- ,iI b n
RESERVOIR
eroptera: 1-131.
Lygaeidae).
*Vylt
Jfrcg.
Zoo!.
7,
I,. (1965). The rrlationships between the brain, corpora allata, and oocyte growth in the Central American locust, Schi~tocercn sp. II. The innervation of the corpora allata. the lateral neurosecretory complex, and ooryte growth. J. Insect Physiol. 11, 271-280. TROMSEN. M. (1954). Xeurosecretion in some H?;menoptera. Dan. Biol. Skr. 7, 1-24. THOMSEN, M. (1965). The neurosrcrctory s:;stcTm of the adult Calliphorrc eq&rocephnlu. II. Histology of the nwrosecretory cells of the brain and some related structures. 2. Zellforsch. Nikrosk. Aurct. 67, 693-717. WIGGLESWORTH. V. B. (1964). The hormonal regulation of growth and reproduction in insects. Arlr~~,r. Insect Ph ysiol. 2, 247-336. &STRONG.
ASD
<‘OMP.4R.4TIVF:
The
16, 574-585
k:SDOCRI401,0~:Y
Neuropilar
( 1971)
Neurosecretory
migratoria
Reservoir
migratorioides
Received September
of
Locusta
R& F
11, 1970
In Locusta migratoria migmtorioides, neurosecretory fibers from the intracerebral portions of the nervi corporis cardiaci interni (NCCI) leave the major axon tracts and ramify through the adjacent neuropile before reentering the same or the opposite tract. The fibers carry droplets of neurosecretion, and the complex forms a “reservoir” of neurosecretory material within the neuropile. It is concluded that reservoir fibers which cross to the opposite NCCI. and subsequently recross the ,midline at the major decussation of the tracts, must originate in median neurosecretory cells which consequently innervate the ipsilateral corpus cardiacum. This conclusion is strongly supported by the distribution of nemosecretion following unila.teral cautery of the pars intercerebralis. Neurosecretion accumulates in the neuropilar reservoir during starvation, and diminishes during subsequent feeding. The reservoir can accommodate large variations in the amount of material within the neurosecretory system, and can explain the absence of clear-cut cycles in the content of neurosecretion within the perikarya of the median neurosecrctory cells.
Descriptions of insect neurosecretory systems sometimes mention the presence of droplets of neurosecretion lying within the neuropile of the protocerebrum, outside the paired axon tracts from the median neurosecretory cells. In Sy72agr& (Parngris) calida, the droplets are confined within fine fibers which leave the main axon tracts and appear to reenter after a short detour (Thomsen, 1954). The fibers are difficult to see in Oncopelfus fasciatus, although the neurosecretory granules are obvious (*Johansson, 1958). Stainable granules in “intercellular spaces” close to the major axon tracts are present in Melanoplus sanguinipes (Dogra and Ewen, 1970). In Carausius morosus, the amount of neuropilar neurosecretion is such that the region is called a reservoir (Herlant-Meewis and Pacquet, 1956). Large amounts of neurosecretion, apparently lying outside the major neurosecretory tracts, are present in Locwta migratoria cinerascens (Girardie, 1967, Figs. 1-3) and have been briefly mentioned by Girardie and Girardic (1967). 574
During the course of experiments designed to inhibit and accelerate the release of material from the cerebral neurosecretory systems of locusts, it was noticed that the median neurosecretory cells of Locusta migmtoria migratorioides reacted differently from those of Schistocerca gregaria. In the latter, starvation (among other treatments) eventually causes a marked accumulation of neurosecretion within the perikarya of the A-cells of the pars intercerebralis, the amount diminishing rapidly on subsequent feeding (Highnam et al., 1966; Highnam, 1967). In L. m. migratorioides, on the contrary, starvation results in no large accumulat’ion of neurosecretion within the perikarya of the A-cells. The protocerebral neuropile of L. ~1. migratorioides contains very much larger amounts of neurosecretion than that of 8. gregaria. The possibility that this neuropilar material functions as a “reservoir” in L. vz. naigratorioides, modifying the bistology of the protocercbral A-cells during periods of accumulation and release of WI!-
IGEUROPILAR
NEUROSECRETORY
rosecretion, has been investigated. The way in which the intracerebral portions of the nervi corporis cardiaca interni (NCCI) are modified to produce the reservoir-like structure is first described, since to our knowledge no detailed account is yet available. MATERIALS
AND
METHODS
Reariblg. Female L. m. migratorioides were rcarcd from fledging with males in glass-fronted cages of 60-liter capacity, at a temperature of 30 t 2°C. and with a 12-hr photoperiod. The animals were fed fresh letture and bran daily until the beginning of the experiments. Stnwutiw and subsequent fee&g. Forty 6- to ‘I--day-old female locusts were placed in a clean cage, provided with tap water in a petri dish. and starved for 4 days. Dead animals were removed to prevent cannibalism. Five animals were sacrificed at the end of this period. Fresh lettuce and bran were then placed in the cage and samples of 5-6 locusts were taken 5, 15, 60, and 120 min after feeding had begun. Females drawn from the same hatching group were allowed to feed normally, and a sample of 8 individuals sacrificed at the same time as the starred locusts. With two replicates, no obvious differences were found between samples. The observations reported in the Results section comprise 20 continuously fed. 11 starred. and 44 starved/fed individuals. Hwtology. The neuroendocrine systems of the locusts were processed histologically and stained with paraldehyde-fuchsin (Highnam. 1962). Each staining rack contained slides from fed and starred individuals, and from each of the starved/ fed samples. Some neurosecretory systems were stained in siiir with periodic acid/Victoria blue and the brains and corpora cardiaca mounted complete (Dogra and Tandan, 1964). Unilateral cautery of the pars itltercerebralis. In 34 6- to g-day-old locusts. neurosecretory cells on the left side of the pars intercerebralis together with some near the midline from the right side (to ensure complete destruction of all cells on the left side) were destroyed with an electric cautery needle (Highnam, 1962; Hill. 1962) ; 9 locusts were treated similarly, except that the needle was not heated when it touched the pars intercerebralis. The locusts were fed fresh lettuce and bran for 4 days. After this recovery period, the animals were starved for 4 days, 25 operated, and 7 shamoperated animals surviving the treatment. The neuroendocrinr s.vstems were stained with paraldehyde-fuchsin ; each staining rack contained sections from oprrated and sham operated individuals.
575
RESERVOIR
Measurement of cell and wxlenr diameters. Neurosecretory cells from histological sections of the pars intercerebralis of starved and continuously fed locusts were projected onto the scrren of a television monitor viq a transist.orized camera channel, giving a magnification of X2400, The cell and nuclear diameters were measured on the acrren with a pair of dividers and read off on a centimeter rule. At least 40 A-cells were measured in each animal. The average volumes of cells and nuclei were found for each individual, and mean and standard errors were calculated for ~a& s:kmple. Estimatio,l of content of neurosewetion witltiu the system. Using an Integrating Mkrodensitomcter, Type GS2 (Barr & Stroud). ahsorhnncc values were measured at fixed positions along each neurosecretory system. The numbers of rcadings in each individual were as follows: nearosrcretory cells, 6 : dorsal wings of reservoir. 4 : uIq,cTr central reservoir, 3; lower central reserl-oir. 3; crossover point, 3; both exit points, 6; both NCCI, 6: corpora cardiaca. 3. Different frame areas were used depending upon the structur:d limits of the neurosecretory syskm in each SYtion, and the instrument readings were corrected for background and scanning area. lifter each *ession of use, any variation in instrument performance was corrected using a standard Zeks Graufilt’er. absorbance value 0.33. Light source wavelengths were 5400 x for counterstained seetions, and 5600 X for sections stained only with paraldehyde fuchsin. The va!ues for the integrated absorhances are given in units of pL2X lo-‘. RESULTS
The Structure of A’eurosecretory
the
Newopilar
Reservoir
Each major axon tract from the two groups of median neurosecretory cells curves anteriorly through the brain, crossing its fellow about half-way along its intracerebral length, and turns posteriorly to leave the ventral surface of the brain as the NCCI. The neuropilar reservoir is restricted largely to the region dorsal to the decussation of the major axon tracts (Fig. 5A). The reservoir is diffuse, composed of many fibers containing beads or elongated droplets of neurosecretion. Its limits are relatively well defined, and the volume contained within these limits is constant between individuals of different ages and de-
Yelopmental conclition. HOWWY, t11v to tlica rcgioll of (l(~(~us>:ttioll. ‘l‘h(~y ~‘21111~8 amount of neurosecrction carried l)y the tlivitlrd into two yet::: tllcb more cbrxntr:il fibers can vary considerably I F~C below 1. axons sl)iral with a steel) I)itcah (Figs. I :~n(t The most dorsal part of the rc>.qt*rvoir lies 5C 1 ; the l>eriphcral axons n-it11 a very sh:~llateral to the median neurosecretorp ~11~ low f,itrh (Figs. I and 51).E,F I. The tloul&~ and abo\Te their yentral limits. Here the slkal is lost ventral to the tlecusaation of reservoir is in the form of two “wings.” the the t’racts, but realq,ears within the dorsal tips of which are 1)ostcrior to each major lobcls of the coqlora rartliacn. C’uriously, the axons coml)osing tlicb tTv0 neurosecretory tract although the greater major tracts spiral in the same direction: part of each wing lies lateral to its own anticlockwise \&en viewed from the dorsal tract (Fig. 5AI. The two dorsal l\.ings surface of the brain (Fig. 1 i, shown parswing downward and anterior to the axon clearly by the t>eripheral axons. tracts, and meet about loo-140 IL above the ticularly The two groups of median neurosecretory region of decussation. The reservoir now forms an inverted triangle, with its allex cells on either side of the midline must consequently be considered asymmetric in this just below the decussation (‘Fig. 5A 1. respect. Since the axons of the neuropilar In the dorsal third of the reservoir, venreservoir originate and terminate in the tral to the dorsal wings, the clensity of fibers major tracts, this asymmetry results in difis uniform, but below this region the fibers ferences in the patterns of the reservoir are contained in two loosely connected sheets which cross in the midline to give a fibers associated with the two tracts. The shallow ljitch of the peripheral axons figure of eight appearance in horizontal section (Fig. 5H). The lool~ of the “8” enclose enables the reservoir fibers to leaye and less dense regions of fibers. The al)ex of the enter the major tracts more or less horizontally. The fibers constituting the right reservoir also contains few fibers (Fig. 5A). dorsal wing of the reservoir leaye the right, The axons composing each major neurotract’ posteriorly, or encircle the tract besecretory tract follom7 a spiral course dorsal
FIG. I. Diagramof t.hemajor axon tracts and neuropilar reservoir fibers in Locusta migrato~ia migratorioides’ as viewed from the anterior surface of the brain. The central (cj) and peripheral (pf) axons in the tracts spiral in the same direction on both sides. The fibers in the dorsal parts of the reservoir (f.i.) enter and leave t,he same tract; those in the central part of the reservoir (f.ii) leave one tract and cross the midline to enter the ot,her; those in the ventral part’ of the reservoir (f.iii) leave one tract, do not cross the midline, but ent.er the other t,rart vetlt,ral to the major decussation. Compare Fig. .5.
NEUROPILAR
XEUROSECRETORT
fore leaving laterally. The fibers loop extensively in the neuropile before returning to the right tract anteriorly. Fibers constituting the left dorsal wing of the reservoir, on the contrary, leave the left tract anteriorly and after traversing the neuropile, return to the tract posteriorly (Fig. 1) , Where the dorsal wings of the reservoir begin to swing anteriorly to the two major tracts, the departing and returning fibers cross each other at right angles lateral to each tract (Fig. 5B). On the right side the departing fibers leave the posterior face of the tract and reenter anteriorly, whereas on the left side the directions of the fibers are again reversed. The asymmetry of the reservoir caused by the similarly spiraling right and left major tracts is most obvious centrally. It will be remembered that here the reservoir is anterior to the major tracts (Fig. 5A). Manv of the fibers in this part of the reservoir leave one of the major tracts and enter the other: fibers leave the lateral face of the right tract, cross the midline in a smooth curve and enter the lateral face of the left tract (Fig. 5G). But the fibers passing in the opposite direction leave the inner face of the left tract, move anteriorly, and loop tightly to enter the inner face of the right tract (Fig. 5H). The different paths of the fibers moving from right to left, and from left to right, give the reservoir its figure of eight appearance in horizontal section. ,Just above the decussation of the major tracts, reservoir fibers move anteriorly and then downward, remaining on the same side of the midline (Figs. 1 and 5A). They then enter the opposite tract after it has crossed its fellow. In the crossover itself, the right tract moves either anterior or posterior to the left tract: in 100 brains selected at random from other L. m. nrigmforiaides material, the former occurred in 52 specimens and the latter in the remaining 48. Depending upon where thev leave and enter the major tracts, t)he fihers of the neuropilar reservoir can thus be divided into three groups (Fig. 1) : (i) those composing the dorsal and lateral wings of the reservoir which leave and enter the same
RESERVOIR
577
tract (Fig. 5B) ; (ii) those forming the central part of the reservoir which leave one tract, cross the midline, and enter the other tract (Fig. 5G,H) ; and (iii) those near the ventral part of the reservoir which leave one tract, do not cross the midline, but enter the other tract after this has crossed over. It is not known whether the neuropilar reservoir is formed from axons which leave and reenter the major tracts more than once during their passage through the brain, or whether only a single diversion occurs. The dense network of reservoir fibers suggests the former, although it is possible that the fibers branch within the reservoir. It is likely, however, that the type ii fibers, crossing from one tract to the other and then recrossing the midline in company with other axons at the major decuesation, originate in neurosecretory cells which consequently innervate their ipsilateral corpus cardiacurn. Type iii fibers would similarl? innervate the ipsilateral gland. This possibility has been tested experimentally. Ipsilateral Innervation of the Corpora Cardiaca by Median Neurosecretory Cells After cautery of the left side of the pars intercerebralis followed by 4 days starvation, the neurosecretory cells on the right’ side contain dispersed aggregates of ncerosecretion, and their axons together with the dorsal wing of the reservoir on this side are normal. No neurosecretory cells are present, on the left side, and axons and dorsal wing of the reservoir are indicated only by a very few droplets of neurosecrction (Fig. 6A). Ventral to the dorsal wing of the reservoir, fibers carrying neurosecretion pass from the right major tract to the remains of the left (Fig. 6B). Below this region, the left tract now contains considerable amounts of neurosecretion, although always less than the right. The tracts decuseate normally, and the right tract (originally left) carries less neurosecretion than the left (originally right) (Fig. SC). The right XCCI and the right dorsal lobe of the corpora cardiaca also contain significant
57x
HIGHSA\I
amounts of neurosecretiou, although less than the left XCCI and the left dorsal lobe of the corpora cardiaca (Fig. 6D). Kcurosecretion is usually evenly dispersed through the unpaired ventral lobe of the corpora cardiaca, although in some sperimcuu the right side of this lobe contains more neurosecret,ion than the left, suggesting that some axons from the NCCI which traverse the dorsal lobes of the gland once more cross each ot,her in the ventral lobe. In the sham-operated individuals, the distribution of neurosecretion throughout the neurosecretory system, including the reservoir, is similar to that in starved but otherwise normal females. The absence of neurosecretion from the proximal parts of the left axon tract and the left dorsal and lateral wings of the neuropilar re.zervoir after cautery of the
FIG. 2. Diagram of the neurosecretory system of a female after cautery of the left side of the pars intercerebralis and subsequent starvation, as viewed from the anterior surface of the brain. Neurosecretion (shown by stippling) is absent from the proximal parts of the major axon t,ract, and the reservoir on the left side, but, appears within the left tract at the level of t,he cent,ral part of the reservoir, and can then be followed into the right NCCI (m) and right lobe of the corpora cardiaca (WC). Compare Fig. 6.
ASI)
\VEST
left groul) of median nr~rlroscc*rc~tory cell>. together with its reappcarancc in the tract at the lcvc~l of the central part of the rcscrvoir (Fig. 2)) strongly supports the nssumption that type ii reservoir fibers cro.ds from one major tract to the other in normal animals. That fibers bearing neurosecretion can then be traced through the major decussation of the t.racts and into the right NCCI and the right dorsal lobe of the corpora cardiaca can only mean that a proportion of the neurosecretory cells on the right side of the brain innervates its ipsilateral corpus cardiacum. Eflects of Starvation and Subsequent Feeding upon the Neuropilar Neurosecretory Resewoil The amount of neurosecretion in the perikarya of median neurosecretory cells of lo- to 11-day-old females which have been starved for 4 days differs little from that in the cells of fed females of the same age (Fig. 3). In similar females starved for 6 days, the mean volume of the neurosecretory cells together with their mean nuclear volume are both significantly less than those of fed individuals (Table 1). Moreover, all parts of the reservoir together with the axon tracts at the points of exit from the brain and the NCCI of starved individuals contain more neurosecretion than those of the fed animals (Fig. 3). The corpora cardiaca of starved animals, on the contrary, contain less neurosecretion than those of fed individuals (Fig. 3). When previously starved females begin to feed, the perikarya of the neurosecretory cells appear to contain more neurosecretion, although sample variation is such that the amounts are not significantly different from those in starved animals (Fig. 4A). The neurosecretory material is clumped together in the cells of the starved animals, and is dispersed through the perikarya in the feeding individuals, although these differences are not sufficiently clear cut to alter significantly the microdensitometer absorhante values. Five minutes after previously starved females begin to feed, the amounts of neuro-
NEUROPILAR
NEUROSECRETORY
TABLE
379
RESERVOIR
1
COMP.IRISON OF CILLLVL.IR MD NtTCLt:.\ti VOLUMES OF THE A-NWROSECRETORY CELLS IN THE BRMNS OF STARVLD .~ND CONTINUOUSLY FED FEMALES OF Locusta migratoria migratorioides Treatment (number of animals) Starved for 6 days prior Continuously fed (9) Comparison
of means:
cellular
Age at sacrifice (days)
to sacrifice
(9)
volumes,
11-14 11-14
p = 0.01-0.001;
secretion decrease significantly in the dorsal wings and ventral (reservoir II) parts of the reservoir, the points of exit from the brain of the axon tracts, and the NCCI (Figs. 4A,B). Neurosecretion does not decrease in the central parts (reservoir I) of the reservoir, the region of crossover of the major axon tracts, and the corpora cardiaca (Figs. 4A,B).
; Q ii
nuclear
Cellular volume ($) ( * SEM)
Nuclear volume w ( f SEW
924 * 52 1199 5 75 volumes,
5.55 f 39 706 5 40
p = 0.0’2-0.01.
Fifteen minutes after the recommencement of feeding, all parts of the reservoir show a large variability in neurosecretory content, although the dorsal wings, the points of exit from the brain of the major tract’s, and the NCCI, still contain less material than those of starved animals (Figs. 4A,B). The corpoka cardiaca at this time have an average content of neurosecretion
Controlr
(
Fed
m
Starred
4 days
f { CdlS
2
Dorsal
Wings
Reservoir
I
Reservoir
II
Crossover
Exit Point
NCC
I
CClrpOr0 Cordiaco
110 loo. 90 . 80 . 70
.
60 50 40 30 -
-I-
20 10 -
FIG. 3. Microdensitometer absorbance values for different parts of the cerebral neurosecretory systems of continuously fed and 4-day starved locusts. The animals were 10-11 days old at sacrifice. The vertical lines indicate the standard error of the mean for 5 continuously fed animals and 5 starved animals. Note that all parts of the neurosecretory system of the starved locusts contain more neurosecretion than those of the fed, except the perikarya of the neurosecretory cells and the corpora cardiaca.
.\SI)
much greater than that of atarvecl aniinals, although there is a large individual variation (Fig. 4B). One and two hours after prcriously starved animals begin to feed, all parts of the neurosecretory system show great variation in their content of material, although the dorsal wings and ventral part of the reservoir, the exit points from the brain of the major axon tracts, and the NCCI all contain less material, on average, than the corresponding regions in the neurosecretory systems of starved individuals (Figs. 4A,B j . DISCISSION
In many insects, histological changes in the median neuroeecretory cells of the brain
\\‘ES’l
are tisrociatctl with :I \.:~ric~tv of clevc~lol~mental or l~hysiological events;, n-hcbrcw in ot11cra no such changes can bc obsc~rvccl (\l?gglesworth, 1964). In I,. rr/,iy~ntoricr in particular, the absence of cyclical change> in the median neurosecrctory cells forms the basis of an elaborate hypothesis on the mode of action of the thoracotropic hormone (Clarke and Langley, 1963 1. In S. gregarin, starvation is associated with the accumulation of material within the cerebral neurosecretory system, including the perikarya of the median neurosecretory cells; subsequent feeding results in a rapid diminution in the amount of this material (Highnam et al., 1966). In L. m. migratorioides, starvation and subsequent,
FIG. 4A. Microdensitometer absorbance values for parts of the cerebral nemosecretory system of starved locusts, and of locusts at different times after the recommencement of feeding. Vertical lines indicate the standard error of the mean of samples of 5 or 6 individuals. Note the sharp decreases 5 min after the beginning of feeding in the dorsal wings of the reservoir, and the ventral part, of the reservoir (Reservoir II). FIG. 4B. As Fig. 4A, but for the remaining parts of the cerebral neurosecretory system. Note the decreaseafter 5 min at the points of exit of t#he NCCI from the brain, and in the NCCI themselves. Note also the temporary increase in the corpora cardiaca 1-i min after the recommencement of feeding.
NEUROPILAR
NEUROSECRETORY
feeding produce no such dramatic changes in the perikarya of the median neurosecretory cells, and a first conclusion would be that the cells in this species react to the treatments in a manner different from those of S. gregaria. However, the present results show that neurosecretion accumulates within the neuropilar reservoir of L. 1)~. migratorioides during starvation, and subsequent feeding is correlated with changes in the content of material within the reservoir and the remainder of the neurosecretory system. When Calliphora erythrocephala is fed upon sugar water, neurosecretion accumulates within the perikarya of the median neurosecretory cells, disappearing when the flies are subsequently fed upon meat (Thornsen, 1965). In this species, the nuclei of the “full” neurosecretory cells are smaller than those of animals of the same age fed upon meat (Thornsen, 1965). In S. gregaria also, the nuclear volumes of “full” neurosecretory cells are less than those of cells containing “dispersed aggregates” of neurosecretion (Highnam, 1966). Assuming that nuclear volume reflects synthetic activity in the neurosecretory cells, it is likely that the synthesis of neurosecret.ion is ultimately depressed when material accumulates within the neurosecretory cells of both C. erythrocephala and I$. gregaria. In L. m. migratorioides, the nuclear volumes of the median neurosecretorp cells are smaller in starved than in continuously fed females. It is suggested that in this insect also, the synthesis of neurosecretion is depressedwhen release is retarded and material accumulates, but that by the time the neuropilar reservoir becomes filled with neuro
RESERVOIR
581
In L. m. ,migratorioides, the amount of neurosecretion in the proximal and distal parts of the neuropilar reservoir diminishes markedly within 5 min after previously starved females begin to feed. Neurosecretion similarly decreases in the axon tracts and NCCI distal to the reservoir. This suggests that feeding stimulates the proximodistal flow of neurosecretion along the system, resulting in a temporary accumulation of material in the corpora cardiaca by the fifteenth minute after the recommencement of feeding. The apparent lack of changes in the central part of the neuropilar reservoir within 5 min of feeding could be due to the replacement of neurosecretion, in this area of dense fibers, by material flowing down from the dorsal wings. Similarly, at the region of decussation of the major tracts, where the axons follow a more direct course, the apparent lack of change could result from the rapid flow of neurosecretion along the axons. By the end of 60 min after the recommencement of feeding, the neurosecretion which had accumulated in the corpora cardiaca has been discharged, and the content of material in the glands has returned to its original value. In the reservoir, the individual variation in amount of neurosecretion in samples taken at 15, 60, and 120 min after the recommencement of feeding increases markedly. It is likely that this variability is due not only to individual differences in the rate of flow of neurosecretion (perhaps due to variations in feeding rate), but also to increases in the rate of synt,hesis of material by the neurosecretory cells. In S. gregaria, feeding after prior starvation produces an initial increase in the amount of neurosecretion in the major axon tracts just proximal to their points of exit from the brain (Highnam et al., 1966). In both S. gregaria and L. m. migratorioides, therefore, the stimulus of feeding causesan increased axonal flow of neurosecretion. In 8. gregaria, the corpora cardiaca of starved individuals contain large quantities of neurosecretion ; the initial increase in the rate of flow of material causesit to “back up” in the distal parts of the axons. In L. m. migratorioides. on the contrary, the corpora
582
FIG. 5. The structure
HIC;HNA.\I AND IVEST
of the neuropilar neurosecretory reservoir of Locusta migratoria migf’alorioides. (A) Whole mount of the pars intercerebralis, stained in .s& with periodic acid/Victoria blue, viewed from the anterior surface. Note that the dorsolateral parts of the reservoir are associated with their respeo tive major axon tracts, but that the central parts of the reservoir connect the two tracts. The lettering indicates levels of horizontal sections shown in B, G, and H. (B) Horizontal section of the right major axon tract (at) at the level a in Fig. .5A. Note that the incoming and outgoing reservoir fibers cross more or less at right angles (arrowed).
NEUROPILAR
NEUROSECRETORY
cardiaca of starved individuals contain less neurosecretion than those of continuously fed animals, so that the increased flow consequent upon feeding can be accumulated within the glands, at least temporarily. The short delay before material accumulates within the corpora cardiaca reflects
RESERVOIR
583
the time taken for neuroeecretion to pass at least from the NCCI, and probably from more distant sites. The rate of flow of neurosecretion along the axons in L. m. migratorioides is thus of the same order as that in S. gregaria (Highnam, 1966). In L. m. migratorioides, each median
FIG. 6. Horizontal sections through parts of the neurosecretory systems of animals whose left. group of median neurosecretory cells has been cauterized. (A) Section just below level a in Fig. 5A. No fibers or neurosecretion can be seen on the left side, although gaps in the neuropile (arrowed) occur. (B) Se&on at, about level 6 in Fig. 5A. Central reservoir fibers swing across from the right tract (compare Fig. 5G), and fibers carrying neurosecretion can now be seen in the position of the left tract (arrowed). (C) Section just below the major decussation of the axon tracts. Although smaller in amount than in the left (originally right) tract, significant amounts of neurosecretion are present in the right (originally left) tract (arrowed). (D) Section through the dorsal lobes of the corpora cardiaca. The right NCCI (m) and the right dorsal lobe of the corpora cardiaca @cc) contain neurosecretion, although in smaller amount than in the left NCCI (In) and left dorsal lobe (ICC). Stained paraldehyde fuchsin. Scales in p.
(C) Vertical section through the origin of left major tract showing central fibers (arrowed). (D) Vertical section through the periphery of the left major tract showing fibers (arrowed) which seem to run almost horizontally. These are parts of the peripheral axons which spiral with a much shallower pitch than those shown in 5C. (E) Vertical section through the right axon tract showing peripheral fibers (arrowed) running from left to right of the photograph. (F) The same section as 5E, but focused through the thickness of the axon tract, to show the peripheral fibers (arrowed) on t,he opposite side; these now run from right to left of the photograph. (G) Horizontal section of the axon tracts and reservoir at the level b in 5A. Fibers (arrowed) leave the outer face of t,he right tract and enter the outer face of the left tract. (H) Horizontal sect,ion of the axon tracts (at’s) at the level c in 5A. Fibers (arrowed) leave the inner face of the left tract and enter the inner face of the right tract. B - H stained with paraldehyde fuchsin. Scales in p.
group
of
cerehl
neuro~wretory
~~~~11s ill-
nervates its ipsilatersl ai n-cl1 ai its contralateral corpus cardiscum. The l)lienomenon is difficult to demonstrate cxperimentally, because partial cautcry of the cell groups is followed by increased activity in the remaining cells (Highnam and Hill, in preparation), and neurosrcretion within the corpora cardiaca can be made obvious only when its release is retarded, for example by starvation, as in the present experiments, Such double innervation of the corpora cardiaca may have important consequences, as when conclusions about the control of the activity of the corpora allata are based upon experiments involving unilateral cautery of the pars intercerebralis (e.g., Strong, 1965). Moreover, sotne axons may again cross the midline within t,he corpora cardiaca in L. m. migratoriaides, as in Melanoplzts snnguinipes (Dogra and Ewen, 19701. Arborizations of fibers associated with t)he roots of the intracerebral portions of t#he NCCI in Periplaneta americana are considered to be dendritic processes of the neurosecretory cells (Adiyodi and Bern, 19681. These arborizations, containing neurosecretory material, bear a remarkable resemblance t’o the neuropilar reservoir in L. m. migratorioides, and were indeed considered to be part of a small reservoir when the neurosecretory systems of our own stocks of P. americana were examined in 10 p thick sections and in whole mounts. In P. awericana, Rlatta germanica, Carausius morosus, and S. gTegaria, some of bhe “arborizing” fibers pass from one major neurosecretory axon tract to the other very like the type ii fibers in L. II). ntigratorioides. In this latter insect especially, the neuropilar neurosecretory rcservoir cannot be composed primarily of dendrit’ic processes, since both the histological and experimental evidence described here points to the carriage of neurosecretory material by the reservoir fibers to the corpora cardiaca. The asymmetry of the spiraling major axon tracts in L. m. migratorioides is of considerable interest,. In iMeZn?lopZzrs sangwinipes, the “medial” groups of ncurose-
cwtory
~11~
n-it11
th+r
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are founcl together on one side of the brain or the other in 10$4 of the inclividuals examined (Dogra and Encn, 19701. The asymmetry of the cells in both sljccies perhaps reflects a unilateral origin during embryogenesis: a detailed esamination of the early stages of devclo1n~~et~t~ of the cells will prove worthwhile. ACKSOWLEDGMENTS We are very grateful to Mr. Roger Webb, made the photomicrographs. Part of the work carried out during the tenure of an S.R.C. search Studentship held by M.R.W.
who was Re-
REFERESCES IL G., AND BERN, H. A. (1963). ?u’curonal appearance of neurosecretor~cells in the pars intercerebralis of Periplanet
NEUROPILAR
NEUROSECRETORY
K. C. (1966). Estimates of neurose-rctory activity during maturation in locusts. Pror. Int. Sump. Insect Endocrinol. BTIIO. V. J. A. Novak. Ed., Prague. HIGHNAM. Ii. C. (1967). Insect hormones. J. Endocrinol. 39, 123-150. HIGHNAM. K. C., HILL. L.. AND MORDUE, W. (1966) Thr endocrirw system and oocvte growth in Schistocerc~~ in relation to starvation and frontal ganglionectom~. J. lrwect Physiol. 12, 977-994. HILL, L, (1962). Xeuroaecretory control of haemolymph protein concentration during ovarian develoljment in the desxt locust. J. Insect Plqsiol. 8, 609-619. Jow.~sssor, A. S. (1958). Relation of nutrition to cndorrinr,-reproduc.tivc functions in the milkwc:~d hu< Oucope1trt.s frrsciat~ca (Dallas) (HrtHIGHNAM.
- ,iI b n
RESERVOIR
eroptera: 1-131.
Lygaeidae).
*Vylt
Jfrcg.
Zoo!.
7,
I,. (1965). The rrlationships between the brain, corpora allata, and oocyte growth in the Central American locust, Schi~tocercn sp. II. The innervation of the corpora allata. the lateral neurosecretory complex, and ooryte growth. J. Insect Physiol. 11, 271-280. TROMSEN. M. (1954). Xeurosecretion in some H?;menoptera. Dan. Biol. Skr. 7, 1-24. THOMSEN, M. (1965). The neurosrcrctory s:;stcTm of the adult Calliphorrc eq&rocephnlu. II. Histology of the nwrosecretory cells of the brain and some related structures. 2. Zellforsch. Nikrosk. Aurct. 67, 693-717. WIGGLESWORTH. V. B. (1964). The hormonal regulation of growth and reproduction in insects. Arlr~~,r. Insect Ph ysiol. 2, 247-336. &STRONG.