Adaptation of certain histological techniques for in situ

455

Adaptation of certain histological techniques for in situdemonstration of the neuro-endocrine system of insects

and other animals

By G. S. DOGRA and B. K. TANDAN

(From the Department of Zoology, University of Lucknow, Lucknow, India)

With 3 plates (figs, i to 3)

SummaryThree techniques for staining the secretory neurones in sections were applieddirectly to the whole brain and/or intact organs of the neuro-endocrine system ofcertain insects, and the whole brain of various invertebrates and vertebrates. Afterminor changes in the original procedures, in situ staining was achieved in thosecomponents of the neuro-endocrine system that are known to contain the neuro-secretory material. With the Victoria blue staining technique, the secretory neurones,the neurosecretory pathway, and the storage-and-release organ were stained satisfac-torily in all the experimental animals, in such a way that observations could be madein whole mounts or suitably dissected portions of the bulk-stained preparations. Withthe aldehyde-fuchsin and aldehyde-thionin staining techniques, the somata and theproximal portion of the axon of the neurones and the storage-and-release organwere usually stained satisfactorily enough for purposes of observation in the in-vertebrate material only. On sectioning the bulk-stained components of the neuro-endocrine system and mounting the sections, the sites known to contain theneurosecretory material were revealed promptly. On comparing the informationderived from mounts of the bulk-stained preparations with that derived from sectionsof similar preparations, and also with that derived from routine histological pro-cedure, no difference was detected.

IntroductionD U R I N G an investigation of the neuro-endocrine (or, for brevity, 'neurocrine')system of dipterous insects, the routine histological procedure of determiningthe topographical distribution and the number of secretory neurones in thebrain proved to be unduly lengthy. Not infrequently, through the loss of oneor more vital sections, at or after oxidation—especially if the oxidant werestrong—the series was ruined and vexation was added to an already lengthyprocedure. Hence the necessity arose to avoid or overcome these difficulties.

The median secretory neurones in the protocerebrum, being superficial,are often visible in the living insect brain. Their study by dark-ground illumi-nation and phase-contrast microscopy is, indeed, the result of this favourableposition. The superficial position of these neurones in the brain and theproperty of the perilemma investing it to permit passage to nutrient andexcretory substances (Wigglesworth, i960), suggested that even in the wholebrain the neurones might respond to the stains that are selective for them insections. In accordance with this line of reasoning, the aldehyde-fuchsin[Quart. J. micr. Sci., Vol. 105, pt. 4, pp. 455-66, 1964.]

456 Dogra and Tandan—Techniques for neuro-endocrine system

technique (Cameron and Steele, 1959) was applied directly to the brain of theflesh-fly, Sarcophaga ruficornis (Fabricius). When such slight changes intechnique were made as were necessary to adapt it to large pieces instead ofsections, it was found that the secretory neurones were stained. Two otherstaining techniques were then applied to the brain of the same species of flesh-fly, with eventual success.

In situ staining of the secretory neurones in the brain of S. ruficornis, andlater in other insects, encouraged similar applications to other components ofthe neurocrine system of insects in their intact state, and also to whole brainsof various invertebrates and vertebrates, with success. Whether or not thebulk-stained components would withstand paraffin embedding for subsequentsectioning was the next logical inquiry. A positive result was obtained.

Since the utility of the bulk-stained preparations far exceeded expectations,the details of the techniques are presented here, with some of the results thatdemonstrate their usefulness.

MethodsUsually several brains or intact organs of the neurocrine system of insects

or brains of invertebrates were processed together. Before oxidation they weredivided into two equal lots. One unoxidized lot served as the blank (control)and the other was oxidized. Occasionally, when the size permitted, the un-oxidized half of a longitudinally divided brain (and/or other components inthe case of insects) served as the blank, while the other half was oxidized forexperiment. The much larger vertebrate brain was usually processed singly.

Staining procedure IPerformic acid \ Victoria blue (VB) staining technique of F. D. Humberstone.This technique for revealing the neurosecretory material in oxidized,

paraffin sections of the brain has not hitherto been published. Mr. Humber-stone has kindly permitted us to give its details to enable workers to investigatethe neurocrine system in the manner presented in this paper.

Mr. Humberstone's technique for demonstrating neurosecretory materialin paraffin sections is a modification, carried out for Dr. J. C. Sloper, of theperformic acid / alcian blue technique of Adams and Sloper (1956) which wasdevised to demonstrate cystine or cysteine in paraffin sections of the hypo-thalamus of man, rat, and dog. The essence of the performic acid / alcian bluetechnique of Adams and Sloper is the oxidation of cystine or cysteine with avery strong oxidant, followed by the demonstration of the resultant cysteicacid with the basic dye alcian blue, at a low pH, 0-2. Because of its highcystine content the neurosecretory material (referred to as the 'posteriorpituitary principles' in Adams and Sloper, 1956) was demonstrated withremarkable specificity throughout its distribution in the hypothalamus of theabove-mentioned mammals.

The performic acid / Victoria blue technique is basically the same as the

Dogra and Tandan—Techniques for neuro-endocrine system 457

performic acid/alcian blue technique, differing from it, however, in thestain—which is an iron-resorcin lake of Victoria blue; this is applied to theoxidized sections to reveal the resulting cysteic acid. Mr. Humberstone con-ceived the idea of using an iron-resorcin lake (or precipitate) of basic dyes forthis purpose, knowing such lakes to be an important group of stains for elastictissue (Lillie, 1954). A few years ago it was indicated by Sloper (1958a) thatHumberstone obtained better staining of oxidized sections by using the preci-pitate from a crystal violet / dextrin / resorcin / fuchsin mixture instead ofalcian blue. In the same year Sloper (19586) recorded that a variety of dyes canbe substituted for alcian blue. When Mr. Humberstone substituted Victoriablue for crystal violet as the basic dye, and used the derived precipitate as thestain, superior results were obtained.

Thus, by using Victoria blue, a dye well known for its special affinity forelastic fibres, Mr. Humberstone adapted beautifully the iron-resorcin lakesof basic dyes, used for staining elastic fibres, for staining the neurosecretorymaterial in oxidized sections.

Mr. Humberstone's staining technique was kindly furnished to us by Dr.Sloper in a cyclostyled form. Acknowledgement is made to Mr. Humberstonefor permitting the first publication of an original technique.

Reagents. The oxidant, performic acid, is prepared according to Pearse(1953), being the same as that used by Adams and Sloper (1956).

Prepare the staining solution thus:

Mix in a flask: distilled water 200 mldextrine 0-5 gVictoria blue 4R 2 gresorcin 4 g

Bring to boil. When boiling briskly add boiling 25 ml of 29% ferric chloride.Boil for 3 min; cool. A heavy precipitate forms. Filter and dry the precipitatein an oven at 50° C. Dissolve all precipitate in 400 ml of 70% alcohol. Whendissolved add 4 ml of concentrated hydrochloric acid and 6 g of phenol. Forbetter results use after two weeks. The stain keeps for months.

In control section(s) omit oxidation.Result. Material rich in cystine appears blue in test section(s) only.Precautions. The H2O2 used in preparing the oxidant should not be kept for

more than 3 weeks after opening the bottle.The slides must be scrupulously clean. For this boil them in a detergent

(sodium lauryl sulphate) or treat with chromic acid.Technique. Fix the dissected brain in 10% formaldehyde-saline; embed in

paraffin wax and section. Bring sections fixed to the slide to xylene, then toabsolute alcohol; allow them just to dry. Place the slide on match-sticks in aPetri dish and drop performic acid solution on the dry section(s). Oxidize, 5min. Wash in distilled water, 15 min. Rinse in 70% alcohol. Stain in stainingsolution, 12 h. Wash in 70% alcohol. (Here you may rinse in tap-water,

45 8 Dogra and Tandan—Techniques for neuro-endocrine system

counterstain in o-i% safranin in i% acetic acid, 5 min, and rinse again intap-water.) Dehydrate, clear, and mount.

The details of our method of modifying Humberstone's technique, so asto make it applicable to bulk material instead of sections, is given below.

Since the organs comprising the neurocrine system of insects and manyother invertebrates are small, the bulk-processing technique is neither incon-venient nor wasteful.

1. Expose the brain or other organ in a partially anaesthetized or unanaes-thetized animal placed in an appropriate physiological saline solution. Forinsects that of Ephrussi and Beadle (1936) is suitable. Fix in situ in 10%formaldehyde-saline (physiological saline 90 ml, commercial formalin10 ml). After 2 to 3 h dissect out the organs required and place in freshfixative; fix for 24 to 36 h; wash well in tap-water for 2 or 3 h; next indistilled water for 10 to 20 min; blot off the water with strips of filterpaper.

2. Oxidize the organs in oxidant until transparent (5 min or more).3. Blot off excess oxidant with filter paper.4. Wash repeatedly in distilled water, 20 to 30 min.5. Transfer through 30% to 70% alcohol.6. Stain in staining solution, 12 to 18 h, the period depending on size.7. Quickly blot off excess stain with filter paper.8. Differentiate in 70% alcohol; change the alcohol repeatedly, until no

more superfluous stain is given off.9. Dehydrate; clear in cedarwood oil, 2 to 4 h. Either mount the whole

brain or intact neurocrine system or dissected components of the latter inCanada balsam (but if so, remove the cedarwood oil with xylene (2 min)before mounting), or embed in paraffin wax in normal manner, cutsections, and mount in Canada balsam. If desired, counterstain withsafranin. Both thin (5 /x) and thick (25 ju.) sections are satisfactory forobservations.

The brain of small vertebrates can be processed whole, but trimming afterfixation avoids waste of reagents. That of larger vertebrates requires trimming.It is advisable to process the pituitary gland separately.

1. Fix the whole brain for 48 h or longer, depending on its size, in 10%formaldehyde-saline. Wash well in running tap-water for about 24 h;next in distilled water, about 1 h.

2. Oxidize brain in oxidant, 20 to 30 min, or until transparent; a smallpituitary requires about 15 min.

3 to 9. As for the invertebrates.Notes and cautions. The storage-and-release organ is revealed at stage 8;

the secretory neurones are revealed at the same stage if heavily loaded, other-wise at stage 9. The appearances in mounted preparations, depending on theamount of contained neurosecretory material, are as follows: somata, blue orgreenish-blue; proximal portion of axons, greenish-blue; the neurosecretory


pathway, light greenish-blue; storage-and-release organ, blue or dark blue.Background, unstained or faint blue.

At stage 2 the brain (especially that of insects, owing to air trapped in thetracheoles) tends to float in the oxidant. To keep it submerged it is weightedwith a thick coverslip, which in turn is kept gently pressed down with abluntly-pointed glass rod. Since the H2O2 ingredient of the oxidant is un-stable, a new bottle, once opened, serves for 2 or 3 weeks only, even if storedin a refrigerator; thereafter it must be discarded (in accordance with Hum-berstone's directions).

The staining solution used at stage 6 was Victoria blue RN 275, not Victoriablue 4R. The ferric chloride ingredient of the solution is hygroscopic; andalthough the hydrated compound serves the purpose, yet it is preferable touse an unhydrated sample. In comparison to a freshly prepared to a 3-month-old staining solution, a solution from 3 to 20 months old gives more brilliantresults and leaves the background unstained.

As oxidation loosens the sheath investing the brain, at stage 8 this sheathis now readily removed. The process of differentiation may thus be hastened.The differentiation of vertebrate brain is very slow; it should be consideredcomplete only when the alcohol is no longer coloured.

Staining procedure IIGomori's aldehyde-fuchsin (AF) technique, modified by Cameron and Steele

{1959)The following technique is applicable to the neurocrine organs of insects

and other invertebrates.Stages 2 to 8 are so arranged as to correspond with those given by Cameron

and Steele.1. Follow, in general, the VB technique for bulk-processing, but fix for

12 to 24 h in Bouin's fluid; wash thoroughly in 70% alcohol; bring downto distilled water.

2. Oxidize organs in oxidant, 2 to 3 min (0-3 g KMnO4 in 100 ml watercontaining 0-30 ml concentrated H2SO4).

3. After blotting off the oxidant with strips of filter paper, bleach with 4%sodium bisulphite; renew the solution once or twice, remove on becom-ing perfectly white (1 to 10 min, depending on size of material).

4. Wash in distilled water, 5 to 10 min.5. Same as for VB technique.6. Stain in staining solution, 2 to 10 min.7. Same as for VB technique.8. Differentiate in 95% alcohol until no more superfluous stain is given off;

change alcohol once or more. If a precipitate adheres to the sheathinvesting the brain, treat with 70% alcohol until the precipitate fadesaway.

9. Same as for VB technique, but for counterstaining sections use therecommended Halmi's mixture.


Notes and cautions. The components of the neurocrine system are revealedat stage 8 or 9, as in the VB technique. The appearances in mounted prepara-tions are as follows: somata, light to deep purple; proximal portion of axons,purple; the neurosecretory pathway, occasionally light or faint purple;storage-and-release organ, dark purple; background, faint purple.

Thorough removal of the picric acid of the fixative at stage 1 is essential,as even the lightest yellow tint in the material vitiates the sharpness of thestained components.

Bleaching at stage 3 is controlled under a stereoscopic microscope, withstrong reflected illumination.

During differentiation at stage 8, pressing the organs gently with the tipsof forceps expels superfluous stain quickly and loosens the sheath investingthe brain, which may be removed either at this stage or in cedarwood oil(stage 9).

Staining procedure III

The aldehydejthionin [A Th) technique of Paget (igsg)The following directions apply to the neurocrine organs of insects and other

invertebrates. Stages 2 to 4 are so arranged as to correspond with those givenby Paget.

FIG. 1 (plate), A, pituitary of the white laboratory rat, mounted with the dorsal surfacetouching the coverslip. VB technique.

B, dissected pars intercerebralis of the grasshopper, Euconocephalus sp., showing the twoNCC I and their crossing over. VB technique.

c, infundibular process of the lizard, Hemidactylus flaviviridis, mounted with the surfacetowards the adenohypophysis touching the coverslip. VB technique.

D, corpora cardiaca of the dragonfly, Bradinopyga geminata, with associated structures. VBtechnique.

E, dissected pars intercerebralis of the grasshopper, Oedaleus abruptus, showing the twoNCC I and their crossing over. VB technique.

F, dissected pars intercerebralis of Bradinopyga geminata, showing the two NCC I and theircrossing over. VB technique.

G, brain of B. geminata, mounted with the anterior surface touching the coverslip. Parsintercerebralis/corpus cardiacum components in the intact state. Corpora cardiaca have beenreflexed anteriorly. VB technique.

H, dissected pars intercerebralis of the red cotton-bug, Dysdercus koenigii, showing 9 + 9secretory neurons. ATh technique.

1, dissected pars intercerebralis of the flesh-fly, Sarcophaga ruficornis, showing about 26secretory neurones heavily loaded with the neurosecretory material. AF technique.

J, same as I, but of a newly-ecloded flesh-fly, showing 15 or 16 secretory neurones only, asall are not active, containing small amounts of the neurosecretory material. AF technique.

K, sinus gland of the fresh-water palaemonid, Macrobrachium sp. (immature). AF technique.L, dissected pars intercerebralis of the bug, Graptostethus sp., showing 5 + 5 secretory

neurones. VB technique.M, same as L, but of the bed bug, Ciinex lectularius, showing 5 + 5 secretory neurones. AF

technique.N, supraoesophageal ganglion of the leech, Hirudinaria granulosa, mounted with the dorsal

surface touching the coverslip. AF technique.ao, aorta; ad, adenohypophysis; ca, corpus allatum; cc, corpus cardiacum; ct, connective

tissue; ipr, infundibular process; ist, infundibular stem; rn, oesophageal nerve.

FIG. I

G. S. DOGRA and B. K. TANDAN

FIG. 2G. S. DOGRA and B. K. TANDAN


1. Same as for the AF technique.2. Oxidize organs in oxidant, 2 to 3 min (0-5 ml concentrated H2SO4 added

to 100 ml of 0-5% KMnO4).3. Same as for the AF technique, but bleach with 2% potassium metabi-

sulphite, 2 to 10 min, until perfectly white.4 and 5. Wash in distilled water.6. Stain for 15 min to 2 h.7 and 8. Differentiate for 2 min in distilled water.9. Same as for VB (and AF) techniques, but for counterstaining sections

use the stains originally recommended, or Halmi's mixture, which isequally satisfactory.

Notes and cautions. The components of the neurocrine system are revealedat stages 7 and 8 or at stage 9, as in the VB and AF techniques. The appearancesin mounted preparations are as follows: somata, blue or dark blue; proximalportion of axons, light blue or blue; the neurosecretory pathway, occasionallylight blue; storage-and-release organ, dark blue; blackground, faint blue.

Bouin's fluid is substituted for Zenker's, because the mercuric chloride inthe latter interferes with dissection.

The sheath investing the brain is removed in accordance with the instruc-tions given under Notes and cautions referring to the AF technique.

To display effectively the secretory neurones and the neurosecretory path-way in whole mounts, it is essential to remove the sheath investing the brain.Although the sheath can be peeled off before fixation, this is not favouredbecause its removal late in the procedure, at stage 8 or 9, protects the prepara-tion from contamination with foreign particles. At stage 9 the brain is dissectedwith fine-pointed needles to expose or isolate the area containing the stainedcomponents. Dissection is performed under double illumination, the sourceof reflected light being a 6- to 8-volt spot-light and that of transmitted lighta 100-watt milk-white bulb. The storage-and-release organ does not requireelaborate dissection.

The bulk-stained preparations can be stored in cedarwood oil for about aweek without apparently affecting the brilliancy of the stained components,but dissection is not possible after about 48 h, as the tissues become brittle.

FIG. 2 (plate), A, partly dissected hypothalamus (left half) of the lizard, Hemidactylusflaviviridis, mounted with the inner ventricle surface touching the coverslip. Optic chiasmaand optic nerve removed. VB technique.

B, dissected hypothalamus of the lizard, Hemidactylus flaviviridis, mounted with the dorsalsurface touching the coverslip. Optic chiasma and optic nerves present. VB technique.

c, portion of the preparation shown in A, at higher magnification, showing the relay ofsecretory neurones. VB technique.

D, undissected hypothalamus (right half) of the lizard, Hemidactylus flaviviridis, mountedwith the outer surface touching the coverslip. VB technique.

E, left-hand side portion of the preparation shown in B, at higher magnification, showing therelay of secretory neurones. VB technique.

me, median eminence; npa, nucleus paraventricularis; nsu, nucleus supraopticus; on, opticnerve.


ResultsObservations on the bulk-stained preparations were made in two ways, as

described below.Examination in whole mounts or mounts of exposed or dissected portions

Fig. 1, G shows the intact neurocrine system of a dragonfly, Bradinopygageminata; the pars intercerebralis/corpus cardiacum components of thissystem, being stained, are quite distinct.

Fig. 1, N demonstrates the distribution of the secretory neurones, in thesupraoesophageal ganglion of a leech, Hirudinaria granulosa.

Fig. 2, A is the left half of the partly-dissected hypothalamus of a lizard,Hemidactylus fiaviviridis, showing the secretory neurones of the paraventri-cularis and supraopticus nuclei (and the distal half of the tractus hypophyseus,together with the median eminence) in their natural positions. This figure alonedemonstrates the inter-relationships of the important components, except theinfundibular stem and process, of the neurocrine system. The terms used forthe components are those given by Sloper (1958&).

In fig. 2, D, showing the right half of the undissected hypothalamus, thesecretory neurones of the nucleus paraventricularis are much clearer. Fig. 3, Fis an enlargement of the area enclosed in the rectangle in fig. 2, D, to show theappearance of the intact neurons in situ.

In fig. 2, B, showing the partly-dissected hypothalamus of H. fiaviviridis,one sees the secretory neurones of the nucleus supraopticus of both sides intheir natural positions, slightly anterior to and overlying the optic chiasma.From the main cluster of the nucleus supraopticus, a relay of neuronesextends towards the median eminence, the latter represented in this figureby the median dark area. The relay is more distinct in fig. 2, c, E.

Thus, in brains mounted whole or after such dissection as is unavoidablefor proper display and mounting, the topographical distribution of thesecretory neurones is revealed convincingly.

Fig. 1,1, J shows the difference in the amount of the neurosecretory materialin the somata of secretory neurones of the flesh-fly in imagos of different ages.As even small amounts of the elaborated material are revealed, phases of itsaccumulation in the perikaryon are well exhibited.

Fig. 1, M shows the dissected brain of the common bed bug, Cimex lectu-larius. This preparation demonstrates that the handicap of its size is overcomeby staining the brain as a whole. Satisfactory preparations showing the secre-tory neurones in the brain of other even smaller insects (mosquitoes, mosquitolarvae, &c.) have been made without much difficulty.

Fig. 1, H, L shows that the secretory neurones can be counted directly, iftheir number is not too large.

Fig. 3, E shows neurones from the cluster of the nucleus supraopticus ofH. fiaviviridis. It demonstrates that they can be isolated and teased for obser-vation. Fig. 3, G is a magnified view of a part of the cluster shown in fig. 3, E,to show the appearance of the neurones in a whole mount.

FIG. 3G. S. DOGRA and B. K. TANDAN


Isolation of neurones presents a considerable advantage, as it has madetheir direct counting possible. Information on the number of secretory neu-rones in the brain might promote an understanding of the physiologicalphenomena regulated by their secretions, one such interesting phenomenonbeing the adaptation of related animals to widely-varying biotopes (seeHanstrom, 1956).

The results given below on the neurosecretory pathway that transportsthis material, and on the storage-and-release organs, are equally informative.

Fig. 1, B, E, F demonstrates unequivocally the crossing over of the twonervi corporis cardiaci I. The figure depicts the secretory neurones (out offocus) and the two NCC I in a brain, in position of dissection. The curvaturein fig. 1, F (slightly out of focus) represents the change in the course of thetwo NCC I from the anterior to the ventral surface of the brain.

Since the discovery of the crossing over of the two NCC I (Hanstrom, 1940),it has been reported in numerous insects of different orders; and although itis an established feature of the pars intercerebralis/corpus cardiacum complex,we have not seen such a convincing demonstration of it in the literature.

Fig. 2, A shows the hypothalamo-hypophysial tract in the brain of H.flaviviridis, from the point marked by the arrow to the attachment point ofthe infundibular stem with the median eminence.

Fig. 1, D shows the corpora cardiaca of the dragonfly, B. geminata, andfig. 1, K the sinus gland of the fresh-water palaemonid, Macrobrachium sp.These are the storage-and-release organs of these two animals.

Fig. 1, A, c shows the infundibular process of the neurohypophysis, alongwith the infundibular stem. Fig. 1, A is the pituitary of the white laboratoryrat in the natural position; in this figure the distinction between pars inter-media and pars distalis parts of the adenohypophysis is not reproduced, butit is clearly evident in the original preparation. Fig. 1, c is the infundibularprocess, detached for mounting separately from the preparation of the brainshown in fig. 2, B.

As the natural relationships are conveyed better in the whole or intact stateof these organs, such preparations have been utilized for the microphoto-graphic demonstration of the results.

FIG. 3 (plate), A, 12 y. sagittal section in wax of a bulk-stained brain of the lizard, Hemidac-tylus flaviviridis. VB technique.

B, 10 ix sagittal section of a bulk-stained brain of the lizard, Hemidactylus flaviviridis. VBtechnique, counterstained with safranin and mounted in Canada balsam.

c, 6 /x transverse section in wax of a bulk-stained brain of the red cotton-bug, Dysdercuskoenigii, showing 3 + 4 secretory neurones. AF technique.

D, 12 fx sagittal section in wax from the same series from which A was taken, showing thesecretory neurones of the nucleus supraopticus. VB technique.

E, some isolated and teased secretory neurones of the nucleus supraopticus of the lizard,Hemidactylus flaviviridis. VB technique.

F, enlarged view of the secretory neurones of the nucleus paraventricularis, enclosed in therectangle in fig. 2, D.

G, magnified view of the secretory neurones of the nucleus supraopticus shown in E.


Examination in sections

Fig. 3, c is a transverse section, in wax, of the brain of a bug, Dysdercuskoenigii, to demonstrate the stained secretory neurones in the pars intercere-bralis of the protocerebrum.

Fig. 3, A, D shows small parts of different sagittal sections of an uninter-rupted series, still in wax, of the whole brain of H. flaviviridis. The sectionshown in fig. 3, D passes through the nucleus supraopticus, and the stainedsecretory neurones are unmistakable in it; that in fig. 3, A passes almostthrough the middle of the neurohypophysis.

Fig. 3, B is a section similar to that in fig. 3, A, but counterstained withsafranin and mounted in Canada balsam. It differs in no way from a finishedsection obtained by routine histological procedure.

The anatomical distribution of the neurosecretory material in the tractushypophyseus and the infundibular process agrees remarkably in fig. 3, A and B.The adenohypophysis became detached from both these brains during pro-cessing.

Thus, on sectioning paraffin-embedded preparations and simply mountingthe sections in Canada balsam, the sites known to contain the neurosecretorymaterial are revealed. Although counterstaining for contrast is desirable, itis nevertheless optional.

RemarksThe VB and AF staining techniques were mostly used, on account of the

stability of the staining solutions—about 20 and 8 months respectively.The three techniques worked well with invertebrates, but only the VB

technique succeeded with vertebrates.Much information on the secretory neurones, the neurosecretory pathway,

and the storage-and-release organ is derivable directly from mounts of bulk-stained preparations. All the information presented here, other than thatobtained from the sections of the bulk-stained preparations, was derived fromsuch mounts. This information, it would be conceded, is in a form that isclearer than that obtained by the usual histological procedure. These qualitiesresult from the intact state of the components in question.

As this mode of investigating the neurocrine system eliminates the necessityfor sectioning, one of the two difficulties stated at the beginning—the lengthynature of the histological procedure—was overcome, and the other—detach-ment and flotation of sections—was naturally not encountered.

To adapt the bulk-stained preparations for subsequent sectioning was alogical outcome. Examination in cedarwood oil makes it possible to select welland darkly-stained preparations, which, being heavily loaded with the neuro-secretory material, are better suited for study in sections. No method of makingsuch an assessment has previously been available. It has only been possible todo it at the end-stage, when sections have been cut, stained, and mounted


indiscriminately, at the expense of much time and labour. Screening of thepreparations before sectioning saves time and fruitless labour.

Thus, bulk-staining makes possible the study of the components of theneurocrine system in whole mounts and also in sections of the same or similarpreparations. Information can therefore be obtained in two unrelated waysand, what is more, can be cross-checked. A comparison with the informationderived from routine histological procedure shows no obvious difference.

Elimination of oxidation of the sections, a step responsible for their detach-ment and subsequent loss by flotation, has made possible the mounting ofserial sections of whole brain of both invertebrates and vertebrates, withoutloss. In these uninterrupted series not only are all sections present, but, inthe sections that contain them, the components of the neurocrine system arein an equally satisfactorily stained state.

Insects being of primary interest to us, it will not be out of place toreport two features of the results with members of this group. These salientfeatures are the outcome of the intact state and clarity of the pars intercere-bralis/corpus cardiacum components, as a consequence of which a comparativestudy of the neurocrine system is greatly simplified.

1. Results with the 3 techniques do not agree completely intraspecifically.Whereas the results with the AF and ATh techniques tend to agree, thosewith the VB technique differ (and the magnitude of this difference variesbetween the species). This lack of intraspecific agreement seems so far to bedue to either (i) the strength of the oxidant in the respective techniques, or(ii) the physiological state of the individual, or (iii) a combination of (i) and(ii), or (iv) intraneuronal factors, hitherto not well understood.

2. Results with one technique on different hemimetabolous insects are notstrictly parallel. So far, inherent morphological differences in the componentsof the neurocrine system, which seem to have received scant attention,appear to be responsible for this divergence.

Specific instances of these two features will be given in full reports on theinsects concerned.

It may be stated, to complete the record, that statement 1 above is broadlyapplicable to members of other classes of arthropods besides insects, and tomembers of other invertebrate phyla.

DiscussionThe idea of staining the nervous tissue in bulk is not new, as it is not

uncommon in neurohistological investigations for this tissue to be fixed first,then stained, and subsequently sectioned (Lee, chapters 39 and 40, 1950).Metallic compounds or histological or cytological stains have been used inthis way. Although unconventional, this method has provided most valuableinformation on the cyto-architecture of the nervous tissue.

The original features of this report and the conclusions derived from themare:

2421.4 I i


1. The in situ staining of the components of the neurocrine system, andthe utilization of bulk-stained preparations for making observations. Thismethod, tested with a variety of animals, has led us to conclude that ex-perimentally produced changes in the anatomical distribution of theneuro-secretory material can be demonstrated rapidly—and possibly moreconvincingly—in such preparations than by the ordinary histological pro-cedure involving the staining of sections. In vertebrates, one-half of thelongitudinally divided brain can conveniently be oxidized and the resultsdemonstrated directly, somewhat as in fig. 2, A, while the other (unoxidized)half serves as a 'blank' or control.

2. The demonstration that a specific technique (VB), hitherto used onsections, can be used with success on whole or gross pieces of nervous tissue.This leads us to conclude that the chemistry of secretory neurones (perhapsof neurones in general), which has hitherto been studied in sections, mightbe investigated by applying appropriate techniques on whole or gross piecesof nervous tissue.

Finally, this simple and direct method of demonstrating the components ofthe neurocrine system has provided students of zoology with a means ofstudying an important organ system that has not previously been so accessibleto investigation.

We are grateful for Dr. J. C. Sloper, of Charing Cross Medical School,University of London, for helpful suggestions and for advising us to try Mr.F. D. Humberstone's staining technique, which is a modification of a methodintroduced by Dr. Sloper; and to Mr. Humberstone, of Dr. Sloper's Depart-ment, for generously permitting us to give details of his previously unpublishedtechnique. We are indebted also to Dr. John R. Baker, F.R.S., for helpfulcomments on the typescript; to Dr. Nitya Anand of the Central DrugResearch Institute, Lucknow, for very valuable help, and to Professor M. B.Lai for his unceasing interest in our work. Thanks are also extended to theDyestuffs Division of the Imperial Chemical Industries, Blackley, Manchester,for the gift of Victoria blue RN 275.

ReferencesAdams, C. W. M., and Sloper, J. C, 1956. J. Endocrin., 13, 221.Cameron, M. L., and Steele, J. E., 1959. Stain Tech., 34, 265.Ephrussi, B., and Beadle, G. W., 1936. Amer. Nat., 70, zi8.Hanstrom, B., 1940. K. svenska VetensAkad. Handl., 18, 1.

1956. Proc. 8th Symp. Colston Res. Soc, 23.Lee, B., 1950. The microtomist's vade-mecum, n th edition. London (Churchill).Lillie, R. D., 1954. Histopathologic technic and practical histochemistry. New York (Blakiston).Paget, G. E., 1959. Stain Tech., 34, 223.Pearse, A. G. E., 1953. Histochemistry, theoretical and applied. London (Churchill).Sloper, J. C, 1958a. Zweites Int. Symp. uber Neurosekretion, Lund, 1957. Berlin (Springer-

Verlag).19586. Internat. Rev. Cytol., 7, 337.

Wigglesworth, V. B., i960. J. exp. Biol., 37, 500.

Adaptation of certain histological techniques for in situ

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