Download Adaptation of certain histological techniques for in situ

455
Adaptation of certain histological techniques for in situ
demonstration 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)
Summary
Three techniques for staining the secretory neurones in sections were applied
directly to the whole brain and/or intact organs of the neuro-endocrine system of
certain insects, and the whole brain of various invertebrates and vertebrates. After
minor changes in the original procedures, in situ staining was achieved in those
components of the neuro-endocrine system that are known to contain the neurosecretory material. With the Victoria blue staining technique, the secretory neurones,
the neurosecretory pathway, and the storage-and-release organ were stained satisfactorily in all the experimental animals, in such a way that observations could be made
in whole mounts or suitably dissected portions of the bulk-stained preparations. With
the aldehyde-fuchsin and aldehyde-thionin staining techniques, the somata and the
proximal portion of the axon of the neurones and the storage-and-release organ
were usually stained satisfactorily enough for purposes of observation in the invertebrate material only. On sectioning the bulk-stained components of the neuroendocrine system and mounting the sections, the sites known to contain the
neurosecretory material were revealed promptly. On comparing the information
derived from mounts of the bulk-stained preparations with that derived from sections
of similar preparations, and also with that derived from routine histological procedure, no difference was detected.
Introduction
D U R I N G an investigation of the neuro-endocrine (or, for brevity, 'neurocrine')
system of dipterous insects, the routine histological procedure of determining
the topographical distribution and the number of secretory neurones in the
brain proved to be unduly lengthy. Not infrequently, through the loss of one
or more vital sections, at or after oxidation—especially if the oxidant were
strong—the series was ruined and vexation was added to an already lengthy
procedure. 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 illumination and phase-contrast microscopy is, indeed, the result of this favourable
position. The superficial position of these neurones in the brain and the
property of the perilemma investing it to permit passage to nutrient and
excretory substances (Wigglesworth, i960), suggested that even in the whole
brain the neurones might respond to the stains that are selective for them in
sections. In accordance with this line of reasoning, the aldehyde-fuchsin
[Quart. J. micr. Sci., Vol. 105, pt. 4, pp. 455-66, 1964.]
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Dogra and Tandan—Techniques for neuro-endocrine system
technique (Cameron and Steele, 1959) was applied directly to the brain of the
flesh-fly, Sarcophaga ruficornis (Fabricius). When such slight changes in
technique were made as were necessary to adapt it to large pieces instead of
sections, it was found that the secretory neurones were stained. Two other
staining techniques were then applied to the brain of the same species of fleshfly, with eventual success.
In situ staining of the secretory neurones in the brain of S. ruficornis, and
later in other insects, encouraged similar applications to other components of
the neurocrine system of insects in their intact state, and also to whole brains
of various invertebrates and vertebrates, with success. Whether or not the
bulk-stained components would withstand paraffin embedding for subsequent
sectioning 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 that
demonstrate their usefulness.
Methods
Usually several brains or intact organs of the neurocrine system of insects
or brains of invertebrates were processed together. Before oxidation they were
divided into two equal lots. One unoxidized lot served as the blank (control)
and the other was oxidized. Occasionally, when the size permitted, the unoxidized half of a longitudinally divided brain (and/or other components in
the case of insects) served as the blank, while the other half was oxidized for
experiment. The much larger vertebrate brain was usually processed singly.
Staining procedure I
Performic 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. Humberstone has kindly permitted us to give its details to enable workers to investigate
the neurocrine system in the manner presented in this paper.
Mr. Humberstone's technique for demonstrating neurosecretory material
in paraffin sections is a modification, carried out for Dr. J. C. Sloper, of the
performic acid / alcian blue technique of Adams and Sloper (1956) which was
devised to demonstrate cystine or cysteine in paraffin sections of the hypothalamus of man, rat, and dog. The essence of the performic acid / alcian blue
technique of Adams and Sloper is the oxidation of cystine or cysteine with a
very strong oxidant, followed by the demonstration of the resultant cysteic
acid with the basic dye alcian blue, at a low pH, 0-2. Because of its high
cystine content the neurosecretory material (referred to as the 'posterior
pituitary principles' in Adams and Sloper, 1956) was demonstrated with
remarkable specificity throughout its distribution in the hypothalamus of the
above-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 the
stain—which is an iron-resorcin lake of Victoria blue; this is applied to the
oxidized sections to reveal the resulting cysteic acid. Mr. Humberstone conceived the idea of using an iron-resorcin lake (or precipitate) of basic dyes for
this purpose, knowing such lakes to be an important group of stains for elastic
tissue (Lillie, 1954). A few years ago it was indicated by Sloper (1958a) that
Humberstone obtained better staining of oxidized sections by using the precipitate from a crystal violet / dextrin / resorcin / fuchsin mixture instead of
alcian blue. In the same year Sloper (19586) recorded that a variety of dyes can
be substituted for alcian blue. When Mr. Humberstone substituted Victoria
blue for crystal violet as the basic dye, and used the derived precipitate as the
stain, superior results were obtained.
Thus, by using Victoria blue, a dye well known for its special affinity for
elastic fibres, Mr. Humberstone adapted beautifully the iron-resorcin lakes
of basic dyes, used for staining elastic fibres, for staining the neurosecretory
material 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. Humberstone
for 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
dextrine
Victoria blue 4R
resorcin
200 ml
0-5 g
2g
4g
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 precipitate
in an oven at 50° C. Dissolve all precipitate in 400 ml of 70% alcohol. When
dissolved add 4 ml of concentrated hydrochloric acid and 6 g of phenol. For
better 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 H 2 O 2 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 to
absolute alcohol; allow them just to dry. Place the slide on match-sticks in a
Petri dish and drop performic acid solution on the dry section(s). Oxidize, 5
min. Wash in distilled water, 15 min. Rinse in 70% alcohol. Stain in staining
solution, 12 h. Wash in 70% alcohol. (Here you may rinse in tap-water,
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Dogra and Tandan—Techniques for neuro-endocrine system
counterstain in o-i% safranin in i% acetic acid, 5 min, and rinse again in
tap-water.) Dehydrate, clear, and mount.
The details of our method of modifying Humberstone's technique, so as
to make it applicable to bulk material instead of sections, is given below.
Since the organs comprising the neurocrine system of insects and many
other invertebrates are small, the bulk-processing technique is neither inconvenient nor wasteful.
1. Expose the brain or other organ in a partially anaesthetized or unanaesthetized animal placed in an appropriate physiological saline solution. For
insects that of Ephrussi and Beadle (1936) is suitable. Fix in situ in 10%
formaldehyde-saline (physiological saline 90 ml, commercial formalin
10 ml). After 2 to 3 h dissect out the organs required and place in fresh
fixative; fix for 24 to 36 h; wash well in tap-water for 2 or 3 h; next in
distilled water for 10 to 20 min; blot off the water with strips of filter
paper.
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 in
Canada balsam (but if so, remove the cedarwood oil with xylene (2 min)
before mounting), or embed in paraffin wax in normal manner, cut
sections, and mount in Canada balsam. If desired, counterstain with
safranin. Both thin (5 /x) and thick (25 ju.) sections are satisfactory for
observations.
The brain of small vertebrates can be processed whole, but trimming after
fixation 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 small
pituitary 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, otherwise at stage 9. The appearances in mounted preparations, depending on the
amount of contained neurosecretory material, are as follows: somata, blue or
greenish-blue; proximal portion of axons, greenish-blue; the neurosecretory
Dogra and Tandan—Techniques for neuro-endocrine system
459
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 the
tracheoles) tends to float in the oxidant. To keep it submerged it is weighted
with a thick coverslip, which in turn is kept gently pressed down with a
bluntly-pointed glass rod. Since the H 2 O 2 ingredient of the oxidant is unstable, a new bottle, once opened, serves for 2 or 3 weeks only, even if stored
in a refrigerator; thereafter it must be discarded (in accordance with Humberstone's directions).
The staining solution used at stage 6 was Victoria blue RN 275, not Victoria
blue 4R. The ferric chloride ingredient of the solution is hygroscopic; and
although the hydrated compound serves the purpose, yet it is preferable to
use an unhydrated sample. In comparison to a freshly prepared to a 3-monthold staining solution, a solution from 3 to 20 months old gives more brilliant
results and leaves the background unstained.
As oxidation loosens the sheath investing the brain, at stage 8 this sheath
is now readily removed. The process of differentiation may thus be hastened.
The differentiation of vertebrate brain is very slow; it should be considered
complete only when the alcohol is no longer coloured.
Staining procedure II
Gomori'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 down
to distilled water.
2. Oxidize organs in oxidant, 2 to 3 min (0-3 g KMnO 4 in 100 ml water
containing 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 becoming 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 sheath
investing the brain, treat with 70% alcohol until the precipitate fades
away.
9. Same as for VB technique, but for counterstaining sections use the
recommended Halmi's mixture.
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Dogra and Tandan—Techniques for neuro-endocrine system
Notes and cautions. The components of the neurocrine system are revealed
at stage 8 or 9, as in the VB technique. The appearances in mounted preparations 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 the
stained components.
Bleaching at stage 3 is controlled under a stereoscopic microscope, with
strong reflected illumination.
During differentiation at stage 8, pressing the organs gently with the tips
of forceps expels superfluous stain quickly and loosens the sheath investing
the 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 given
by Paget.
FIG. 1 (plate), A, pituitary of the white laboratory rat, mounted with the dorsal surface
touching the coverslip. VB technique.
B, dissected pars intercerebralis of the grasshopper, Euconocephalus sp., showing the two
NCC I and their crossing over. VB technique.
c, infundibular process of the lizard, Hemidactylusflaviviridis,mounted with the surface
towards the adenohypophysis touching the coverslip. VB technique.
D, corpora cardiaca of the dragonfly, Bradinopyga geminata, with associated structures. VB
technique.
E, dissected pars intercerebralis of the grasshopper, Oedaleus abruptus, showing the two
NCC I and their crossing over. VB technique.
F, dissected pars intercerebralis of Bradinopyga geminata, showing the two NCC I and their
crossing over. VB technique.
G, brain of B. geminata, mounted with the anterior surface touching the coverslip. Pars
intercerebralis/corpus cardiacum components in the intact state. Corpora cardiaca have been
reflexed anteriorly. VB technique.
H, dissected pars intercerebralis of the red cotton-bug, Dysdercus koenigii, showing 9 + 9
secretory neurons. ATh technique.
1, dissected pars intercerebralis of the flesh-fly, Sarcophaga ruficornis, showing about 26
secretory 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, as
all 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. 2
G. S. DOGRA and B. K. TANDAN
Dogra and Tandan—Techniques for neuro-endocrine system
461
1. Same as for the AF technique.
2. Oxidize organs in oxidant, 2 to 3 min (0-5 ml concentrated H 2 SO 4 added
to 100 ml of 0-5% KMnO 4 ).
3. Same as for the AF technique, but bleach with 2% potassium metabisulphite, 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 is
equally satisfactory.
Notes and cautions. The components of the neurocrine system are revealed
at stages 7 and 8 or at stage 9, as in the VB and AF techniques. The appearances
in mounted preparations are as follows: somata, blue or dark blue; proximal
portion of axons, light blue or blue; the neurosecretory pathway, occasionally
light blue; storage-and-release organ, dark blue; blackground, faint blue.
Bouin's fluid is substituted for Zenker's, because the mercuric chloride in
the latter interferes with dissection.
The sheath investing the brain is removed in accordance with the instructions given under Notes and cautions referring to the AF technique.
To display effectively the secretory neurones and the neurosecretory pathway 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 favoured
because its removal late in the procedure, at stage 8 or 9, protects the preparation from contamination with foreign particles. At stage 9 the brain is dissected
with fine-pointed needles to expose or isolate the area containing the stained
components. Dissection is performed under double illumination, the source
of reflected light being a 6- to 8-volt spot-light and that of transmitted light
a 100-watt milk-white bulb. The storage-and-release organ does not require
elaborate dissection.
The bulk-stained preparations can be stored in cedarwood oil for about a
week 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, Hemidactylus
flaviviridis, mounted with the inner ventricle surface touching the coverslip. Optic chiasma
and optic nerve removed. VB technique.
B, dissected hypothalamus of the lizard, Hemidactylusflaviviridis,mounted with the dorsal
surface 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 of
secretory neurones. VB technique.
D, undissected hypothalamus (right half) of the lizard, Hemidactylusflaviviridis,mounted
with the outer surface touching the coverslip. VB technique.
E, left-hand side portion of the preparation shown in B, at higher magnification, showing the
relay of secretory neurones. VB technique.
me, median eminence; npa, nucleus paraventricularis; nsu, nucleus supraopticus; on, optic
nerve.
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Dogra and Tandan—Techniques for neuro-endocrine system
Results
Observations 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, Bradinopyga
geminata; the pars intercerebralis/corpus cardiacum components of this
system, being stained, are quite distinct.
Fig. 1, N demonstrates the distribution of the secretory neurones, in the
supraoesophageal 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 paraventricularis and supraopticus nuclei (and the distal half of the tractus hypophyseus,
together with the median eminence) in their natural positions. This figure alone
demonstrates the inter-relationships of the important components, except the
infundibular stem and process, of the neurocrine system. The terms used for
the components are those given by Sloper (1958&).
In fig. 2, D, showing the right half of the undissected hypothalamus, the
secretory neurones of the nucleus paraventricularis are much clearer. Fig. 3, F
is an enlargement of the area enclosed in the rectangle in fig. 2, D, to show the
appearance 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 in
their natural positions, slightly anterior to and overlying the optic chiasma.
From the main cluster of the nucleus supraopticus, a relay of neurones
extends towards the median eminence, the latter represented in this figure
by 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 unavoidable
for proper display and mounting, the topographical distribution of the
secretory neurones is revealed convincingly.
Fig. 1,1, J shows the difference in the amount of the neurosecretory material
in 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 its
accumulation in the perikaryon are well exhibited.
Fig. 1, M shows the dissected brain of the common bed bug, Cimex lectularius. This preparation demonstrates that the handicap of its size is overcome
by staining the brain as a whole. Satisfactory preparations showing the secretory neurones in the brain of other even smaller insects (mosquitoes, mosquito
larvae, &c.) have been made without much difficulty.
Fig. 1, H, L shows that the secretory neurones can be counted directly, if
their number is not too large.
Fig. 3, E shows neurones from the cluster of the nucleus supraopticus of
H. fiaviviridis. It demonstrates that they can be isolated and teased for observation. 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. 3
G. S. DOGRA and B. K. TANDAN
Dogra and Tandan—Techniques for neuro-endocrine system
463
Isolation of neurones presents a considerable advantage, as it has made
their direct counting possible. Information on the number of secretory neurones in the brain might promote an understanding of the physiological
phenomena regulated by their secretions, one such interesting phenomenon
being the adaptation of related animals to widely-varying biotopes (see
Hanstrom, 1956).
The results given below on the neurosecretory pathway that transports
this material, and on the storage-and-release organs, are equally informative.
Fig. 1, B, E, F demonstrates unequivocally the crossing over of the two
nervi corporis cardiaci I. The figure depicts the secretory neurones (out of
focus) and the two NCC I in a brain, in position of dissection. The curvature
in fig. 1, F (slightly out of focus) represents the change in the course of the
two 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 it
is 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 of
the infundibular stem with the median eminence.
Fig. 1, D shows the corpora cardiaca of the dragonfly, B. geminata, and
fig. 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, along
with the infundibular stem. Fig. 1, A is the pituitary of the white laboratory
rat in the natural position; in this figure the distinction between pars intermedia and pars distalis parts of the adenohypophysis is not reproduced, but
it is clearly evident in the original preparation. Fig. 1, c is the infundibular
process, detached for mounting separately from the preparation of the brain
shown in fig. 2, B.
As the natural relationships are conveyed better in the whole or intact state
of these organs, such preparations have been utilized for the microphotographic demonstration of the results.
FIG. 3 (plate), A, 12 y. sagittal section in wax of a bulk-stained brain of the lizard, Hemidactylusflaviviridis.VB technique.
B, 10 ix sagittal section of a bulk-stained brain of the lizard, Hemidactylusflaviviridis.VB
technique, 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, Dysdercus
koenigii, showing 3 + 4 secretory neurones. AF technique.
D, 12 fx sagittal section in wax from the same series from which A was taken, showing the
secretory neurones of the nucleus supraopticus. VB technique.
E, some isolated and teased secretory neurones of the nucleus supraopticus of the lizard,
Hemidactylusflaviviridis.VB technique.
F, enlarged view of the secretory neurones of the nucleus paraventricularis, enclosed in the
rectangle in fig. 2, D.
G, magnified view of the secretory neurones of the nucleus supraopticus shown in E.
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Dogra and Tandan—Techniques for neuro-endocrine system
Examination in sections
Fig. 3, c is a transverse section, in wax, of the brain of a bug, Dysdercus
koenigii, to demonstrate the stained secretory neurones in the pars intercerebralis of the protocerebrum.
Fig. 3, A, D shows small parts of different sagittal sections of an uninterrupted series, still in wax, of the whole brain of H. flaviviridis. The section
shown in fig. 3, D passes through the nucleus supraopticus, and the stained
secretory neurones are unmistakable in it; that in fig. 3, A passes almost
through the middle of the neurohypophysis.
Fig. 3, B is a section similar to that in fig. 3, A, but counterstained with
safranin and mounted in Canada balsam. It differs in no way from a finished
section obtained by routine histological procedure.
The anatomical distribution of the neurosecretory material in the tractus
hypophyseus and the infundibular process agrees remarkably in fig. 3, A and B.
The adenohypophysis became detached from both these brains during processing.
Thus, on sectioning paraffin-embedded preparations and simply mounting
the sections in Canada balsam, the sites known to contain the neurosecretory
material are revealed. Although counterstaining for contrast is desirable, it
is nevertheless optional.
Remarks
The 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 bulkstained preparations. All the information presented here, other than that
obtained from the sections of the bulk-stained preparations, was derived from
such mounts. This information, it would be conceded, is in a form that is
clearer than that obtained by the usual histological procedure. These qualities
result from the intact state of the components in question.
As this mode of investigating the neurocrine system eliminates the necessity
for sectioning, one of the two difficulties stated at the beginning—the lengthy
nature of the histological procedure—was overcome, and the other—detachment and flotation of sections—was naturally not encountered.
To adapt the bulk-stained preparations for subsequent sectioning was a
logical outcome. Examination in cedarwood oil makes it possible to select well
and darkly-stained preparations, which, being heavily loaded with the neurosecretory material, are better suited for study in sections. No method of making
such an assessment has previously been available. It has only been possible to
do it at the end-stage, when sections have been cut, stained, and mounted
Dogra and Tandan—Techniques for neuro-endocrine system
465
indiscriminately, at the expense of much time and labour. Screening of the
preparations before sectioning saves time and fruitless labour.
Thus, bulk-staining makes possible the study of the components of the
neurocrine system in whole mounts and also in sections of the same or similar
preparations. Information can therefore be obtained in two unrelated ways
and, what is more, can be cross-checked. A comparison with the information
derived from routine histological procedure shows no obvious difference.
Elimination of oxidation of the sections, a step responsible for their detachment and subsequent loss by flotation, has made possible the mounting of
serial sections of whole brain of both invertebrates and vertebrates, without
loss. In these uninterrupted series not only are all sections present, but, in
the sections that contain them, the components of the neurocrine system are
in an equally satisfactorily stained state.
Insects being of primary interest to us, it will not be out of place to
report two features of the results with members of this group. These salient
features are the outcome of the intact state and clarity of the pars intercerebralis/corpus cardiacum components, as a consequence of which a comparative
study 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, those
with the VB technique differ (and the magnitude of this difference varies
between the species). This lack of intraspecific agreement seems so far to be
due 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 not
strictly parallel. So far, inherent morphological differences in the components
of 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 the
insects concerned.
It may be stated, to complete the record, that statement 1 above is broadly
applicable to members of other classes of arthropods besides insects, and to
members of other invertebrate phyla.
Discussion
The 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 in
this way. Although unconventional, this method has provided most valuable
information on the cyto-architecture of the nervous tissue.
The original features of this report and the conclusions derived from them
are:
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Dogra and Tandan—Techniques for neuro-endocrine system
1. The in situ staining of the components of the neurocrine system, and
the utilization of bulk-stained preparations for making observations. This
method, tested with a variety of animals, has led us to conclude that experimentally produced changes in the anatomical distribution of the
neuro-secretory material can be demonstrated rapidly—and possibly more
convincingly—in such preparations than by the ordinary histological procedure involving the staining of sections. In vertebrates, one-half of the
longitudinally divided brain can conveniently be oxidized and the results
demonstrated 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 on
sections, can be used with success on whole or gross pieces of nervous tissue.
This leads us to conclude that the chemistry of secretory neurones (perhaps
of neurones in general), which has hitherto been studied in sections, might
be investigated by applying appropriate techniques on whole or gross pieces
of nervous tissue.
Finally, this simple and direct method of demonstrating the components of
the neurocrine system has provided students of zoology with a means of
studying an important organ system that has not previously been so accessible
to 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 method
introduced by Dr. Sloper; and to Mr. Humberstone, of Dr. Sloper's Department, for generously permitting us to give details of his previously unpublished
technique. We are indebted also to Dr. John R. Baker, F.R.S., for helpful
comments on the typescript; to Dr. Nitya Anand of the Central Drug
Research Institute, Lucknow, for very valuable help, and to Professor M. B.
Lai for his unceasing interest in our work. Thanks are also extended to the
Dyestuffs Division of the Imperial Chemical Industries, Blackley, Manchester,
for the gift of Victoria blue RN 275.
References
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