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8-1-1957

A study of the differentiation of neurosecretory cellsin Rana PipiensWilliam H. WoodsAtlanta University

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Recommended CitationWoods, William H., "A study of the differentiation of neurosecretory cells in Rana Pipiens" (1957). ETD Collection for AUC Robert W.Woodruff Library. Paper 672.

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1. ~flva’se Section tIwtm~i the Diamqtalrni at a___p$j;i Lnva Shimtrzg k Ilect C~3a....4 * a *n-n.e am. nete.• 22

2. Higier ~wd.fieatfm at Fig~we One......454.44,4aeCsee~e .a.e*,s 22

3. Sagtttal Section through the zaacØzalnn at an Animal with~tcnial ftiDs Showing a Mect Get aioqg a Rypcytiyseal Elect!

a... s.e.ecaa,en~sa4c,.q~ 22

Ig. Nmwceecretcry LLaba’ial in the Cerebral Reci~tere. of aYoung kbilt~ 22

S. A ?rasvne Section ~Uutugh the Real of a N~,y Ratchet! laneStowing One Mast Get bebaas t~ lateral Edge of the BrainaM the Right ~w ad Another in the Downi Area at the

236, Maw et~ Cells in the Vaztro4at.eral Preqtic Area of ma

Animal. ‘with Zzt1ec’z~t OiI4~,.. a*a*aseaa ø*OS*ea*-Øs a *e.a..oa..pe*fl~ 23

7, Rigwr Magniflcabjcaa of Figure~ Szawing Tao Mtiveij Secreting~ 23

8. Sagittal Section taarough the Diaieqtalon at a’ Animal wit~rnvt,~ Stowing Keawosecrctuzy Material nec the

- Saltenular Miriam ad Uae 1~ta’~’i~, ~aaure.-, . ,.•.~. .. . .. ... 2k

% Right Magnification of Figure~ *ee#*tflwoan eec...,, 2k

ZOo Trerawe Section through the !flm~qflhalen of at Animal with~tternal Oi3is Showing a Ri*bnred Itit. Cell....,..444. macacasn.e 21i

lie ?ranavase Sectisi through. the Diascqtalcn. at at Ardant ‘withStat Idth.awls Sual.ng tctezt Seerotcwy Calls in the VentralFloor of the Ryp~helnE,..4,.,.,,.,.,...,.4_ ns*am*s*s.s*.esea.~ 2k

12* A tflatad” Ner~ Fiber Rztatag Vatro4ata’afly fran thePreqtic IAmbus of a Young AdiAlt Axzlaaal.. m•eiasn a a4e~ S.....~ 25

33. Sigher Magnification of Figure~ S

1k. tratavase Section thröEji the Bypothalms at an Adult AnimalStewing Two Mtj.tely Sec bS CeOis In the à’eoptic Maclam.. ,ae 25

IS. Idjier Magnification of P~.Sik, Stewing One of tim Two -.

Secreting Can5.n..a.~n.......e..,..,._,._,,,*,._......,.,,,..,.,. 25

iv

OHAFTEE I

INTRODUcTION

It is commonly kio~n that the nervous syst~n is con~o~ed of a grot~ of

highiy specialized cells called neurons or nerve cells. Neurons are specially

designed to receive nerve impulses and eonv~y- them to other cells. In addi—

tion to these ftnction~ some nerve cells eha~r functional ~charaitsristics of

gland celia • This functional activity- has been accepted by mar~r investiga

tors as neurosecretion. Neurosecretjon was first clearly described by Speidel

(‘22) in his observation of secretory celis in the spinal cord of the skate.

Its foundation has been based a.t exclusively on cytological observations.

The products eal rated by neurosecretory cells have been variously referred

to as neurosecretory products, neuroaecretory material or neurosecretory ccl—

bid (Smith ‘!l) • In this investigation the products w~ be designated for

the most part as netiroseeretory material.

The purpose of this sti4y- was to describe the difference in appearance

of nerosecret~ory material in both larval arid adtt ia pipiens. An attea~t~

was also made to determine the stage at which this activity first appeared in

larval animals.

I

CHAPTER II

REVIEW ~ LITERATURE

Neirosecrstjon as a process has oni~r recentiy been observed and therefore

still remains in an almost entirei~j des ptive state. Nerve cells character~.

ized by glandt~lar activity have been described in most animals of both the

vertebrate and invertebrate grc~s~; however, investigations carried out on

vertebrates have been much more extensive and nunerous.

According to Scharrer and Soharrer (S is), the aunelid worms of the inver

tebrate groups have been observed to possess neuroecretory cells. They

pointed out that the poiychaete worm, Nereis, was found to possess two sym

metrically situated groups of secreting nerve celis located in the dorsoca’udal

area of the cerebral ganglion. Scbmicl (‘347) demonstrated neurosecretory ac

tivity in the earthworm, Lumbricus terrestris. The results of his study showed

that most of the ganglia of this worm contained secreting cells. Masses of

the cells were seen concentrated along the dorsal margin of the ganglia, as

had been pointed out by Scharrer and Scharrer (134~).

.Arthropods have been studied in relation to the process of neurosecre

tion. Scharrer (‘14la~ ~1i2b) described neuroglandular cells in representatives

of the orders Orthoptera and liphosura, In urn ins, a mether of the order

Xiphosura, she pointed out that neurosecretory cells were present in neariy

all parts of the central nervous system, greater concentrations of these cells

were found in the abcjomjrjaj. ganglia and circumesophageal z~i.ng. I:n the order

Orthoptera certain species of cockroaches (Leucophaea maderae~, Blaberus

cre~iztffer~, and Periplanet~a americana) seem to show a rather typical location

of neuroglandular activity for arthropods. Neurosecretory cells in these ani-~

mals are localized in the pars interoerebrsjis and the subesophageal genglia.

2

3The irert~ebrates have been studied extensively for the presence of neuro

secret~ activity and the presence of this activity has been confirmed in

animals from all vertebrate groups (Bargniauzi and Stharrer ‘~1) • According to

Speidel (‘22), D~iigren was the first investigator to observe neiirosecretory

cells in the spinal cord of the skate a. He observed that these cells

were veiy conspic~n~ in that they were large, I gcar~ nultimicleated ~id

glandular in nature, Speidel (‘22) studied the nerve cells of ela~mnobranchs,

teleost, and ganoida aM. confirmed the findings of Dahigren ~o had described

the cells as being secretoz~ in nature. in these ce11~ Speldel pointed out

that at first the granular ~rtoplasm was homogeneous and chromatin was die-.

tributed throughout the nucleus in the form of large granules. He character..

ized the cytoplasm of an active cell by the presence of vacuoles containing

granules i~hich varied in size. Closer observation revealed that the granular

material had been discharged from some vacuolee. Frequetrtly, the granules

were seen as deeply stained bodies idiich were distributed throughout the grey

matter of the cord.

Up until 1928 very little attention was given to the secretory activity

of the nerve cells described by Speidel.. At this tinc, according to •S~ax~

(‘i~O), a series of studies were made by then on such fIshes as Thutoga onitis~

FunduUia heteroclltus and many others. Esserrbialiy the same information was

obtained from these studies as was found by- Speidel. Studies on fishes showed

that the preoptic nucleus of the hypothaiirnrn constituted the major area of

secretory activity, Observations by Scharrer (‘1~l) on the nucleus preopticus

in Fundulus heteroclLtne have cleariy shown cells of neurosecretion. A simi.

lar study made by Palsy (tL~3) on the eatfishes (Noturus I Lay~, Ameiurus

nebulosus and I~meiur-as moles) revealed the presence of secreting nexve cells.

In this study it was noted that the nucleus lateralis tuberis was the center

k

of secretory activity. Histological studies of sections through this region

were characterized by cells with po~morphic nuclei. These cells showed

changes that were correlated ‘with a nuclear secretion.

The conspicuons neuroglandular ee).le found in the pre~tin nucleus of

fishes were also obeerv~ed in this area in ribjans. Schaz~er and Scharrer

(t1~O) ~aninecI the nucleus preopticus of the toad (Bufo) and found that this

area CofltLained large muI.tinueleated cells with masses of colloidal material

lying between them. According to Rarguiazm end Scharrer (‘51), Hild observed

that the colloidal masses secreted by the cells of the preoptic nuèleus in

the frog moved down the hypophyseal tract toward the pituitary gland. He

concluded that this material was pr~oduced in the hypothalmi nucleus and

transported by the perdpheral movenent of axoplasm to the posterior lobe of

the pftuitax~.

Bar~uann and Scharrer (‘51) reported that neurosecrotory material in

reptiles, birds and m~nals was generafly found in the supraoptic and para-.

ventricular nuclei,. along the s~reoptico p e~ tract and in the pars

nervosa The findings by- Do Greet ( t57) on the brain of the rat imre contrary

to those of Bar~nann and Stharrer (‘51). He observed the appearance of neuro-.

secretory material in the hippoca~nis, the hthemilar nucleus,, blood yeas els

and in the hypothaim 4 opheal systen. Scharrer (‘52) has described neu—

rosecretory material in the supraoptic and paraventricu~ar nuclei of the

snake (Thas~nopi~ ~.). In these nuclei there were seen mai~r colloidal gran...

ules and droplets which varied in size. In addition to these nuclei he also

foirn neurosecretory material distributed in the dorsal region of the dien~

cephalon which was directed toward the comuia aura pallii postericr and the

paraphysia. He frequently observed that the material collected in the com~

xnissura paflii poa~toi~,

The supraoptic and paraven tyular nuclei of man have been ~d~eusivel~y

studied by Scharrer and Scharrer (t)~O), The two nuclei in man ~which they

described as being homologous with similar regions in lower vertebrates

showed characteristics of secretor~y activity. These observers ooncluaed that

the supreoptic and paraventricrnlar nuclei in man, like the preoptic nucleus

in lower vertobrates, possess a glandular function.

Fruitful attenpts have been made to correlate neu secretory cells with

stages of various secretory cycles. Scharrer and Scharrer (‘U~) related the

appearance of neurosecretory granules with three cellular components: (1) the

Nissl substance, (2) the baa~~i)jc portion of the cytoplasm and (3) the

nucleus~ Scharrer, Palsy and Nilges (‘lt5) denonstrated a relationship. between

neuresecretory granules and the Nisel substance of neurosecretory ceTh.

These investigators e~qlained that this relationship was observed in neuro

secretory cells found in fishes, amphibians, reptiles and mammals. The cycle

was first characterized by the presence of peri~,heraIly located Nisal bodies

~which soon b~eeame intermingled with fine colloidal gianules. The secretory

granules gradually increased in size1. whereas the, Nissi bodies se€mGd to have

diminished. As the granules increased in size they often filled the entire

cytoplasm, and in some ee:L13 were seen to ~rtend into the axon hillock. The

granules were later erpelled into the intercellular spaces~. This represented

the end of this particular cycle.

Scharrer (‘hi) worked on the fish Fundulus heteroejitus and Scharrer

(‘lila) worked on cockroaches and showed that the neurosecretory material in

these animals tended to ere~,iify a close relationship with the appearance

of a vacuolated cytoplasm. In these cells large granules were seen contained

in distinct vacuoles along with a few colloidal droplets. According to then,

the droplets resulted from a fusion or coalescence of the numerous small

6

granules. The end of this cycle was represented by cells with a homogeneous

cytoplasm which contained only a few ~ty vacuoles, The discharged col:Loidal

droplets were generally thserved outside of the cells or distrthuted along the

2d5 cylinders..

It has been reported that some animals have produced nettrosecretory gram..

ii3.es in the call nucleus. Palsy (?)~3) ~j~ ~ji~n~ ~ralis of cat—

fishes (Noturus flavus, fl~eiurus nebulosus and Ameittrus melas) and found that

the secretory process in these animals began with the appearance of scattered

naurosecretory granules within the nuclei, These granules were noted to have

increased in size until the nuclei of the cells appeared crowded. This cytle

teiwinated with the granules being discharged directly from the nuclei of the

cells,

The physiological role of neurosecreti.on has been studied by many ixwes~

tigators • According to Scharrer and Scharrer (‘iSO), two methods o± attacking

this ~roblen have been ~Ioyed. The first was an att.enØ to correlate nan

rosecretory activity with the life cycle of some animals. The second was

that of an endocrinologleal approach. Scharrer and Scharree (‘~) demon..

atrated the first method by euccessftliy using it in the life cycle of the

honeybee. They found that the brain of this animal contained the greatest

quantity of colloidal droplets during the middle of its life cycles whereas

the young and aged bees showed little or no neurosecretion.

Kopec (‘22) used the endoarinologicai. approach in his study of larvae of

~yinantria dispar L., and demonstrated that the brain of this insect secreted

a homnonea-like substance necessary for metsmor~iosis. In doing this the

brains or supraesophageal ganglia of the larvae were removed during their

last molt, Later observations showed that only a small number of the larvae

pupated. The majority of the animals remained in their larval stages until

7

death, According to Itopec this indicated that there was a substance (or aub-.

stances) in the brain of this animal that influenced iaet~norphosis; he sag~~

gested that it should be considered a gland of internal secretion,

Another type of neurosecretory function has been found in the central nez’~

vous system of Liinulus (Brown and Cunningham, ‘131). Extracts from the central

nervous system of this arthropod,. when in3eoted into lice~ were shown to

influence pigment concentration in the chromataphores of this animal. By

separating e,ctracts from various portions of the central nervous system of

limulus, the chroma’bophorjc principle was found to be highiy concentrated in

the posterior portion of the cireirnzesaphageal nerve ring. About the same time

these observations were made, evidence was presented by Stharrer (‘1~) which

indicated that the location of neuroseerctc~ry cells in Mmulus corresponded

aimost identicaliy with the distribution of chroniat*oric material found by

Brown and Cunningham. Consequently, it was conclnded that the source of

chromatophorebropjc material was the neuroeecret1c~ry cells found in the central

nervous system of this animal.

According to Scharrer and Scharrer (tIiO), nourosecretoz7 activity can be

correlated with the life cycle of only one vertebrate. An earlier stuck by

one of them showed that a fish, ~finea vulgaris, ~thibited a high degree of

neurosecretory activity during winter months. Later studies on other fishes

revealed no such activity (Palsy, t1~3).

Successful atterz~ts have been made to correlate the functional role of

neurosecretjan with endocrine activity in vertebrates. Axons from cells of

the preoptic, laberelis tuberis, st~raoptic and paraventricular nuclei in

vertebrates extend through the infundibulum and innervate the pars nervosa

or posterior pituitary gland (Scharrer and Scharrer, ‘k)~). Neuroaecretary

granules and droplets have been traced along the tracts formed by these axons

•auow1.wqLtW~1.3t4Td~a~teq~sodet~JOu’~2~~zGet~eq~q2~i

TetrntuQ~odh~et~q.~sq~pse~ze~aq~,~o.zjpepn~~uoo~~•uie~s.

8W~.W~~9t~0UOT~flU~LoD~U~pa9U.~DbXQ~2A30a)~e’4zrçQ~ZCZe

‘ptwt~1zeqq~oet~q.uQ•~urgtieeeat~jo~a~stues~t~od~eti~tq

tr~e~iLzoe-8o~zuauJo~io’~etc1ap~eiet~q.UG.tp.~t~ep~pe’4~ou

~I‘UO~S~X~~ULt~4~.tIeq~&JOSuaae8teae~eq~S~J

-q~oeçqus£q~~o~ego.meu~o~n~zrt£etIJ,•s~e~rnw~u~o~.pe~

UO’ç~e.t3eSGxfleu~(t~a)~pu~~u2~z~~qpe~sa~u~t~eq~

•(~‘~e~u8~~‘z~t~pu~uevz~fttt~e~uet~sPUE

~JO~°t~zoeq~sodet~UTq~~oaoq.ptmo;e.ze~i.pt~

9

CHAPTER III

MATERIALS ANT) ~TH0DS

The animojs used in this study were larval and adt~lt stages of Rena

pie~e. The first gro~of an. used was obtained from the L~.mburger

~a~any, Qshkosh, Wisconsin~ The group contained tadpoles measuring from

appriae3y 20 to 50 nan. in leng~. Since much younger and consequently

sma:tler nimals were also needed i.n this investigation, a second group of

animals was obtained These animals were coflected from .smafl ponds and

marshy areas near Little Rock, Arkansas. This latter group contained a great

diversity of stages, including freshly fertilized egg masses and adult frogs.

All animals used were divided into eight : ., according to their de...

velopmental stages, The groups and stages were as foflowet

Grç~ g~ ~~uatelan~bh

I Hatching 6 - 8 mm.U FSrternal ill 9 —12mm,

In Hind 1iü1 bud ~3 -l9nrnt.I! Hirllith 20 —29mm.V Foreaodhd~. 30 —30mm.

VI Four legged Yaris~le (disappearing tail)VU 1mm adult Vari~le

VIII Adult 2 — 3 inches

The animals which were not used diat1ely were kept under normal labo

rstory coniitions~ The tadpolss were kept in a lar~e aquariun ithich cone.

tamed at one end firmly packed dirt ~th a few~ green plants grouing in it

and at the other end pond waters During the larval period, the animals were

fed a diet of boiled lettuce or spinach. After metamorphosis they were fed

ants and small cockroaches. A si~ly of pond water (obtained from ponds where

animals were collected) was kept on band, The water in the aqun urn was

changed frequently to r~iiove waste and decaying food material as well as to

9

10

afford a fresh supply of oxygen. This e~rviroriment was appropriate for the

ai.thsequerrb metemorphosing of these an:buals.

The egg masses eofleet~ed were placed in ~0, 000 cc. containers nearly

filled with pond waters The water was changed once a day- during the hateh~

ing and early larval stages0 After this period it was changed twice each

week. Animals used from this group were selected as they reached the stages

desired.

The hypothalmic nuclei of the ciience1~i~løn were examined histologicaijy

in all eight stages. These examinations were facilitated by the procedures

indicated below. The entire brain was conveniently removed from the adult

specimens, whereas the infeasibility of brain removal in larval stages ran..

dered it necessary to use the whole animal.

In removing the adult brain, a large pair of scissors was insert~ed in

the corner of the axdmalis rno~dbh, and the cranium was swiftly cut off with a

transverse incision a shorb distance behind the eye. Lateral incisions were

them made rostrad -with a pair of esceedingly sharp pointed scissors. Care

was taken net to damage the brain. The olfactory- and optic nerves were then

cut making it possible to eaeilçy lift cub the brain with a pair of forceps.

The brain was then fixed in Bbtth~1s fluid1 en~edded in paraffin and sestioned

at !~“microns.

The tadpoles were fixed, en*,~edded and aectioned~ -using the same proca..

dures ~ were e~loyed for the preparation of the adult brain. There was

one e~ccep~ion, tadpoles measuring more than 12 nmi.~ (Groups III, IV, V and

VI) were placed in decalcifying fluid from 1 o~ Li. daye, depending on the do...

gree to which the skeleton had hardened.

AU sections were stained in chrome..aium haemataxylin.-phioxine as cia-.

scribed by Goinori (t141).

‘a1. Sections were hydz’abed and refixed for 211 hours in Benin~s fluid.

2. The picric acid was then removed from sections by washing them for

about 0 minuses in ntnning tap water.

3. They were tr&~ted for one rninu!~e in a solution containing equal paits

0,3% potassium pat~anganaba and 0.3% sulfuric acid.

11~ They were decolorized in a 5~ sodium bisulfite so1~tion and ire hed

for 10 nthmtes in running tap water,

5. Sections were stained in chr ne-alum h atxlin-~hio~cjne (at least

two da~ old and not more tha 5) 30 to 115 minutes,

6. They were treated one minute in 1% hyth’ochloric acid and washed in~er

ronning tap water fron 30 to 115 ninutes.

7, Sections were conuterstained for 5 ninutee in a fresh 0.5% phloxi.ne

B sobition and rinsed in distilled water.

8. They were treated one mimzbe in 5% ~ospbctungstic acid, then washed

15 minutes in running tap water and differentiated in 95% alcohol.

9. They were then dehydrated in two changes of 100% elcóhol,~ cleared in

~rj~l and mounted in balsan.

CHAPTER IV

~PERIMEi~L RESULTS

General observations.~Neurosecrebory material (granules, droplats and

masses) stained deep blue to biue~mblacko Some of the mteria2.cccasiona3i~r

presented a greenish hue. A few other str~actures also stained the same way

in which neitrosecretory material did. These included cells and bz’anes

which were frequently located in parts of the brain other than the dienc~,h—

alon. Some of the cells referred to here appeared like mast cells and were

very prevalent in larval animal (figs • 1 and 2). These cells sometimes

stained brawn~h black to black. ~tch of the n isecretory material was

seen in and near the blsod vessels in different parts of the brain, parbicu—

lar)~r the h~rpopbyseal blood vessels (fig. 3). in rn~ instances a few blue-

black masses were observed in regions of the cerebral hemispheres (fig. 14).

Large droplets and masses were often observed in the dorsal diencephalic

area, in the region of the habenuler nucleus and posterior connnissure (figs.

Band9).

Gr~p~ (Hatching stage)...-.A thorough er~nination of the entire brain of

these animals showed no defindte neurc secretory material. host cells in the

hypoth~nic nuclei of the diencephalon appeared normal. A few large mast—

like cells were observed which appeared to be entirely covered with dark gram.

tiles. These cells were always observed outside of the hypothaindc area, gem.

eraily- in the dorsal part of the diencephalon and between the eye and the

lateral edges of the brain surface (fig. ~).

Groi~ II (External gill stage) .-~~Sections through the diencephalon dif-~.

fared widely from those observed in anim~s of the preceding group. Large

cells filled with deeply stained granules were found in the preoptic nucleus

~13

and in the ventral floor of the bypcthalmus. Irregular shaped blue-black

masses were also found in the preoptic nucleus. Some cells in the ventr~—

lateral preoptic area could be seen extruding deeply stained masses from

their nuclei (figs. 6 and 7). A section fr~n one animal showed a mass of

neizrosecretory material near the ventral floor of the tkird ventricle and

another in the pituitary. A few masses were also found in either close

proximity to pituitary blood vessels, or within them, Blue-black masses

were occasionally seen in the dorsal dienc~,haJ.ic area near the posterior

conmiissure (figs. 8 and 9), A few mast-like calls were usually present in

different areas in the d•ence~ia1on, occasion Liy some of them appeared

ruptlu*ed (fig • 10).

Group III (Hind limb bud) .—The preoptic nuclei of the hypothalzmis showed

very little similarity to the seine area observed in the previous group, in.

that the nuclei contained no deeply stained, granular celia or blue-black

neurosecretory masses.. A few cells in the ventral floor of the hypothalmus

appeared to be in a latent secretory stage (fig. U). A smali group of nerve

fibers was obse extend~ig venbrc:4ateraliy from cells in the preoptic nu

cleus. These fibers seened granulated, presenting a ~beaded” appearanx~o.

Rarely were there observed de€ply stained masses in any part of the hypothalmo...

hypophyseal system.

Group IV (Hind limb) .—Glose observation of sections throu~i the diencoph—

elan of animals comprising this group revealed no appreciable change that

could be correlated with an increase or decrease in neurosecretoxy activity.

A few celia in the ventral preoptic area of the hypotha:bnna, similar to those

observed in Group In, showed a latent or very little seóretory activity. A

few blue-black stained masses which appeared greenish in. hue were found in

the ventro—lateral region of the hypotbalmus.

1k

Grai42 V tore and hind mb) .—Nellrosecretox3r cells in the hypabhalmus

were at a miMmrnn as observed in Graaps III and IV. in most cases very little

or no secretory activity could be detected~ The dorsal part. of the diencaph...

alan showed masses of neurosecretory material which were blue-black and green.-.

ish black in color. These masses corresponded closebr to the masses found in

aninals observed previously which showed a high rate of secretory activity.

Group VI (Four legged) ..-A thorough erseinatian was made of sections

through the diencephalon. The major change observed in these aninals was

charaot,erized by the ~pearance of a few granules, droplets and masses in the

preoptic nucleus. Gd~mular~1ike masses which stained a deep blue were often

seen~ particularly near the third ventricle. A number of the droplets end

masses were seen in regions of the cerebral henispherea. Granules of varying

sizes were frequently noticed along the fibers which projeeted toward the veii—

tral floor of the hypothaimas. Mast-l~ce cells were relatively numerous in

the ventro.4aberal hypotha)ic region. These cello were extrenely granular

and often appeared to be extruding their granules fromi a ruptured surface.

Group VII (Very yoimg athilts)....m..The hypothaimic nuclei showed an abr~t

increase in neurosecretory activity. Numerous blue-black granules which

varied in size were seen within and lateral to the c,~1ls of the preoptic nu

clei. These blue to blue-black masses appeared to be both irregular and

spherical in shape. Upon closer observation these masses appeared to b e ag~

gregations of spiral sb~ed granules. The preoptic area proper revealed mar~

blue to blue-black masses which were dint~ ad throughout it. Rostral3y,

they extended as far as the nuclei of the hippocaspus and caudally, a good

distance b~iind the optic chiasma where they were directed toward the in...

fnndibulrnn. Many fine granules could be clearly seen in s~e of the masses.

The lateral bypothaloic area disclosed blue to blue-.black drriplets and

granules along “beaded” nerve fibers (figs. 12 and 33). Thee fibers passed

ventrally toward the inf nd~u1mn where ~n occasional blue...black mass was

ncticed. A few drcplets and granules appeared to ~tend ashozt distance in-.

to the axons of some cells.

Group Viii (4dult ~rogsJ.—Neurosecrctory aetivfty in most respects was

similar to tha~, observed in young adult frcge. The primary difference was

in the fact. that the secretory products (granuLes, droplets and masses) ware

almost entirely restricted to the hypothalmus • The cells in the preoptic

nucleus were seen actively secreting blue~-biack granules (figs. 1k and 33).

Frequently h1ue-~black masses were found near or within vascular areas of the

dienee~ialon. The granules and dror lets were generally located within the

cytoplasm of cells in the pr ic nuclei. Some droplets and masses were

seen ventral to the third ventricle and occasional1~y- could be traced into the

pituitary stalk. A few fibers which passed Ye riaberafly from cells in

the preoptic nuclei were granulated and showed the s~ene “beaded” effect which

was observed in some of the earlier stages of development. Keener observation

of these granules showed a close resanblance to most of the other neurosecre—

tory products,. which were directed toward the pituitary,

CHAPTER V

DISCUSSION

In this study as in similar studies by other inv~estigators (Bargmann

and Scharrer ‘51, Sloper ‘55 and Do Groot ‘57), h~ipotha1mic nenrosecretory

material when stained with chrome~.a1uza haetnatozylin-.phloxjne, colored a deep

blue to blseeblack. The material has also been occasionally observed by

Sloper (‘55) and Do Groot (‘57) to stain a greenish blue color. This cor

responds closely with the gre~iish hue of some ma rosecretory material foimd

in this investigation. Observations of neuroaecrotoz7 material outside of

the h~ipotbalso-4iypophys.eal system parallel previous findings by Scharrer

(‘52-) who traced macsecretory material along -nerve fibers in the dorsal

region of the diencephalom toward the eommissura p~i1i1 posterior and the

paraphysia. He noted that the material aectinulated in the comrniast~ra p~ThT1

posterior, Observations resenthljng these were made in the dorsal region of

the diencephalon and posterior coimniss-are of some animals used in this in...

vestigs~ion. De Groot’s (‘57) observations of neurosecretory material in

the hippocamptis, the habenular ntzcle~rn and hypophyseal blood vessels in the

rat correspond identically with findings reporbed here in ~na pipiena. His

description of mast cells in these areas. which frequently e~thibited a rap.

tured appearance also correlated with the mast-’iIke cells reported in this

study. Similar findings were made by Sloper (‘55) in the dog and cat, lie

stated that some granular celia took on a brown coloration when stained with

chromed..alum haematoxylin...phloxLne. This was attribnted to a variation in

staining intensity. The brownish black to black mast—like celia observed in

this investigation may be explained on this baeis,

16

17

In comparing the cells observed in the hy othalsnrn of Group! (hatching

stage) with those in Group IX (external gin stage) it was apparent that the

first significant manifestation of neiu’osecretory a tivity occurred in the

latter group. Groups III (hind limb bud stage),. iv (hind limb stage) and V

(fore and hind limb stage) showed a sharp decrease and almost disappearance

of neurosecretory material when co~ared with those in Group II.

The decreased ameunb of neurosecratory material observed in these ani

mals rnay be accounted for in one of two ways: (I) the material could have

been improperly fixed; (2) the quantity of neurosecretory material found in

different stages may have varied according to~tIE functional requirements of

the animal. The latter assui~tion ee~d more likely in that there appeared

to be a decrease only during stages (Groups III, IV and V) where the primary

ch~e was an increase in growth. An increase in neuroseerebory material

was noted in animals poss easing four legs (Group Vi) • iThiming this period it

is well known that these animals undergo a marked functional and structural

transition from aquatic to terrestrial life, therefore the increase in nen—

rosecrebory material may well be associated with these changes..

The localized distribution of neirosecretory material within the hype

thalme-4~ypophyseaI system of alult animals. correlated with previous findings

by Smith (‘Si) in other species of the frog (Rana catesbiana and Rana

grylio). As described by him, blue-black spherical and irregular shaped

masses, and fine granules were observed in the preoptic nucleus and along

the preoptico.4~ypopkyseal tract. Some nerve fibers of the tract occasion—

ally presented a “beaded” appearance. Numerous blue—black masses were also

found in the pars nervosa and in close association with hypophyseal blood

vessels.. According to Bargiaann and Scharrer (S 51), Hild reported similar

findings in the frog. No mention was made of the particular species used

18

in Hild’s study.

Studies made on the h othalinus of rat,s (Srnith’~l; De Groot

snakes (Scharrer ‘~2) dogs and cats (Sloper ‘≤~) showed a aTm~fl ar pattern of

distribution of neurosecretory material as found in adult Rana pipiens in

this investigation. These investigators reported the presence of neuro—

secretory material (blue.”biack granules, droplets and masses) in the supra—

optic and paraventricular nuclei which could be traced along the axons that

extended from these nuclei and terminated in the infundibulum. “Beaded”

axons were also reported to have been. seen, as wail as blue-black masses in

the pars nerv-osa. One of thesi (Smith) described the granular masses as bOa.

ing tangled and vermiform~ This description was in accord with the spiral

shaped granular tnasses observed in arthuals in Groups VII (yoimg adults) and

VIII (adults) in this study.

~AP~ER VI

STTh~ARY AM) CO~LUSION3

1, Larval and adnib stages of Ran pipiens have been studied for nenre

seerebory activity.

2. The preoptic nucisus -peered to be the center of neorosecretoi~r ~.

tivity in these an1rn~,

3. The b~oth popby~eal systeu seens to have contained the greatest

quantity of nenrosecrebory material. The material was. also occauiona~J.

found in the dc~l region of the dience~ialon, the h oa~ns and in

or either closely associated irith b p~ seal blood vesso3s,

k. Neurosecretory activity seemed to have made its first significant ap.M

pearance in animals ~itb external gills (Group II).

!5~. Animals ithich comprised Groups Ifl (hind limb bud stage), XV (hind limb

stage) and V (fore and hind limb stage) showed a marked decrease in

neurcsecretcz’y material, ~thereas those in Groi~ VI (fonr legged stage)

sho~d an increase in m~terial. This m~r have been due to a variation

in the functional requi ants for the material during different develop...

mantel Stages.

6. Neurosecretory mat~ial w~ found to be elatively abundant in Gromps

VII (young adult stage) and Vlfl (adult stage)j however, the dist~.ibu—

tion of thin material in the latter group a~eared to be confined pri—

manly to the hthabo~ hyseal system.

LtT~RATUP~ GIThD

Bargmenn, W. and E. Schan~er 1%1 The site of origin of the homnones of thePosterior pituitary. kner. Scientist, 39: 2~—2g9.

Brcmi, Frank A., Jr., and 0. O~zem 191~1 Upon the presence and distribution of a chroinatophorotropic princ~lo in the central nervous systemof Ltmulus. Biol. Bull., 81: 80..9S.

Be Grost, J., Z9~7 Neuroeecretion in experinental conditions. Anat. Rec.,127: 201—217.

Gomori, 0., 191s1 (~servations with differential stains on htriuan islets oflangerbans. Am. Jour~ Pathol., 17: 39S~wW)6..

ICopec, ~tefan 1922 Studies on the necessity of the brain for the inceptionof insect meb~rphosis, Biol. Thifl., i~: 323-3k2.

Palsy, Sanford L., 191j3 Neurosecretjon. V. The origin of neurosecretöz~granules from the nuclei of nerve cell, in fishes. Jour. 0oxx~, Near.,79: 2k7..27~,

Scharrer, Berta 19141a Neurosecretion, II. Nourosocretory coils in thecentral nervous system of cociroaches, Jour. Conp. Near., 71~:93—108.

_________ l91~1b Neuroneerotion. IV. Localization of neurosecret1ory cefls

in the central nervaus system of limulus. Biol. Bull., 81: 96~1Ci~.

Seharrer, Ernst 191~1 Neurosecz’etion. 1. The nucleus preopticus of Fundulusheterocitus. Jour. Co~. Near,, 7k: 81—92.

_________ 19~2 Nearosocretion. I. A relationship between the paraphysis

and thepa~a~ventrjcular nucleus in the garter snake (Thamnophis ~ia•)~Biol. Bull., 101: 106—113.

Scharrer, Ernst and Berta Scharrer 191K) Secretory cells within the bypo~.tha3mus, flea. Pubi. Ass. Nerr. 1~ent. Din., 20: l70-.i91~.

__________ l91d~ Neurosocretion. VI. A comparison between the intercere

braiis.acardj~.~jj~~ system of the inseats and the h~potha3ino-.hypophyseal system of the vertebretea~ Biol. Bull.., 87: 21~2a~2~l.

_________ i91~5 Neurosecretion, Thysiol. Rev., 2!~: 171—181.

Scharrer, Ernst, S. L. Pa]*y and R. 0. Nilges 191i5 Neurosecretion. VIII.The Nissi substance in secreting nerve cefls • .Anat, flee., 92: 23~30.

20

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