- Email: [email protected]
Hormonal content and ultrastructure of the disconnected neural lobe of the grass frog (Rana pipiens)
GENERAL
ASD
(‘OMP.\RATIVE
~KDOCHIN0I~~C.T
Hormonal Content Neural Lobe
15,
272-288
(1970)
and Ultrastructure of the Grass Frog
Receivrd
Decembt~r
of the Disconnected (Rana pipiens)’
29. 1969
Based on changes in ultrastructure and vasotocin content, t\vo postol’erativc phases can be distinguished in t,he neural lobes disconnrctcd from l’hr hypothalamus. During the first phase (first 2 days after transcrtion) only very few axons start. t,() degenerate. and the vasotocin content doc,s not differ from that of the, l,ontrol glands. Axons filled with tubular formations are more num(lrous than in th? controls. The second phase (6th to 9th postoperative days) is characterisrd by thr dt~gc~ncrat.ion of most of thr nnurosecretory axons and by :I dramatic fall in 111~ \-asotociu content of the glands. During this ljhase. the number of Golgi complexrs in the. pituicytes increases considerably. Simultaneously. the appearance of well organized c::tnaliculi lined by pituicytcs is obsc>rvId. A second cell type. in addition to pituicytes. is observed in the second phasca. Thesc, cells have a characteristic nucleus and a cytoplasm mainly occupi(,d by rough cmdoplasmic reticulum and lipid droplets. The pituicytk; reorganization and the’ a~,pearance of a new type of eel1 indicate i hat the chronically disconncc*tr,d neural lob<’ is not a dead formation but a struct,urcx that mny play some unknown functional role The rapillaries of the disconnected neural lobr arp characterized by the Lvideninp of the endothelial fenestrations and tht, disappcLar;mce of f,heir membrane. nhicah I’+ sults in a direct communication between thr c.apillary lumc>n and 111~ ~)(~rivnsc.ular space. White blood cells are densely packed against the capillary walls. ;md most of these cells have processes that, prnrtrak the rxndothrlial openings and contac,t (11~ perivascular basement, membrane.
The morpho180gical changes that take place in the neural lobe of the hypophysis after interruption of the hypothalamohypophyseal nervous pathway have been mainly st,udied in amphibian species. I,ight microscopical studies reporting a decrcasc in the amount of stainable material begin’ This investigation was support)ed by grants 5ROl NB 06641 NEUB and 5ROl NB 07492 front the National Institute of Health and b,v the, Space Sciences Research Center of the Unirc>rsit> of Missouri. ‘Fellow of the Consejo National de Invf3tig;lciones Cientifiras y Tkcnicas dc la Rcl)fibli,,:l Argentina. 3 Present address: Instituto de Histologia >. Embriologix. Casilla de Corrco 56. Mcndoza. Argc,ntina.
ning t(w) days after tht> tlisc~onnectiot~ of the neural lobe have been reported by Iturriza and Restelli (1967) and Dellmann and Owsley (1968) . Ultrastruc*tural studic:: of t.he disconnected neural lobe,, with spcrisl emphasis on the pituicytw. harc~ been carried out, by Sterba and Briickncr I 1967. 1969) and Dcllmanu and Owsley i 1969 1. Preliminary studies of the hormollnl VOW tent of cxtracta including hot tr t.hc median cmineuce and neural lobe aftw tiiwounc~c*tion from the hypothalamus have* bc(>11[IUIJlished by Dcllmann ef nl. ( 39138). The pwscnt paper clcwrihrs thts llltr:r*tructural changes of tlw disconnectcxcl new rai lobe, with special referenw to the ncrv(’ fibers. and the hormonal content of this lobe :tt tliffcrwit postopclrativc intervals.
l’HAttMACOL06Y
AND
MORPHOLOGY
That morphology and hormonal content of t!htB disconnected median eminence bal:c becbn described in a separate paper (Dellm:tnn and Rodriguez, ~~L~TF.RIAI,S sil,Y/irlrl
1970).
AND
METHODS
I’TOrerl~~rrs
ITron January to Map the hypot,halamo-hypo~hyscal tract of 63 frogs (group I) was t,ransrrtfd at the lrvcl of the anterior c>nd of thf, m(,lli:in ~~minenc~c~(Fig. 1) following tht, technique
l’tc. 1. Schematic representation of the frog hypophyseal region showing the site of transection (arrow) and the area extracted for hormonal determinatiorl and fixed for the morphological study (striped zone) which corresponds to the neural lobe. IK, infundibular recess; I, infundibulum; ME, median eminence; PI, pars intermedia; PD, pars dist,alis. tlrscribetl by DAlmann and Onsley (1968) An&hesi:t was in 0.1% MS 222 (Sandoz). Thr median cminrncr of 7 frogs (group II) WI* vSposed as for t rimsection, but transrcation was not l,~~rformcd. Theat animals were considerrtl :W .~I1:lm-ol)c,r;lted, and used as controls for the morphologic~;d study. A group of 15 nortnnl frogs (group III) did not, rrcrivc any treutmWt and wt’rf’ us~l as controls for the r)h:rrmncologic,:~l study. The animals of the thrrc groups were fed OIIU ;L wcv~k with tadpoles and kept, at a constant trmpc~rat,ur’e (lS”C), humidity and light (light : rl:+rF; ratjo 12 : 12). The animals \vf‘cre kepl in whitr c~)ni:linc~ra. f /LI,).II/OCOlOyicnI Procerlur’w il&,rtls. 42 transected frogs divided into 12 group;; and 18 normal frogs divided into 5 groups lycrc, killed h,v decapitation without anesthesia
OF
DIHCOXXECTED
XEITRAL
LOBE
“73
(for distribution of groups s.‘c Fig. 2) TOP brains wcw rrmowd :is soon as po&Ae (in 1 to 3 minut’es) and the neural lobes wcrc carrfull~ SC,Nrated from the median rminences (Fig. 1). Estrnction of Glu~ls. Tlrr neural lobrs of :111 the animals in each groul) \vcrc‘ ~~)oletl and homogenized in 0.25% acetic acid. The homogI%natcs tvrrc obtained with :L Teflon pclstlr turning for 2 minutes at equal spryd for all c,stracts. TIN> homogr~natrs were hrated in il boiling-watclr I)ath for 3 minutc,s imd thrxn c~c~ntrifuged :it 3000 r[>m for 10 minutes (Hcll~~r anti I,ctleris. 1959). TIw Aupc‘rnatants \vrrc’ kept &fy)ly frozen and :~ssayc~l within thr s:mw day. I-trsopresso,~ il.s.scr,~. Thr Ijrclysor activity of ttlc extracts was measured hy the method of D[xk:mski (1952) with some modlficutions. The rats were :me*thctizrd with 12% ethanol gircn t lrrough :I stomach cannula. A carotid artery and :I jugd:rr \-tin were cannulatrd and trachrotomy was [“‘rformed. Dihrnzylinr, 300 ~g/lOOp, was given intravenously to block sympathetic activity. A (2 + 2) assay design was used. E:I& of the values in Fig. 2 w<‘rc obtained from t,hrre (2 + 2) y;cts (12 points). Pitrrssin (Pa&r,. Davis & Co.) was used a:: standurd prq)aration. Twenty-one transectrtl and 7 shilnl-ol’t~r;ltc~tl frogs wcr~ distributed into seven groups which were killed at 6. 12, 24, and 36 hours. 2. 6, and 9 days after t,he operation. The animals were killed by decapitation without anesthesia. The hypophyseal region was fixed I)?; injection of a a-fold nldehyde mixturr into the cerebral ventricles (Rodrigurz. 1969). The glands were postfiscd in I’/ osmium trt,rosido. stained P?Xbloc with uranyl 1967) and embrdded in awtatc~ (Farnovsk,.. Bralditp (Glauert and Glnuert. 1958). For further information on the rlectron microscol)ic:d nlrthods WV Rotlrigur,x and Dcllmann (1970).
Content of the A701”VlMll Disconnected .Yeuml Lobes
Vasotocin,
and
As shown in Kg. 2, the vasot,ocin content of normal neural lobes extracted between .January and May ranges between 42 and 82 mC/gland.
Twent,y-four
and
48 hours
aft’er the transection, the vasotocin content, of the neural lobe did not, show any substantial change (Fig. 2). Sine days aft’er the transcct’ion, the hormonal content of the neural lobe was reduced to about 10% of thr normal values. One month aft,er the
RODRiGl-EZ
AKD
operation, no hormonal activity xx,; clctected in the rliaconiit&2d tieural lohr.
The ultrastructurc of the normal neural lobe will be described briefly since it 1~13 already been reported by other :tuthorLs iOota and Kobayashi, 1963; Zambrano and DC Rohertis, 19681. With thr method usect, the neural 101)~has t,hrcc arms n-hich havcx a distinct,ive :q)pcar:mce. Subependymnl region. That nerve fibers in this area are frequently separated by large spaces filled by a fine granular riiaterial of moderate electron density. These fillt~l spaces also occur hctwcen ncrvt’ fibers alttl qmtdytnal ~11s and only occasi’onally bvtweert rpcttdyrnal ~11:: (Fig. 3 1. Thc~;c~itt-
DKLLMANN
PHARMACOLOGY
ASD
MORPHOLOGY
OF
DISCONNE:
TED
NEURAL
LOBE
I:[(;. 13. Ependymal and sabependymal regions of the neural lobe of a normal frog. IR; infundibrkr of the third ventric4e; RL, basemeltt, membrane which is preszllt at. the jlmctiorl hetweell the nerlral 1, hr net~r:rl lobe: the arrows point to filled spaces between neruxsecretory aso~~s. X8700.
tlcnsc~ hodics (Fig. ,5I Intercellular are only occasionally seen.
spaces
‘I%c~ ultrastructural changes that take l)lacc in the neural lohe following the transection can clearly Iw grouped in two ]hWS.
“7 r,
stalk
recess and
the operation. Six hours after the transection, no changc~ wrrc ohserred. After 12, 24, and 33 houra of transection the most! evident modification was a considerable cnlargen~cnt of the intcrccllular space in the sul);el)c~rldymal and hilar regions (Figs. 6-8) and of the space fwtwccn epcndymal cells (Fig. 7). In the animals killed 36 hours after the operation. the ncrvc fibers and ircrve cntlings containing tubular for-
276
E’l( elect.1 Frc UI-“\l inter<
ItODHiGI~EZ
AND
DELLMANK
with I lxrge tilld
PHARMACOLOGY
FIG c*ellul:
11
AND
6. Kenral l&e of an animal spaces in the hilar region.
MORPHOLOGY
killed
24 hours
PVR, perivascular
OF
DISCONNECTED
NET-RAL
LOBE
aft)er the transection showing the presence region; C, capillary. X 10,000.
inatic D11s appear to lbe more numerous than in th e c,ont’rols. The tubular formations also seem :o have a larger diameter than those obsel:v,ed in the control glands (Fig. 9). In xdtlit ,lIan to tubular formations, thest fibers
277
of lnrg e iriter-
contain several large dense bodie s and lamcllar bodies and only few neuroses :retory granules and mitochondria. As a whole these endings resemble the fibers obIserved in the stamp distal to the transection at the
l’HAHMAC!OLOGT
FIG. 9. I’erivascldar vGt.h numerous tubular to intrrcell~dar spaces:
AKD
MORPHOLOGY
OF
DISC!ONPiECTED
XETWAL
LORE
region 36 hours after transection showing the presence of a nerve ending (S) formations, dense bodies, and a few neurosecret,ory granules CG). The arrows PI’S, perivascular space; C, capillary. X25,000.
FIG. 8. Sllbependymal region observed bet weru the epcndyma
at the level where a basement membrane is abseut. A large and a Herring body. ZR, infundibular recess. X 10,000.
filled
filled point
space
is
280
RODRf(;I-EZ
AND
same postoperative period (36 hours I (3~ Dellmann and Rodriguez, 1970). The first degenerative changes of the neurosecretory fibers are observed 48 hours after transection. The first detectable change in the degenerative process is generally the engulfing of the axons by the pituicytes. The first evident change in the
DEI,I,I\IANK
axons themselve,5 consisted of a d.enser packing of the ncuaosccretory gra ,nules within the axon and the appearance of a fine granular dense material in t’he axoplasm between the neurosecretory gral nules, all of which give these axons a darke Br appearance t’han the neighboring ;axons (Fig. IO).
PHARhIA(‘OLOGT
FIG.
engulfed YT, rough processes pituicyte.
ASD
MORPHOLOGk-
OF
DIS(‘OS\‘NECTED
NEI:RAL
LOBE
281
11. Hilar region of an auimal killed after 48 hours of Iranseci,ion. Several neurosecretory axons are by a pituicyte. 1, II, III, IV, and 1’ represent the possible sequence in the degenerative process. endoplasmic reticulum; r, free ribosomes; m, mitochondria; the small arrows indicate cytoplasmic of the pit.uicJ-tc, and the large arrow points to loose neurosecret,ory granldes being engulfed by the X 1.‘3,000.
282
FIG
pituic arrow
RODRiGUEZ
AND
DEI,I,MASN
PHARMACOLOGY
FIG.
jetting of unkt spaces.
AXD
MORPHOLOGY
13. Netual lobe !I days after the opetxt.iott. Tao mic :rovilli (mv) into it. The nuclear and cytoplasmic are clearly shown. G, Golgi apparat’us; 1OW n natttre Xl 10,000.
OF
DISCONNECTED
IGEVRAL
LOBE
283
pituicyt,es are seen fornlittg a cana &cult 1s and 1X0differences between pituicytes an d one of the ( :ells T, tight junction: the R~TOWY indic :at,e it ztercelll ular
284
ItODHiGl-EZ
AND
After the axons have been engulfed, thtly degenerate following a well established pattern already described by Sterba and Briickner (1969) and Dellmann and Owsley (1969). Part of such “pattern of dcgeneration” is clearly shown in Fig. 11. A variant to this pattern seems to be given by axons wh’ose membrane is dissolved before the
DELLMASS
ongulfing by the pituicytcv. which results in the, presence of loose ntvrosecret ory granult~~ in the intercellular space. HowCIW. theac granules are finally engulfed by the pituicytcs and dig&cd in the samme way as the engulfed axons (Fig. 11 I. Only few axon.5 showed degcncrative changes after 48 hours of transcvtion.
PHAKMA(‘OLOGY
AND
MORPHOLOGY
Phase 11. Six and 9 days after the transection, the degenerative changes of the neurosecretory fibers and the cellular reaction arc very pronounced, so that the ‘origillal organization of the neural lobe is no longer recognizable (compare Fig. G and 12). Only few neurosecretory fibers are c>xtrapituirytic and they very likely are fibt>rs which were not transected during the operation (Fig. 12). The “filled” intercclIIilar spaces are present throughout the gland (Fig. 12). Within the pituicytes some consistent changes with respect to those observed during the first phase arc seen. The engulfed axons are in very advanced >lages of degeneration and in many in~tanccs it is impossible to recognize their original structure. Transitional forms be1U-ecn degenerating fibers and large lipid droplets arc observed (Fig. 13). The moat intc>rcsting change during thii: I~lias~~seems t,o be the appenrancc of canalicbuli linctl by t,wo or more pituicytes (Figs. 12-14). Longitudinal s&ions ‘of these canaliculi show that they are really tubular, not ,jrlst, saccular formations. Generally, the c~:malic~lli are laterally sealed by tight junctions tFigs. 13 and 14). The pituicytes I)roject, short microvilli into the lumen of thr canaliculi. Sometimes t#liesc microvilli are slender and of a lmiform dian&er (Fig. 14j, whercxas in other occasions they are globular am1 occupy t)hc canalirular lumen completely (Figs. 12 and 13). Few coated pinocytic vesicles arc gencrnllv seen to oI)cn mto the lumen of the canaiiculi (Fig. 141. -4 considcrablc increase in the number of Golgi complexes, especially in t,he neighborhood of t’he canaliculi, is observed in the pituicptes during this second postoperative pllase (Figs. 12-14), During t’his phase a seoond cell type, in acldition to pituicytes, is observed. They have a characteristic structure: the chromatin is dcnscly packed against the nuclear envelope and t,he cytoplasm is mainly occupied by rough endoplasmic reticulum and lipid droplets which, with the method of fixation and embedding used, have a ring of dense material surrounding a light core Wigs. 12 and 13). A few large and small lamellar bodies are generally present in tlicaae cell+. These cells have numerous
OF
DISC’ONNECTED
NEITRAL
LOBE
285
elongated and slender cytoplasmic processes projecting into the “filled” spaces. Degenerating axons have not been observed in the cytoplasm of these cells. This observation and the fact that there arc not intermediate forms between these cells and pituicytcs suggest that they are a new cell type which appears in the neural lobe at a certain postoperative period. The origin, nature and fate of these cells remain t,o be det’crmined. Vascular changes. The first3 vascular changes are already present in .the first group of glands studied (6 hours after transection) and they consist in the widening and opening of the endothelial fencstration and the packing of white blood cells against the capillary walls. These two changes become more prominent with the passage of the postoperative time. Six and 9 days after t’he transection it is commonly observed that almost a complete ring of white blood cells is interposed bet,ween the cndothclium and the erythrocyt’es (Fig. 15). The lcukocytcs generally have numerous vacuoles localized near to the cell membrane, and cytoplasmic processes which either contact the endothelium or penetrate through the endothelial openings and cont,act the perivascular basement membrane (Fig. 15). P. E. Budtz (personal communication) has observed a similar vascular reaction in the disconnected median eminence of the toad. The present investigation confirms previous reports (Sterba and Briickner, 1969; Dellmann and Omsley, 1969) concerning the beginning of degenerative changes in the disconnected neural lobe about 2 days after the operation. In the present material only a minorit’y of axons start degenerating after 48 hours of transection, and ,most of them look normal. This morphological appearance of the gland during this period is in complete agreement with the hormone bioassays which show that the vasotocin content of the disconnected gland remains unchanged during the first 2 days after the transection. These results are not in agreement wit’h the preliminary observations of Dellmann et al. (1968) of an increased
286
FIG. 15. Neural lobe 9 days aft,er (WC) arranged against t,he endothelium endothelial fenestrations and contact vascular space.’ X9000. Inscrl: High &he perivasrular basement) membrane
RODRiGUEZ
ASD
DELLMANN
transection showing a capillary containing sever:~l white bl()c,rl (:&. (E). One of I hese cells has t.wo procctsses iarrc~ws j which penct rat,e the perivadcular basement, membrane. EC, red blood cells; PF-,S, I)crimagnificaf ion pict,ure showing leukocyte (14yC’~ processes contact illg inrrow). E, endolheli~~m; /‘TV& perivasicldxr space. x 33,000.
PHAR.MACOLOGY
AND
MORPHOLOGY
vasopressor activity in the disconnected neural lobe during the first 2 days after transection. This disagreement may be due t’o the fact that the earlier study did not consider the large variations in the hormonal content of the neural lobe of this species, which may be in the range of 50% (we Fig. 2j. ()ur present observations strongly suggo& that during the first two postoperative days the neurosecretory axons and their hormonal content are not considerably affectcd by their disconnection from the perikarya. This, and the fact that t,he va~ular supply to the gland is not interrul&cd by the “neural disconnection,” would suggest that the disconnected neural lobe, during the first 2 postoperative days, is a good experimental model for the st’udy of some of the mechanisms involved in the release of neurohypophyseal hormones. For example, would the disconncctctl neural lobe release its hormones as a consequence of a humornl stimulus such as dehydration or an int,ravascular injection of hypertonic saline or calcium chloride? -in intriguing question is what inducts t,ht) pituicytes to start engulfing the “normal-looking” axons 2 days after the transection? Why does the engulfing not take place earlier after t,hc transection or in the normal glands? 1)ellmann and Rodriguez (1970) have olj.-erved that within the first 2 days following transection that distal stump has numcrous fibers loaded with tubular formations and that the extracts of t’hese stumps IIAT~Can increased vasoprrssor activity. The authors po&lated that this increased vasotoein content was due to a backflow of ~~xtragranular hormone, from the neural lobe to the distal stump and that the tubular format,ions might play a role in this flo~v. The prcsencc of similar tubular fornultion:: in some nerve fibers and nerve endings of the neural lobe observed in the sanle postoperative period would support that hypothesiw. As in the first phase, the pharmacological ant1 morphological events taking place during the rccond phase (between 6th and 9th po>toperativc days) are in complete agree-
OF
DISCONNECTED
SEURAL
LOBE
287
ment. The ultrastructural study shows that after 9 days of transection only a minority of the fibers does not undergo degeneration, and the bioassays reveal that the glands extracted at the same postoperative interval only contain about a tenth of their normal hormone content. The “filled” intercellular spaces which are 401bserved in the subependymal region of the normal glands and throughout the disconnected neural lobes may be artifacts produced by the hyperosmolarity of the aldehyde mixture (about 1200 mOsM ; for details see Rodriguez, 1969), which might promote the transport of substances from the nerve fibers into the intercellular space. The preferent,ial subependymal localizati80n of these spaces in the normal glands may be due to the fact t,hat the fixative was injected intraventricularly. The generalized localizat’ion of t’he spaces in the experimental glands could be due to the fact that the transected axons are more easily affected by a hyperosmolar fixative than the intact fibers. The integrity of the elementary neurosecretory granules in fibers surrounded by filled spaces, and the high development of these spaces in glands with practically no hormonal act,ir&y would indicate t’hat the material filling t’he spaces does not have any hormonal activity. The striking changes presented by t,he pituicytes during the second phase, namely the considerable increase in the number of Golgi complexes and t)he formation of well organized canaliculi strongly suggest’ that, the reorganized pit,uicytes may have some new function(s) after t’hey have finished the process of engulfing and degradation of the disconnected axons. This pituicytic transformation, and the simultaneous appearance of a second t’ype of cell seem to indicate that’ the chronically disconnected neural lobe is not a dead st’ructure but. that it may have some functional significance. What exactly this significance is appears as an interesting t)ask for future investigations. REFERENCES J. (1952). The quantitative assay of vasopressin.Btit. J. Pharmncol. 7, 567-572.
DEKANSKI,
H.-D., AND OWSLEY, P. A. (1WX) ln\ estigations on the hypothalamo-nerrrolI?;pophysial neurosecretory system of the grass frog (Ram &Gens) after transection of the I~rc)uirnal nuerohypophysis. I. Light microscopic findinpb in animals kept at 18°C environmental Icwrl~,~r:rtUrr. %. Zellforsch. Mikrosk. 4nut. 87, 1 -Ifi. I~LMANN, H.-D., AND OWSLEY, P. A. (1969). lnt11c vcstigations on h?potlral:lmo-llerr~olr~poyhysial neurosecrctory r;ystem oE the grxs~ frog (~~anrc pipiens) aftc,r trxnsection oi‘ thr. proximal neurohypophysis. II. T,ight ant1 vlw.Won microscopic findings in the tlia~onne~tc~d distal ncurohypophysis with special c’nrlllr:i,+ on thr I)ituicytes. Z. Zellfo7xzh. ilfikl-osl; .l,r~ii. 94, 325-336. DIII,IX.~NN, H.-D.. .~NLI RODR~GCEZ. E. M. (1070). Inwstigntions on the liypothalnmo-ncurolr?-pophysial nfwrosccrctory system of t,he gri~b> lrog (Rtrnfr TGpierw) after transection of the, proximal nc,uroh~pclpll3-sis. III. Ultrastrurlurr~ and hormone contt,nt of the dist:rl strrmp, I,r “Aspects of Neuroc,ndocrinology” (W. Rargmnnrr and B. Schnrrer, Eds.) Munic*h. Bcrgmann. hX.l~~l.~NS. H.-D., T~.~r,r~. IT. E.. C~HLIIY. I’. ~1.. ASD T~~r,r~~r~)~r~. T,. 1:. (196s). ~foq)hologic:d and func,tionnl changes in the Ili;;tnl lr~~)~)ill:~l:r~r~c)neurohypophysinl system of tlrc gr:ws frog (IL)ow +[~~cJw) after trnnswtinn of Ihr I~rosimnl rl~rrrolrvpol)il?-sis. Esp~rir~~tl~r 24, 383-3X6. GI,AUERT, ,4. M.. ASI) GL.\~~;RT, R. H. (19.5~~. i1raldit e as an embedding medium for elrct.ron rriic7wxopy. J. Biopl~~ir. Biochcm. (Yytol. 4, 191194. &I.IJ:R. H.. .
ASD
(‘OMP.\RATIVE
~KDOCHIN0I~~C.T
Hormonal Content Neural Lobe
15,
272-288
(1970)
and Ultrastructure of the Grass Frog
Receivrd
Decembt~r
of the Disconnected (Rana pipiens)’
29. 1969
Based on changes in ultrastructure and vasotocin content, t\vo postol’erativc phases can be distinguished in t,he neural lobes disconnrctcd from l’hr hypothalamus. During the first phase (first 2 days after transcrtion) only very few axons start. t,() degenerate. and the vasotocin content doc,s not differ from that of the, l,ontrol glands. Axons filled with tubular formations are more num(lrous than in th? controls. The second phase (6th to 9th postoperative days) is characterisrd by thr dt~gc~ncrat.ion of most of thr nnurosecretory axons and by :I dramatic fall in 111~ \-asotociu content of the glands. During this ljhase. the number of Golgi complexrs in the. pituicytes increases considerably. Simultaneously. the appearance of well organized c::tnaliculi lined by pituicytcs is obsc>rvId. A second cell type. in addition to pituicytes. is observed in the second phasca. Thesc, cells have a characteristic nucleus and a cytoplasm mainly occupi(,d by rough cmdoplasmic reticulum and lipid droplets. The pituicytk; reorganization and the’ a~,pearance of a new type of eel1 indicate i hat the chronically disconncc*tr,d neural lob<’ is not a dead formation but a struct,urcx that mny play some unknown functional role The rapillaries of the disconnected neural lobr arp characterized by the Lvideninp of the endothelial fenestrations and tht, disappcLar;mce of f,heir membrane. nhicah I’+ sults in a direct communication between thr c.apillary lumc>n and 111~ ~)(~rivnsc.ular space. White blood cells are densely packed against the capillary walls. ;md most of these cells have processes that, prnrtrak the rxndothrlial openings and contac,t (11~ perivascular basement, membrane.
The morpho180gical changes that take place in the neural lobe of the hypophysis after interruption of the hypothalamohypophyseal nervous pathway have been mainly st,udied in amphibian species. I,ight microscopical studies reporting a decrcasc in the amount of stainable material begin’ This investigation was support)ed by grants 5ROl NB 06641 NEUB and 5ROl NB 07492 front the National Institute of Health and b,v the, Space Sciences Research Center of the Unirc>rsit> of Missouri. ‘Fellow of the Consejo National de Invf3tig;lciones Cientifiras y Tkcnicas dc la Rcl)fibli,,:l Argentina. 3 Present address: Instituto de Histologia >. Embriologix. Casilla de Corrco 56. Mcndoza. Argc,ntina.
ning t(w) days after tht> tlisc~onnectiot~ of the neural lobe have been reported by Iturriza and Restelli (1967) and Dellmann and Owsley (1968) . Ultrastruc*tural studic:: of t.he disconnected neural lobe,, with spcrisl emphasis on the pituicytw. harc~ been carried out, by Sterba and Briickncr I 1967. 1969) and Dcllmanu and Owsley i 1969 1. Preliminary studies of the hormollnl VOW tent of cxtracta including hot tr t.hc median cmineuce and neural lobe aftw tiiwounc~c*tion from the hypothalamus have* bc(>11[IUIJlished by Dcllmann ef nl. ( 39138). The pwscnt paper clcwrihrs thts llltr:r*tructural changes of tlw disconnectcxcl new rai lobe, with special referenw to the ncrv(’ fibers. and the hormonal content of this lobe :tt tliffcrwit postopclrativc intervals.
l’HAttMACOL06Y
AND
MORPHOLOGY
That morphology and hormonal content of t!htB disconnected median eminence bal:c becbn described in a separate paper (Dellm:tnn and Rodriguez, ~~L~TF.RIAI,S sil,Y/irlrl
1970).
AND
METHODS
I’TOrerl~~rrs
ITron January to Map the hypot,halamo-hypo~hyscal tract of 63 frogs (group I) was t,ransrrtfd at the lrvcl of the anterior c>nd of thf, m(,lli:in ~~minenc~c~(Fig. 1) following tht, technique
l’tc. 1. Schematic representation of the frog hypophyseal region showing the site of transection (arrow) and the area extracted for hormonal determinatiorl and fixed for the morphological study (striped zone) which corresponds to the neural lobe. IK, infundibular recess; I, infundibulum; ME, median eminence; PI, pars intermedia; PD, pars dist,alis. tlrscribetl by DAlmann and Onsley (1968) An&hesi:t was in 0.1% MS 222 (Sandoz). Thr median cminrncr of 7 frogs (group II) WI* vSposed as for t rimsection, but transrcation was not l,~~rformcd. Theat animals were considerrtl :W .~I1:lm-ol)c,r;lted, and used as controls for the morphologic~;d study. A group of 15 nortnnl frogs (group III) did not, rrcrivc any treutmWt and wt’rf’ us~l as controls for the r)h:rrmncologic,:~l study. The animals of the thrrc groups were fed OIIU ;L wcv~k with tadpoles and kept, at a constant trmpc~rat,ur’e (lS”C), humidity and light (light : rl:+rF; ratjo 12 : 12). The animals \vf‘cre kepl in whitr c~)ni:linc~ra. f /LI,).II/OCOlOyicnI Procerlur’w il&,rtls. 42 transected frogs divided into 12 group;; and 18 normal frogs divided into 5 groups lycrc, killed h,v decapitation without anesthesia
OF
DIHCOXXECTED
XEITRAL
LOBE
“73
(for distribution of groups s.‘c Fig. 2) TOP brains wcw rrmowd :is soon as po&Ae (in 1 to 3 minut’es) and the neural lobes wcrc carrfull~ SC,Nrated from the median rminences (Fig. 1). Estrnction of Glu~ls. Tlrr neural lobrs of :111 the animals in each groul) \vcrc‘ ~~)oletl and homogenized in 0.25% acetic acid. The homogI%natcs tvrrc obtained with :L Teflon pclstlr turning for 2 minutes at equal spryd for all c,stracts. TIN> homogr~natrs were hrated in il boiling-watclr I)ath for 3 minutc,s imd thrxn c~c~ntrifuged :it 3000 r[>m for 10 minutes (Hcll~~r anti I,ctleris. 1959). TIw Aupc‘rnatants \vrrc’ kept &fy)ly frozen and :~ssayc~l within thr s:mw day. I-trsopresso,~ il.s.scr,~. Thr Ijrclysor activity of ttlc extracts was measured hy the method of D[xk:mski (1952) with some modlficutions. The rats were :me*thctizrd with 12% ethanol gircn t lrrough :I stomach cannula. A carotid artery and :I jugd:rr \-tin were cannulatrd and trachrotomy was [“‘rformed. Dihrnzylinr, 300 ~g/lOOp, was given intravenously to block sympathetic activity. A (2 + 2) assay design was used. E:I& of the values in Fig. 2 w<‘rc obtained from t,hrre (2 + 2) y;cts (12 points). Pitrrssin (Pa&r,. Davis & Co.) was used a:: standurd prq)aration. Twenty-one transectrtl and 7 shilnl-ol’t~r;ltc~tl frogs wcr~ distributed into seven groups which were killed at 6. 12, 24, and 36 hours. 2. 6, and 9 days after t,he operation. The animals were killed by decapitation without anesthesia. The hypophyseal region was fixed I)?; injection of a a-fold nldehyde mixturr into the cerebral ventricles (Rodrigurz. 1969). The glands were postfiscd in I’/ osmium trt,rosido. stained P?Xbloc with uranyl 1967) and embrdded in awtatc~ (Farnovsk,.. Bralditp (Glauert and Glnuert. 1958). For further information on the rlectron microscol)ic:d nlrthods WV Rotlrigur,x and Dcllmann (1970).
Content of the A701”VlMll Disconnected .Yeuml Lobes
Vasotocin,
and
As shown in Kg. 2, the vasot,ocin content of normal neural lobes extracted between .January and May ranges between 42 and 82 mC/gland.
Twent,y-four
and
48 hours
aft’er the transection, the vasotocin content, of the neural lobe did not, show any substantial change (Fig. 2). Sine days aft’er the transcct’ion, the hormonal content of the neural lobe was reduced to about 10% of thr normal values. One month aft,er the
RODRiGl-EZ
AKD
operation, no hormonal activity xx,; clctected in the rliaconiit&2d tieural lohr.
The ultrastructurc of the normal neural lobe will be described briefly since it 1~13 already been reported by other :tuthorLs iOota and Kobayashi, 1963; Zambrano and DC Rohertis, 19681. With thr method usect, the neural 101)~has t,hrcc arms n-hich havcx a distinct,ive :q)pcar:mce. Subependymnl region. That nerve fibers in this area are frequently separated by large spaces filled by a fine granular riiaterial of moderate electron density. These fillt~l spaces also occur hctwcen ncrvt’ fibers alttl qmtdytnal ~11s and only occasi’onally bvtweert rpcttdyrnal ~11:: (Fig. 3 1. Thc~;c~itt-
DKLLMANN
PHARMACOLOGY
ASD
MORPHOLOGY
OF
DISCONNE:
TED
NEURAL
LOBE
I:[(;. 13. Ependymal and sabependymal regions of the neural lobe of a normal frog. IR; infundibrkr of the third ventric4e; RL, basemeltt, membrane which is preszllt at. the jlmctiorl hetweell the nerlral 1, hr net~r:rl lobe: the arrows point to filled spaces between neruxsecretory aso~~s. X8700.
tlcnsc~ hodics (Fig. ,5I Intercellular are only occasionally seen.
spaces
‘I%c~ ultrastructural changes that take l)lacc in the neural lohe following the transection can clearly Iw grouped in two ]hWS.
“7 r,
stalk
recess and
the operation. Six hours after the transection, no changc~ wrrc ohserred. After 12, 24, and 33 houra of transection the most! evident modification was a considerable cnlargen~cnt of the intcrccllular space in the sul);el)c~rldymal and hilar regions (Figs. 6-8) and of the space fwtwccn epcndymal cells (Fig. 7). In the animals killed 36 hours after the operation. the ncrvc fibers and ircrve cntlings containing tubular for-
276
E’l( elect.1 Frc UI-“\l inter<
ItODHiGI~EZ
AND
DELLMANK
with I lxrge tilld
PHARMACOLOGY
FIG c*ellul:
11
AND
6. Kenral l&e of an animal spaces in the hilar region.
MORPHOLOGY
killed
24 hours
PVR, perivascular
OF
DISCONNECTED
NET-RAL
LOBE
aft)er the transection showing the presence region; C, capillary. X 10,000.
inatic D11s appear to lbe more numerous than in th e c,ont’rols. The tubular formations also seem :o have a larger diameter than those obsel:v,ed in the control glands (Fig. 9). In xdtlit ,lIan to tubular formations, thest fibers
277
of lnrg e iriter-
contain several large dense bodie s and lamcllar bodies and only few neuroses :retory granules and mitochondria. As a whole these endings resemble the fibers obIserved in the stamp distal to the transection at the
l’HAHMAC!OLOGT
FIG. 9. I’erivascldar vGt.h numerous tubular to intrrcell~dar spaces:
AKD
MORPHOLOGY
OF
DISC!ONPiECTED
XETWAL
LORE
region 36 hours after transection showing the presence of a nerve ending (S) formations, dense bodies, and a few neurosecret,ory granules CG). The arrows PI’S, perivascular space; C, capillary. X25,000.
FIG. 8. Sllbependymal region observed bet weru the epcndyma
at the level where a basement membrane is abseut. A large and a Herring body. ZR, infundibular recess. X 10,000.
filled
filled point
space
is
280
RODRf(;I-EZ
AND
same postoperative period (36 hours I (3~ Dellmann and Rodriguez, 1970). The first degenerative changes of the neurosecretory fibers are observed 48 hours after transection. The first detectable change in the degenerative process is generally the engulfing of the axons by the pituicytes. The first evident change in the
DEI,I,I\IANK
axons themselve,5 consisted of a d.enser packing of the ncuaosccretory gra ,nules within the axon and the appearance of a fine granular dense material in t’he axoplasm between the neurosecretory gral nules, all of which give these axons a darke Br appearance t’han the neighboring ;axons (Fig. IO).
PHARhIA(‘OLOGT
FIG.
engulfed YT, rough processes pituicyte.
ASD
MORPHOLOGk-
OF
DIS(‘OS\‘NECTED
NEI:RAL
LOBE
281
11. Hilar region of an auimal killed after 48 hours of Iranseci,ion. Several neurosecretory axons are by a pituicyte. 1, II, III, IV, and 1’ represent the possible sequence in the degenerative process. endoplasmic reticulum; r, free ribosomes; m, mitochondria; the small arrows indicate cytoplasmic of the pit.uicJ-tc, and the large arrow points to loose neurosecret,ory granldes being engulfed by the X 1.‘3,000.
282
FIG
pituic arrow
RODRiGUEZ
AND
DEI,I,MASN
PHARMACOLOGY
FIG.
jetting of unkt spaces.
AXD
MORPHOLOGY
13. Netual lobe !I days after the opetxt.iott. Tao mic :rovilli (mv) into it. The nuclear and cytoplasmic are clearly shown. G, Golgi apparat’us; 1OW n natttre Xl 10,000.
OF
DISCONNECTED
IGEVRAL
LOBE
283
pituicyt,es are seen fornlittg a cana &cult 1s and 1X0differences between pituicytes an d one of the ( :ells T, tight junction: the R~TOWY indic :at,e it ztercelll ular
284
ItODHiGl-EZ
AND
After the axons have been engulfed, thtly degenerate following a well established pattern already described by Sterba and Briickner (1969) and Dellmann and Owsley (1969). Part of such “pattern of dcgeneration” is clearly shown in Fig. 11. A variant to this pattern seems to be given by axons wh’ose membrane is dissolved before the
DELLMASS
ongulfing by the pituicytcv. which results in the, presence of loose ntvrosecret ory granult~~ in the intercellular space. HowCIW. theac granules are finally engulfed by the pituicytcs and dig&cd in the samme way as the engulfed axons (Fig. 11 I. Only few axon.5 showed degcncrative changes after 48 hours of transcvtion.
PHAKMA(‘OLOGY
AND
MORPHOLOGY
Phase 11. Six and 9 days after the transection, the degenerative changes of the neurosecretory fibers and the cellular reaction arc very pronounced, so that the ‘origillal organization of the neural lobe is no longer recognizable (compare Fig. G and 12). Only few neurosecretory fibers are c>xtrapituirytic and they very likely are fibt>rs which were not transected during the operation (Fig. 12). The “filled” intercclIIilar spaces are present throughout the gland (Fig. 12). Within the pituicytes some consistent changes with respect to those observed during the first phase arc seen. The engulfed axons are in very advanced >lages of degeneration and in many in~tanccs it is impossible to recognize their original structure. Transitional forms be1U-ecn degenerating fibers and large lipid droplets arc observed (Fig. 13). The moat intc>rcsting change during thii: I~lias~~seems t,o be the appenrancc of canalicbuli linctl by t,wo or more pituicytes (Figs. 12-14). Longitudinal s&ions ‘of these canaliculi show that they are really tubular, not ,jrlst, saccular formations. Generally, the c~:malic~lli are laterally sealed by tight junctions tFigs. 13 and 14). The pituicytes I)roject, short microvilli into the lumen of thr canaliculi. Sometimes t#liesc microvilli are slender and of a lmiform dian&er (Fig. 14j, whercxas in other occasions they are globular am1 occupy t)hc canalirular lumen completely (Figs. 12 and 13). Few coated pinocytic vesicles arc gencrnllv seen to oI)cn mto the lumen of the canaiiculi (Fig. 141. -4 considcrablc increase in the number of Golgi complexes, especially in t,he neighborhood of t’he canaliculi, is observed in the pituicptes during this second postoperative pllase (Figs. 12-14), During t’his phase a seoond cell type, in acldition to pituicytes, is observed. They have a characteristic structure: the chromatin is dcnscly packed against the nuclear envelope and t,he cytoplasm is mainly occupied by rough endoplasmic reticulum and lipid droplets which, with the method of fixation and embedding used, have a ring of dense material surrounding a light core Wigs. 12 and 13). A few large and small lamellar bodies are generally present in tlicaae cell+. These cells have numerous
OF
DISC’ONNECTED
NEITRAL
LOBE
285
elongated and slender cytoplasmic processes projecting into the “filled” spaces. Degenerating axons have not been observed in the cytoplasm of these cells. This observation and the fact that there arc not intermediate forms between these cells and pituicytcs suggest that they are a new cell type which appears in the neural lobe at a certain postoperative period. The origin, nature and fate of these cells remain t,o be det’crmined. Vascular changes. The first3 vascular changes are already present in .the first group of glands studied (6 hours after transection) and they consist in the widening and opening of the endothelial fencstration and the packing of white blood cells against the capillary walls. These two changes become more prominent with the passage of the postoperative time. Six and 9 days after t’he transection it is commonly observed that almost a complete ring of white blood cells is interposed bet,ween the cndothclium and the erythrocyt’es (Fig. 15). The lcukocytcs generally have numerous vacuoles localized near to the cell membrane, and cytoplasmic processes which either contact the endothelium or penetrate through the endothelial openings and cont,act the perivascular basement membrane (Fig. 15). P. E. Budtz (personal communication) has observed a similar vascular reaction in the disconnected median eminence of the toad. The present investigation confirms previous reports (Sterba and Briickner, 1969; Dellmann and Omsley, 1969) concerning the beginning of degenerative changes in the disconnected neural lobe about 2 days after the operation. In the present material only a minorit’y of axons start degenerating after 48 hours of transection, and ,most of them look normal. This morphological appearance of the gland during this period is in complete agreement with the hormone bioassays which show that the vasotocin content of the disconnected gland remains unchanged during the first 2 days after the transection. These results are not in agreement wit’h the preliminary observations of Dellmann et al. (1968) of an increased
286
FIG. 15. Neural lobe 9 days aft,er (WC) arranged against t,he endothelium endothelial fenestrations and contact vascular space.’ X9000. Inscrl: High &he perivasrular basement) membrane
RODRiGUEZ
ASD
DELLMANN
transection showing a capillary containing sever:~l white bl()c,rl (:&. (E). One of I hese cells has t.wo procctsses iarrc~ws j which penct rat,e the perivadcular basement, membrane. EC, red blood cells; PF-,S, I)crimagnificaf ion pict,ure showing leukocyte (14yC’~ processes contact illg inrrow). E, endolheli~~m; /‘TV& perivasicldxr space. x 33,000.
PHAR.MACOLOGY
AND
MORPHOLOGY
vasopressor activity in the disconnected neural lobe during the first 2 days after transection. This disagreement may be due t’o the fact that the earlier study did not consider the large variations in the hormonal content of the neural lobe of this species, which may be in the range of 50% (we Fig. 2j. ()ur present observations strongly suggo& that during the first two postoperative days the neurosecretory axons and their hormonal content are not considerably affectcd by their disconnection from the perikarya. This, and the fact that t,he va~ular supply to the gland is not interrul&cd by the “neural disconnection,” would suggest that the disconnected neural lobe, during the first 2 postoperative days, is a good experimental model for the st’udy of some of the mechanisms involved in the release of neurohypophyseal hormones. For example, would the disconncctctl neural lobe release its hormones as a consequence of a humornl stimulus such as dehydration or an int,ravascular injection of hypertonic saline or calcium chloride? -in intriguing question is what inducts t,ht) pituicytes to start engulfing the “normal-looking” axons 2 days after the transection? Why does the engulfing not take place earlier after t,hc transection or in the normal glands? 1)ellmann and Rodriguez (1970) have olj.-erved that within the first 2 days following transection that distal stump has numcrous fibers loaded with tubular formations and that the extracts of t’hese stumps IIAT~Can increased vasoprrssor activity. The authors po&lated that this increased vasotoein content was due to a backflow of ~~xtragranular hormone, from the neural lobe to the distal stump and that the tubular format,ions might play a role in this flo~v. The prcsencc of similar tubular fornultion:: in some nerve fibers and nerve endings of the neural lobe observed in the sanle postoperative period would support that hypothesiw. As in the first phase, the pharmacological ant1 morphological events taking place during the rccond phase (between 6th and 9th po>toperativc days) are in complete agree-
OF
DISCONNECTED
SEURAL
LOBE
287
ment. The ultrastructural study shows that after 9 days of transection only a minority of the fibers does not undergo degeneration, and the bioassays reveal that the glands extracted at the same postoperative interval only contain about a tenth of their normal hormone content. The “filled” intercellular spaces which are 401bserved in the subependymal region of the normal glands and throughout the disconnected neural lobes may be artifacts produced by the hyperosmolarity of the aldehyde mixture (about 1200 mOsM ; for details see Rodriguez, 1969), which might promote the transport of substances from the nerve fibers into the intercellular space. The preferent,ial subependymal localizati80n of these spaces in the normal glands may be due to the fact t,hat the fixative was injected intraventricularly. The generalized localizat’ion of t’he spaces in the experimental glands could be due to the fact that the transected axons are more easily affected by a hyperosmolar fixative than the intact fibers. The integrity of the elementary neurosecretory granules in fibers surrounded by filled spaces, and the high development of these spaces in glands with practically no hormonal act,ir&y would indicate t’hat the material filling t’he spaces does not have any hormonal activity. The striking changes presented by t,he pituicytes during the second phase, namely the considerable increase in the number of Golgi complexes and t)he formation of well organized canaliculi strongly suggest’ that, the reorganized pit,uicytes may have some new function(s) after t’hey have finished the process of engulfing and degradation of the disconnected axons. This pituicytic transformation, and the simultaneous appearance of a second t’ype of cell seem to indicate that’ the chronically disconnected neural lobe is not a dead st’ructure but. that it may have some functional significance. What exactly this significance is appears as an interesting t)ask for future investigations. REFERENCES J. (1952). The quantitative assay of vasopressin.Btit. J. Pharmncol. 7, 567-572.
DEKANSKI,
H.-D., AND OWSLEY, P. A. (1WX) ln\ estigations on the hypothalamo-nerrrolI?;pophysial neurosecretory system of the grass frog (Ram &Gens) after transection of the I~rc)uirnal nuerohypophysis. I. Light microscopic findinpb in animals kept at 18°C environmental Icwrl~,~r:rtUrr. %. Zellforsch. Mikrosk. 4nut. 87, 1 -Ifi. I~LMANN, H.-D., AND OWSLEY, P. A. (1969). lnt11c vcstigations on h?potlral:lmo-llerr~olr~poyhysial neurosecrctory r;ystem oE the grxs~ frog (~~anrc pipiens) aftc,r trxnsection oi‘ thr. proximal neurohypophysis. II. T,ight ant1 vlw.Won microscopic findings in the tlia~onne~tc~d distal ncurohypophysis with special c’nrlllr:i,+ on thr I)ituicytes. Z. Zellfo7xzh. ilfikl-osl; .l,r~ii. 94, 325-336. DIII,IX.~NN, H.-D.. .~NLI RODR~GCEZ. E. M. (1070). Inwstigntions on the liypothalnmo-ncurolr?-pophysial nfwrosccrctory system of t,he gri~b> lrog (Rtrnfr TGpierw) after transection of the, proximal nc,uroh~pclpll3-sis. III. Ultrastrurlurr~ and hormone contt,nt of the dist:rl strrmp, I,r “Aspects of Neuroc,ndocrinology” (W. Rargmnnrr and B. Schnrrer, Eds.) Munic*h. Bcrgmann. hX.l~~l.~NS. H.-D., T~.~r,r~. IT. E.. C~HLIIY. I’. ~1.. ASD T~~r,r~~r~)~r~. T,. 1:. (196s). ~foq)hologic:d and func,tionnl changes in the Ili;;tnl lr~~)~)ill:~l:r~r~c)neurohypophysinl system of tlrc gr:ws frog (IL)ow +[~~cJw) after trnnswtinn of Ihr I~rosimnl rl~rrrolrvpol)il?-sis. Esp~rir~~tl~r 24, 383-3X6. GI,AUERT, ,4. M.. ASI) GL.\~~;RT, R. H. (19.5~~. i1raldit e as an embedding medium for elrct.ron rriic7wxopy. J. Biopl~~ir. Biochcm. (Yytol. 4, 191194. &I.IJ:R. H.. .