Electron microscope radioautography of amino acid incorporation by supraoptic neurons of the rat

GE:ENERAL AND COMPARATIi’E

ENDOCRISOLOGY

15, 477-495

(1070)

NOTES

Electron

Microscope

Incorporation

Radioautography by Supraoptic

Neurons

of

Amino of the

Acid Rat

Electron microscope radioautography of the neurosecretory supraoptic nucleus am1 neural lobe was conducted after intracisternal injection of three tritiated amino acids: cysteine, arginine, and glycine. into dehydrated rats. Cytoplasmic uptake and localization are similar for the three amino acids: first over the endoplasmic reticulum and later over the Golgi region and elementary granules. Considerable activity was also found in the nucleus. After a longer period of time, act,ivit,y was detected over some nerve fibers in the neural lobe. Current concepts of subcellular events that occur during the secretory process in protein-secreting cells are based in part on radioautographic studies conducted at the electron microscope level on the exocrine cells of the pancreas (Caro and Palade, 1964; Jamirson and Paladc, 1966, 1968). Observations were later extended to other protein-secreting ccll~:, and also to ordinary neurons (Droz, 1969). However, radioautographic st’udies on the specialized protcin-sccrcting neurosccretory neurons haoc been pursued only at the light-microscope level in a few vertebrates and invertebrates (Sloper et al., 1960; Tijrk et al., 1966; Flament-Durand, 1967; Lcatherland and Dodd, 1969). Insufficient resolution at this level prevents visualization of the intracellular pathway of synthesis and the fat’e of the neurosccretory material. In view of this deficiency, we have employed radioautographic techniques at the level of the electron microscope in order to follow the incorporation of radioisotope-labeled amino acids into the subccllular components of the supraoptic neurosecretory neurons of the dehydrated rat. Three amino acids present in arginine vasopressin, a hormone known to be synthesized by these neurons (Sawyer, 1967), 477

were used: n-cystine-3H (1.0 Ci/mmole) later reduced to cysteine according to Sachs (1963) ; n-arginine-3H (10.0 Ci/ mmole) ; and glycine-3H (4.6 Ci/mmole) In order to achieve a high rate of incorporation, water was withheld from adult male Fischer rats, weighing 180-220 g, 48 hours prior to injection of the isotope. One millicurie of a single amino acid (cyeteine = 120 pg, arginine = 17.4 pg, glycine = 16 pg, each in 0.1 to 0.15 ml aqueous solution) was injected intracisternally into 7 rats under light, ether anesthesia according to the method of Lindbcrg and Ernster (1949). Three rat’s, one from each isotope group, were killed 15 min after injection; 3 ot’hers were killed after 1 hour; and one injected with arginine was killed after 6 hours. All rats were given 95% oxygen-5C/c carbon dioxide to aid recovery from the state of shock which ensued within a few minutes after isotope injection. This condition was most pronounced and of longest duration in the cysteine-injected rats. Supraoptic nuclei and neural lobes were fixed by immersion of the hypothalamic region in 2% paraformaldehyde and 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.ij, for 18-24 hours, at room tcmper-

478

479

SOTEH

ature. After post’fixation in cacodylatebuffered osmium tetroxide, the tissues were embedded in Epon. Thick sections were coated with Ilford K-5 emulsion and exposed for 1-12 weeks. Silver to golden sect,ions (8OCrlOOOX thick) were mounted on naked grids, coated wit’h Ilford L-4 emulsion according to Caro and Tubergcn (1962), and exposed for 1-8 weeks. Thick sections from arginine- and cyst,eine-injected animals revealed a high concent’ration of grains over the supraoptic neurons, but considerably less over t’he optic chiasm, adjacent nonneurosecretory TABLE ~ISTRIHTTIOK

OF &I.VEK

Glf.41Ns

Ovlir(.

the nucleus, some associated with the nucleolus. In rats exposed to label for 1 hour, many grains were evident over the Golgi membranes and associated developing neurosecretory granules, although a considerable number of grains was also present, over the endoplasmic reticulum (Figs. 4 and 7). Neurosecretory axons near the supraoptic cell bodies and other nerve fibers were only lightly labeled. Table 1 gives the distribution of radioautographic silver grains over various cell components after the administration of cpsteine and arginine. 1 COMPONICNTS

OF SUPKAOI~TIC

VP Grains

Label

I>llrat,ion labeling

of

Number of grains count,ed

~-EcKONS

overlying -

Nluzleus

XucleohW

Endoplasmic reticulum

Golgi and neurosecretory

elementary granules

Arginine-“H Arginine-3H Cyst,eine-3H Cysteine-“H ‘b Nllcleolus

15 1 1.5 1 present,

min hour min horlr oldy

959 887 723 670 in one-third

20

3

58

21

5

35

19 39

22 25

3 4

65 40

31

of micrographs

neurons, glia, and capillaries (Figs. 1 and 2). After glycine injection, the concentration of label over the neurosecretory neurons was also intense; however, other hypot’halamic neurons, glia, and connective tissue cells were also labeled appreciably. In electron micrographs, heaviest grain deposit:: were over the endoplasmic reticulum with a comparatively small number in the Golgi regions in the neurosecretory cells of rats exposed t,o label for 15 min (Figs. 3, 5 and 6). Many grains were present over

10

couuted.

Whereas thick sections of the neural lobe from animals exposed to labeled arginine for 1 hour showed only a few scattered grains, primarily over the nuclei of pituicytes, a higher concentration of grains was seen around capillaries of the rat exposed for 6 hours (Fig. 8). Electron microscope radioautography of the neural lobe demonstrated that, some neurosecretory axons showed radioactive label in the elementary neurosccretory granules and clear smal I vesicles 5OOk in diameter (Fig. 9).

thick section coated with Ilford K-5 and exposed for 12 weeks. Note strongly radioact,ive supraoptic neurons !SOS). OC, optic chiasm; ZC, branch of internal carotid. X71. Fro. 2. Methylene bllle-aaure-stained, 1 p thick section of supraoptic neurons of a rat, labeled for 6 hours with arginineJH and exposed for 1 week. Note heavy grain deposit over nucleoli and variation in density of grains over s~lpraoptic neurons. X475. Fro. 3. Electron micrograph of supraoptic neuron labeled with arginine-3H for 15 min, coated with Ilford L-4, and exposed for 6 weeks. Most grains occur over endoplasmic reticulum (BR). G, Golgi systems; L, lysosomes; I\‘, nuclells. FIG. 4. Electroll micrograph of supraoptic neuron labeled with arginirle-3H for 1 hour and exposed for 6 weeks. i\Iany grains are associated with Golgi systems (G), but many grains also still occur over the endoplasmic retic~dum iERj. N, nucleus; *VU, nucleolus.

NOTES

Early attempts to study incorporation of labeled amino acids into brain proteins resulted in only minimal incorporation after the label was injected intravenously. However, when amino acids were injected intracisternally, much greater incorporation occurred (Friedberg et al., 1948)) suggesting that the blood-brain barrier inhibits the transport of amino acids into t’he brain. This inhibition of transport thwarted our initial efforts to label ncurosecretory neurons for elect,ron microscopy. IIre have t ricd in vivo methods (intrapcritoneal and internal carotid artery perfusionj and in vitro methods (incubation of hypothalamic slices in various cult’ure media), but failed by any of these methods to achieve enough incorporation of label to allow rlectron lnicroscopc radioautography. The activity of xupraoptic neurons resulting from intra(*isternal injection is probably due mainly to incorporation of the labeled amino acid into newly synthesized protein, because free amino acids would be washed out in lhe course of tisruc processing for electron microscopy. Caro and Pnlade (1964) report, that, 91-96s of newly formed pan(areatic acinar proteins are retained under these conditions. Even taking into account the llo~sibility of some nonspecific binding of labeled amino acids (Peters and Ashley, 1967): the changing ratio of uptake by the perikaryon of 15min and l-hour I:~hc~lctl rats and the few grains evident over the neural lobe of the shorter term rats, suggest that nonspecific binding alone is unlikely. All the amino acids showed a similar pattern of uptake and localization. Incubation of cysteine-labclctl tis$uC in

481 thioglycolate did not affect the distribution of grains. Thus, cysteine incorporation into protein is presumably not by disulfide bonding but by peptide linkage, as should be true for arginine and glycine. These preliminary results suggest that the schema of endoplasmic reticulum and Golgi apparatus participation in secretory granule formation, already demonstrated in pancreatic acinar and other cell types, also holds for neurosecretory supraoptic neurons. Previous studies on ordinary neurons showed a similar pathway after administration of labeled amino acids or galactose (Droz, 1967; Droz and Koenig, 1969). Our limited data arc in accord with the pattern of synthesis of neurosecretory materials presented by Sachs et cd. (1969). However, at the present time, it, is impossible to state whether the activity seen in the cytoplasm is associated with synthesis of the neurohormonc argininc vasoprcssin, or with the carrier protein neurophysin, or with both, inasmuch as both entit,ics contain t,he three amino acids used hcrc+l (Uttenthal and Hope, 1970). Scott et al. (1970) found high levels of saline incorporation by the cat hyy)othalarnoneurohJ-pophyseal system at the light microscope level and suggested that this amino acid could seryc as an indicator of neurophysin synthesis. Our present results do not agrc’e with t’hose of Roux (1969) , who found incorporation of labeled arginine throughout the neurosccretory tract even after 15 min, as opposed to the perikaryal localization of cyst&e. The 23-29s uptake by nuclear comI)onentP c(>rtainly reflects synthe+ of ma__..

VI{:. 6. E;lectroIl micrograph of supraoptic lIeuron labeled with arginine-SH for 1.5 min and exposed for 8 weeks. Grains occur over the endoplasmic reticulum (ER) (cf. Fig. 3). G, Golgi system; N, nucleus. Fro. 7. Electron micrograph of supraoptic neuron labeled with arginine-3H for 1 hour and exposed for 6 weeks. Many grains occur over Golgi cisternae (G) and developing element,ary nerlroserretoly gratlules ! YG). Some grains occur over the endoplasmic ret,iculum (ER). Flc:. 8. Methylene blue-azure-stained, 1 r-thick sect.ion of neural lobe of a rat labeled for 6 ho~lra with nrginine-“H and exposed for 12 weeks. Clusters of grains occur around capillaries (C) where neurosecretory axons terminate. X500. E’I~. ‘3. Electron micrograph of neural lobe of a rat labeled for 6 hours and exposed for 8 weeks. Silver grains occur over axons containing elementary neurosecretory granules (IVG). C, citpillary; P, pituicyt,e; SB, small vesicles.

482

NOTES

terials other t,lran ncurosecrctory products themselves. Accordingly, part, of the activit,y detected in the cytoplasm may bc the result of incorporation of labeled amino acids into ribonucleoprotein or membrane components, or their transformation into membrane phospholipids. Edstriim et nl. (1961) have shown that individual supraoptic neurons of the rat, shon- twice ah much RNA after dehydration a~ do unstimulat.cd neurons. Nevertheless, the similarity of t,he pattern with all three amino acids and t,he changes with time suggest important incorporation into the hormonc~ and carrier prot8ein. Persis:tent, activity over the cndoplasmic rcticulun~ OIW or mow hours after tritiated amino acid injection is to ~J(J expected from the continuing UIJtake and incorporation of the label from the ccrebrospinal fluid. Periods of incubation shorter thall thaw usccl lrcrcin :ulcI pulse-chasing cqcriments ill taifw ahoultl be uwful in clarifying the pattern of incorporation by t,hc, various ccl1 cornponcnts. The finding of appreciable amounts of axons in the grains over ucuroscrrctory neural lobe aftor 6 hours indicates that the transport of labeled mat’erinl from t.hc soma may br faster than was wident in cxpcriincnts with light-miaroprcviow scope radioaut,ography (Slopw et al., 1960). Pickering autl Jones ( 1970 I h:trc fouutl that, after int,racisternal injection of labelctl tyrosine, labeled vasopressin and oxytocin begin to arrivck in the neural lobe aftcl. about 2 hours. The

olwcrvatioii

that

not

nil

the

neuro-

secrct,ory axoi~r showed labeled m&ri:tl after 6 hours of argininc injection supports previous results of Howe (1959) wit’11 histochemical tests for arginine and suggests the possibility that vasopressin and osptocin are produced and secreted by diffrrent neurons (Hellcr, 1961); inasmuch as present in vasothis amino acid i:: only prcssin. The lack of labeled material in some

ncuroscrwtory

terminals

can

also

be

attributed to :I lower activity of somts neurons andj’or discharge of labeled wcretion prior to termination. Thc~ pwscncr of

a&vity

over

the

cltar

small

vcsic~lw;.

483

NOTES Caldeyro-Barcia and H. Hellcr. eds.), pp. 3-23. Pergamon Press, London. HOWE. A. (1959). The distribution of arginine in the pituitary gland of the rat, with particular reference t,o its presence in “neurosecretory” material. J. Physiol. (London) 149, 519-525. JAMIESON, J. D.. AND PALADE, CT, E. (1966). Role of the Golgi complex in the intracellular trnnsport of secretory proteins. PWC. Ntrt. Acad. Sri. U. S. 55, 424-428. JAMIESON, J. D., :AND PALADE, G. E. (1968). Intracrllular transport of secretory proteins in the pancreatic exocrinr cell. III. Dissociation of intracellular transport from protein synthesis. J. Cell Biol. 39, 580-588. ~~ATHERL~.VD, J. F.. .~ND DODD, J. M. (1969). Aciirity of the hypothalamo-neurohypophysial complex of the european eel (Anguilla anguilla I,.) assessed by the use of an in sit11 staining tc~c~hnique and by autoradiograph~. Grn. Camp. Et1doc1.ino/. 13, 45-59. LINDBERG. 0.. ASD ERNSTER, I,. (1949). The turnover of radio:uti\-e phosphate injected into the subarachnoid space of the brain of the rat. Biochem. J. 46, 43-47. PETERS, T.. AND ~IILEY, C. A. (1967). An artifact, in radioautography due to binding of free amino acids t,o tissues by fixat,ives. J. Cell Biol. 33, 53-60. PICKERING. R. T.. AN) JONES, C. MT. (1970). The isolation of labeled neurohypophysial hormonrs from rat neural lobes after an intracistcrnal injection of 13Hltyrosine. J. Endocrixol. 42, vi. Roux. M. (1969). IZtude histo-autoradiographique de l’incorporation de ‘H-arginine dans l’hypot,halamus neuroskcretoire et le lobe nerveux de l’hypophysc chez la souris albinos normale et deshydratee. Arch. Anat. Microsc. dlorphol. Erp. 58, 311-318. .SACI~S, H. (1963). Vasopressin biosynthesis. II.

Incorporation and protein

of [““Slcysteine into vasopressin associated with cell fractions. J. Neurochenz. 10, 299311. SACHS. H.. FAWCETT, P., TAKABATAKE, Y.. .~ND PORTANOVA, R. (1969). Biosynthesis and release of vasopressin and ncurophysin. Recwt f’rogr. Horm. Res. 25, 447-492. SAWYER. W. H. (1967). Evolution of antidiuretic hormones and their functions. Amer. J. Med. 42, 678-686. SCOTT. D. E., WEINDL, A., AND JOT-XT, R. J. (1970). Autoradiography of the hypothalamoneurohypophyseal system. Anut. Rec. 166, 375. SLOPER. J. C.. ARNOTT, D. J., AND KING, B. C. (1960). Sulfur metabolism in the uituitarv and hypothalamus of t,he rat: a study of radioisotope-uptake after the injection of “‘S DLcpsteine. methioninc. Xld sodium sulf:&. /. Ewlocrinol. 20, 9-23. T&K, I.. AROS, B., KISS, J., AND VIGH. H. (1966). Morphological examination of the metabolism in the secretory cells of the nervous system. I. Autoradiographic study of the incorporation of ““S-labellrd cysteine and methionine into the murosrcretory system of the earthworm (E&e& joetida, Lumbricus hercules). Acta Biol. (Hung.) 17, 185198. UTTESTHAL, L. 0.. AND HOPE, D. B. (1970). The isolation of three neurophysins from porcine posterior pituitary lobes. Biochem. J. 116, 899909. RICHARD S. NISHIOKA DAVID ZAMBRANO HOWARD A. BERN Department Research University Received

of Zoology and its Cancer Genetics Laboratory of California, Berkeley 94720, JzLne 14, 1970