Article on enlargements of active stalactite tips

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Fig. 1. Enl::irgcmcnts of active stalactite tips. (A) Drop on staiactitc tip· (diamcicr ·

11 mm). (13), (C), and (D) Enlargements of (A) (X ~). Striped areas indicate calcium carbonate.

tites, and other limestone structures. of crystals formed in

a

long arc, along With a special microscope, mounted hyphac which connected at both ends

horizontally on an adjustable bracket, with the stalactite (Fig. 1 A). Thus the "sliding along a v.;rtical steel bar, stalac- hyphae function both as crystallization tites were observed i11 situ. As in tclc- nuclei and as attachment, without

scopes, the eye piece on -the horizontal which· individual crystals would be microscope was attached at a right eliminated by the falling drop. In one angle by means of a prism, making it ease a cotton fiber was attached to the possible to observe even in tight spots. tip of a stalactite, and all along its A flashlight was mounted near the length, as far as it was coiled up· and

microscope to illuminate the stalactites. immersed in the drop, a row of crystals Along the limestone surface of all covered the fiber: From it, sc'tondary

active stalactites, short very thin hyphae strings of crystals emerged, apparently

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---growth of stalactites, which, if entirely

dependent upon accretionary growth of

preexisting crystals, do r.ot grow straight. clown, as found in the case of

hclictiics.

Cultures were made on nutrient agar

of the microorganisms living in and on active and inactive stalactites. Cultures

from the terminal drop and the active

rim of the stalactite gave, in five out of six cases, pure cultures of Cephalo-.l'fJOri11m lwndlaecola F. E. Smith, a slow-growing fungus with very fine

white mycclium; in one case bacterial

colonies developed in addition to the fungus. In another test, 42 out of 46

cultures from stalactite tips produced Ceplwlosporium. Of the I 00

micro-organisms developing on hclictites, not

a single one was Ceplwlosporium. In the active stalactites investigated in Lehman Caves the same fungus was

associated with. them, and · no other microorganisms, and certainly no bac-teria, can be i_nvolv.cd if1 their growth. The nutrition of these fungi must de-pend on organic materials that are leached from soil and move clown with the calcium bicarbonate solution

seep-ing into the cave. All calcium

carbon-ate deposits of known biological origin

in oceans (corals, algae, ::ind molluscs) are attributed to the activity of an

en-zyme, carbonic anhydrasc, produced by these organisms .

F. W.

WENT Desert Research ./nstitllte,

University of Nevada System, Reno 89507

Note e-".tcnded perpendicularly (Fig. ID). along hyphae.

Some were branched and had co·nidio- It is conceivable that tl1e presence I. Supported by National ·rark Service.

13 ,\lay 1969; revised 26 June 1969

was found only in the surface layer of

phores. On dissolving the limestone l ) f esc hyphae ::illows the orderly

with dilute acetic ::icid, the m y c e l i u m h ~ the stabctites, and only sparingly. Some

hyphac extended into the surface o f J /

.Brain

Lesions in an

Infant Rhesus Monkey

w::itcr drop hanging from the stalacxit

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Whereas some of the growth of

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Treated

with

Monosodrnm

Glutamate

stalactite surface was obviously by

ex-tension of the existing surface crystals

downward into the drop surface, a l::irge

number of smaller and larger crystals and crystal aggrcg::ites were dispersed. in the drop surfocc, having no continu-ity with the crystals of the stalactite

(Fig. 1,

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::incl C). Yet in some way the crystals were attached: 'When individual

stalactites were observed over a 24-hour period, the unattached crystals

ret;1ineJ the same relative position (1

to 20 ft distance) to the stalactite tube. In a number of cases hyphae were seen to connect these crystals to the body of

the stalactite, and in other cases strings 386

Abstract. 111 an infant rl1esu.1· monkey brain dalllage resulted from

subcutane-ously administered 111011osodi11m gl11ta111ate. Although a relatively high dose of mo110.wcli111i1 gllltamate was used, the i11fa11t was asympto111atic for a 3-hour ob-servatio11 period during which ti/Ile hypothalamic neurons were 1111dergoi11g a process of acute cell death. IVitlz the electron microscope it was observed that dc11drites a11d cell bodies of neurons are the tissue col/lponents prinwrily allected

i11 brai,1 dalllage induced by 1110nosocli11m glutamate.

Susceptibility of the developing cen-tral nervous. system to damage from

subcutaneously administered mono-so_dium glutamate (MSG) has been ob-served in every species of experimental animal tested thus far-mice (/, 2), rats (2, 3), and rabbits (4). In mice,

which have been studied more exten-sively for MSG-induced brain damage than other species, the lowest cfTcctivc

dose for the baby a.nim;il (0.5 g/kg)

was approximately one-tenth that for

the adult (5 g/kg) (2). Additional

studies arc needed to clarify mcch::i-SCIENCE, VOL. 166

nisms t1nderlying the J\JSG effect and to elucidate the basis of enh:1nccd vul-nerability on the part of the immature ncrvot1s system. In the meantime, the

question arises whether glutamate could

have- an occult etiologic involvement in any of the unexplained brain damage syndromes occurring in the course of human ontogcncsis and whether the

widespread practice of feeding

gluta-mate-enriched diets to human infants is a wise one (5). The feasibility of studying these questions in the primate·

is suggested by our e\'idcnee that the

infant rhesus monkey (Macaca nz11/ata)

is susccp_tiblc to glutamate-induced

brnin damage.

Our report is based on ouly · one test subject because we were unable to

ob-tain additional baby monkeys at this

time. However, the pattern of neuronal necrosis induced in the hypothalamus

of experimental animals by MSG is

highly selective for certain· cell types

and has a very distinctive appearance.

Furthermore, as a frame of reference,

we have extensive light ancl electron

microscopic data pertaining to the

evo-lution of this type of lesion in the

retina (6) and the hypothalamus (7)

of numerous rats and mice. The fact

that the margins of the MSG lesio·n

arc sharply demarcated was helpful for evaluating fixation variables with the

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electron microscope because nonn::il,

well-fixed cells of every kind typical for a given region could be examined, just beyond the m:irgin of the ·lesion, for

comparison with degenerating cel!s

within the dari1agccl area.

\Ve separated an infant rhesus

mon-key from its mother S_ hours after birth';

the infant was an alert, healthy-app e:u-ing male with an active cry and appro-.

priate spontaneous motor activity.

How-ever, it weighed only 260 g and

mea-sured 16.5 cm fr~m crown to ri.Jmp, so that, judged by size, it would prob-ably be classified as :i premature infant

(8). Glutavcne, a commercially

avail-able preparation of 1v1SG in 25 percent aqueous solution, was injected

sub-cutaneously in a volume of 2.S ml, the

total dose being 0.7 g or 2.7 g per

kilogram of body weight. The treated

infant was then held and cared for

in a maternil manner (but. not

pro-vitkd with food} for a 3-hour·

observa-tion period; d\1ring this time there ,verc

. no manifestations of a central nervous

system disturbance. Three hours after

treatment the infant was given l mg

· of Sernylan (Parke, Davis) intra

-muscularly, which provided excellent anesthesia characterized by a deep sleep ·with loss of responsiveness to painful stimuli but with retention of full rhyth -mical respirations. Thoracotomy was

then performed so that a cannula could

be clamped into the ascending aorta,

Fig. 1 (top). Cross section of the ventral hypothalamus cutting through the infun-dibular stalk. The lesion (LES) affects

the pcriventricular-arcuate regions bil::lter-ally, giving these areas a rarefied appear-ance. A "Swiss cheese effect" is crcatccl by the dilatation of de.nclritic processes. The

larger holes and open spaces arc dilated bloocl vessels resulting from perfusion fixation (X 50).

Pig. 2 (bottom left). An electron micro-graph showing a massiYcly dilated dcn-dritic process (d) in syn:1ptic contact with

a normal-appe_aring axon tcrmin:11 (a).

The internal content of the clcnclritc con-sists primarily of cliITusely distributed particulate debris. The axon is not swollen

and ·contains numerous synaptic vessicles

and normal-appearing mitochondria (X

10,300).

Fig. 3 (bottom right). An electron

micro-graph of two degenerating neurons (a and

b) illu~trnting alteration of nuclear chro-matin pattern (,1rrnw, a) and disinteg

ra-tion of cytoplasmic components. The mem -brane system comprising the endoplasmic reticulum has degenerated beyond recog-nition and mitochondria hnvc either

rup-tured or become completely spheric-al ( X

6000). 387 I: I

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.nd perfusio11 of tlie br:1in was begun within 30 ~cc:onds. The perfu~atc con-sisted of 3 percent glutaraldehyde in 0.IM cacodybte buffer and 0.02 per-cent Caci;. After 20 minuks of perfu-sion, the br,iin, pituitary [:land, eyes, and optic ncr\'CS were removed and placed ·in jars containing the perfusion fluid. Areas of special interest were dissected out from these tissues which were then fixed further in osmium tctroxide for 2 hours, dehydrated in graded ethanols, and embedded in Araldite after an intermediate stage in toluene. Sections

1 /J.m thick were cut witi1 glass knives (0.95 cm) and stained for light mi-crosc:opy (9). Sites of lesion formation identified by light microscopy were ex-amined 'with the electron microscope in ultrathin sections prepared from the same block.

A lesion affecting the

periventricular-arcuatc region of the hypothalamus and essentially identical in light microscopic appearance to the form of pathology seen in mouse brain after. MSG treat-ment (2, 7) was readily apparent (Fig.

1). Electron microscopic examination e5tablished that the cellular constituents primarily afiected were dendrites and cell bodies of neurons. Many synaptic complexes could be found in which the postsynaptic (dendritic) component was massively dilated (Fig. 2). These processes were either empty or con-tained degenerating organelles and dif-fusely distributed particulate debris. 1l1e prcsynaptic component (axonal) of such complexes was usually unaffected, as were axon bundles passing through the region of injury. Many neuronal cell bodies were swollen with intra-cellular edema and, in ~ome, 'the cyto-plasmic organelles appeared to have undergone a Jytic process, while nuclei showed marked alterations in chroma-tin pattern (Fig. 3). A mild

intracel-lular edema of the cpendyma was evi-dent, but this was not accompanied by degenerative changes in either nuclei or intracellular organelles, and no altera-tions were noted in the appearance of· junctional complexes between epen-dymal cells. No structural alterations

· were detected in glial or vascular

com-ponents to suggest involvement of these elements in the pathologic:il process. The lack of symptoms in this primate infant during the time when a small pcrcent.-igc of its br:1in celJs were being destroyed is evidence of a subtle proc-ess of brain damage in the develop-mental period, which could easily go unrecognized were it to occur in the

388

human infant under routine circum0

stances. However, a high dose of MSG was used to produce brain damage in this neonatal monkey, an<l it was

ad-ministered by the subcutaneous rather

than oral route. Thus, while we have demonstrated susceptibility of a pri-m:itc species to the mechanism of the glutamate efTcct, it remains to be seen

whether this mechanism can be

trig-gered by any set of naturally occurring circumstances. Presumably, an elevated blood concentration of glutamic acid is an important prerequisite to lesion formation.

In attempting to evaluate the risk of

glutamic acid blood concentrations rising high enough to produce brain damage in· the human infant, it is im-portant to recognize that the oral dose of :MSG is but one among several potential determinants of glutamic acid concentrations in the blood. Other· factors, such as circadian periodicity (10), viral infection (11), immaturity -of enzyme systems, _rapid . absorption from an empty gastrointestinal tract, and individnal variations in metabolic capabilities could act in concert with a high glutamate diet to produce much

higher conccnfrations of glutamic aciJ in an infant's blood than might be ex-pected were such. factors O\'erlookcd.

Jo!IN

w.

OLNEY LAWRENCE G. SIIARl'E

Department of Psychiatry, Washington University School of

M cdicine, St. Louis, lv!is.wuri 63 I 10

Rdcrtnccs aud l\'otcs

I. D. R. Lucas and J. P. Newhouse, Arel,.

Optlialmol. SS, 193 (1957).

2. J. W. Olney, Scimce 164, 719 (1969).

3: J. K. Freedman and A. M. ·Polls, lm·cst.

Op/11ha/111ol. 1, ! IS (1962).

4. J. \V. Olney, unpubli,hed data.

S. Monosodium glutamate is the sodium salt

of glutamic acid, an amino acid found as

a protdn con~titocnt in the nurm:il did.

It is also added as a navoring agent to a

variety of commercially prepared foods,

in-cluding nearly ati brands of baby' food.

6. J. \V. Olney, J. Ne11ropatlwl. Exp. Ne11rol.

28, 455 (1969). 7. - - , in prc.-paration.

8. S. R. Napier, and P. 11. Napier, A

llqnd-book of Lfring Primates (Ac:idC"mic Press,

New York, 1967).

9. K. C. Richardson, I.. Jarrett, E. H. Finke,

Stai11 Tec/1110/. 35, 325 (1960). ·

10. R, D. Fcigin, Amer. J. Dis. Child. 117, 24

(1969).

11. - - , R. F. Jaeger, R. \V. McKinney, A.

C. Alcvintos, Amer. J. Trop. Med. Jiyg. 16,

-769 (1967). . •

12. Supported in part by PI!S grants MH38S94,

!-,!H07081, and J\lHL\002. We thank Dr. E.

Robins for advice in prepar3tion of the

manu-script.

29 July 1969 ·

_•

Lutciniziug Hormonc-Rclensing Acfo

,

Hy

in

Hypophysial Stalk

Blood

and Elevation by Dopamine

·

.Abstract. Pituitary halves inc11bat1•d. in pituitary stalk plasma release more luteinizing hormone than their opposite halves incubated i11 plasma from periph-eral blood. Glands incubated in stalk plas111a from dopamine-treated rats re-lease 111ore luteinizing hor111one than glands incubated in stalk plas111a from un-treated controls. L11tehzizing lzor111one-rcleasi11g activity in stalk plamw may be d11e to the luteinizing hormone-releasing factor, and the secretion of l11teinizi11g lzor111011e-relcasing factor 11111y be controlled by a dopa111inergic 111ccl1ani:im.

Adrcncrgic and cholinergic mecha-nisms arc thought to be involved in the regulation of gonadotropin release from

the anterior pituitary (1). For example,

monoamine oxidase (2) and cholin-esterase (3) activities and the mono-amine content ( 4) of the hypothalamus vary during the estrous cycle and at other times when there arc changes in the production of ovarian or testicular steroids, as during pregnancy and after castration. It has been shown by means of a histochemical lluoresccncc tech-nique (5) that monoamines arc pres-ent in high concentrations in several regions of the mammalian nervous sys-tem and that adrcnergic nerve termi-nals arc especially dense in the hypo-thalamus near the median eminence

(6). Recent results indicate that dop-amine stimulates the release of gonado-tropins from pituitaries incubated with hypothalamic tissue in vitro (7).

\Ve observed that in rats the con-centration of luteinizing hormone (LH) in systemic blood increases after the injection of dopamine into the third ventricle of the brain (S). When the anterior pituitary was perfused direct-ly with dopamine by means of a micro-c:rn nu la inserted into a pituitary stalk p_ortal vessel (9), so as not to involve the hypothalamus, LH release was un-affected ( S). We now report that dop-amine increases LH rclc:ise by stimu-lating the secretion of luteinizing hor-mone-releasing factor (LRF).

Jrypophysial stalk blood was