Neurons in Rhesus Monkey Visual Cortex: SystematicRelation between Time of Origin and Eventual Dispositi

Abstract. Autoradiographic evidence after injection of tritiated thymicates that cell position in the laminae of the monkey visual cortex is systrelated to time of cell orgin. The earliest-formed neurons, destined for t,stratum, arise at about embryonic day 45, and the last ones, destined formost cell stratum, form at about day 102; cells of intervening layers areat intervening times. No neocortical neurons are produced in the last twmonths or after birth. Compared to cortical neurons in rodents, th(monkey arise earlier, and the "inside-out" relation of cell position torigin is more rigid.

Morphological and physiologicalstudies of the visual cortex in the mon-key have increased our understandingof the organizational properties of thecerebral cortex in general (1). It is im-portant to determine how such a com-plex and ordered system develops.Almost 100 years ago, it was demon-strated with the use of the Golgi methodthat cellular development of the mam-malian cerebral cortex follows a con-sistent pattern, with large pyramidalneurons of the lower layers taking alaminar position and differentiatingearlier than neurons destined to besituated more superficially (2). More re-cently, it was shown by autoradiographywith [3H]thymidine ([3H]dT) that thedeeper cortical neurons in rodents aregenerated earlier than the more super-ficial neurons (3). In addition, thesestudies provided direct evidence thatcortical neurons originate in prolifera-tive zones close to the ventricular sur-face and migrate to the cortical plateonly after final division of the precursorcells.

It is essential to establish whetherthe neocortex in primates develops ac-cording to the same principle as inrodents and to determine the time spanduring which cortical neurons are gen-erated in a slowly developing gyrence-phalic brain similar to that of man. Inaddition, important questions concernthe generation and migration rates ofcortical neurons, the relation betweenlaminar position and neuronal type-with reference to time of cell genesis,and the possible interaction of youngneurons with each other and with the-specific afferents with which they willmake synaptic contacts.

Several properties of the primary vis-ual cortex (area 17) of the rhesus mon-key make it a suitable model for studyof these problems. The horizontal strat-ification of neurons into separate layersin this species is more precise than inmost mammals, and area 17 can be dis-1 FEBRUARY 1974

tinguished from adjacent are'tively early embryonic stages.size of monkey fetuses allowrfixation for electron micrcvascular perfusion (4), andtracted span of developmentthe resolution of temporal se(

neurogenesis (5). The preseistration of the time of neuroneach of the six cortical la

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first step in the more detailed analysisof neocortical cell genesis in primates.

on Ten pregnant monkeys weighing 6 to10 kg were each injected once intrave-

idine indi- nously with [3H]dT (5 or 10 mc pertematically kilogram of body weight) at 40, 45, 54,he deepest 62, 70, 80, 90, 102, 120, and 140 daysthe outer- of gestation. Females were caged withgenerated males from day 11 to 15 following

Po prenatal onset of estrus, and the time of con-ose in the ception was estimated on the assump-lo time of tion that ovulation occurred on day

12. Pregnancy in the rhesus monkeylasts 165 days. All fetuses were

as at rela- delivered normally and killed 2 to 5The large months after birth (the "juvenile" pe-

s adequate riod, when most cortical cells have at-iscopy by tained their final position). Two addi-the pro- tional monkeys were injected 2 and 18

t increases days after birth, respectively, and killedquences in 3 months after birth. All brains werent demon- processed for autoradiography (5, 6).iorigin for With the aid of a Zeiss microscopeLyers is a equipped with a drawing tube, the posi-

tions of heavily labeled neurons wererecorded within a strip of visual cortex

" approximately 10 mm long situated inthe depth of the calcarine fissure be-

*;Vil tween the two arrows in Fig. 1A. Onlyheavily lalbeled cells are considered tohave had their "birthday" on the dayof [3H]dT injection (5, 6). [In the in-terpretation of autoradiographic data,the term "birthday" is empirically de-fined as the last day on which nuclearDNA is replicated in a given cell line(6).] The minimum number of grains

4; for classification of cells as "heavilylabeled" was arbitrarily determined foreach specimen as half of the grain count

>̂ neurons with maximum radioactivity.Brodman's nomenclature of layers was

VI adopted (7).'t No heavily labeled neurons were seenkX( in the visual cortex of the 3-month-old

monkey that had been exposed to [3H]-.. I..,

Fig. 1. (A) Coronal section of the oc-IVB cipital lobe of a 3-month-old monkey.* The two arrows indicate a strip of visualz cortex about 10 mm long in the depth4 of the calcarine fissure, for which the

time of neuron origin was analyzed inthis study. The area in the rectangle isenlarged on the left side of Fig. 2. The30-,um section was stained with cresylviolet (x 1.2). (B to H) Autoradiogramsof the visual cortex in juvenile monkeys

*_t-~- that had been injected with [PH]dT atvarious embryonic (E) days: E45 (B),

f{^ E62 (C), E70 (D), E80 (E), E102 (Fand G), and E120 (H). Roman numerals

;4-:_ indicate cortical layers according to Brod-man's classification (7). Arrows point toheavily labeled cells; M, solitary pyramidof Meynert; WM, white matter (x 650).

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dT on embryonic day 40 (E40). How-ever, a few lightly labeled neurons, lo-cated exclusively in the deepest part ofthe cortex, were found in more than 50sections examined. Since heavily labeledcells were found elsewhere in the brain,these lightly labeled cells were probablythe products of several cell divisionssubsequent to the time of injection andit is unlikely that any visual corticalneurons were born at E40.The first heavily labeled neurons were

detected in the monkey injected at E45.They were localized in a narrow zonein the deeper portion of layer VI (Figs.1 B and 2A). Scattered neurons in the

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white matter below layer VI were alsolabeled in this specimen. In most fields,a number of lightly labeled cells weredetected superficial to the heavilylabeled cells, an indication that cellsgenerated later take up more externalpositions. This was confirmed by thefinding that heavily labeled neurons inthe animal injected at E54 were locatedsomewhat more superficially, althoughstill within layer VI (Fig. 2B). Themajority of neurons generated at E62were later situated in the upper two-thirds of layer VI, while some~ werelocalized in layer V (Figs. IC and 2C).Cells with axons passing from area 17

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Fig. 2. Diagrammatic representation of the positions of heavily labeled neurons inthe visual cortex of juvenile animals that had been injected with [H3]dT at variousdays of gestation (indicated at the top of each vertical line). On the left is a photo-micrograph of a 30-,um cresyl violet-stained section photographed at the same magnifi-cation used for plotting labeled neurons with the drawing tube. Horizontal markers

on each vertical line except G indicate positions of all heavily labeled neurons en-

countered in a randomly selected strip (2.5 mm long) of the calcarine cortex. The

three labeled neurons represented in G were found only after examination of 80 areas

of calcarine cortex, each 2.5 mm long, in 40 autoradiograms of a-single specimen.Roman numerals indicate cortical layers according to Brodman's classification (7);

LV, obliterated posterior horn of the lateral ventricle.

426

to the midbrain, the so-called giantsolitary pyramidal neurons of Meynert(8) which are situated in layers V andVI in the rhesus monkey (Fig. IC),were also labeled by injection at E26.

At E70, the number of labeled neu-rons per unit length of visual cortexreached a maximum (Fig. 2). Injectidnat this stage labeled mainly neuronsthat later were located in layer V, andalso many cells in layer IVC (Figs. 1Dand 2D). Neurons generated at E80became distributed over the entirewidth of layer IV, with the highest con-centration in layer IVB (Figs. 1E and2E); a few radioactive cells were situ-ated in layer III. Injection at E90labeled neurons in both layers III and II(Fig. 2F).By E102, almost all neurons in the

visual cortex had been born, since fewneurons (less than 1 in 106) at theborder between layer II and the cell-sparse layer I were la,beled in the 3-month-old monkey that had been in-jected on El02 (Figs. IF and 2G).However, in this specimen, some smallnuclei situated mainly in the deeperhalf of the cortex were radioactive;these were classified as glia on thebasis of small nuclear size, absence ofNissl substance, and satellite positionwith respect to neurons (Fig. IG). Thiscorrelates with the time of appearanceof numerous astrocytes in the depth ofthe fetal monkey visual cortex as as-sessed by the Golgi method (9). Injec-tions at El 20, later during gestation, orafter birth did not label neurons of thevisual cortex. However, numerous glial(Fig. I H) and endothelial cells werelabeled in the cortex in these specimens.The neurons in the plexiform layer I

were not labeled in any of the speci-mens studied. These neurons eitherwere generated before E40, or theyarose during a relatively brief intervalbetween the ages sampled by injectionof [:'H]dT in this series of animals.

As shown in Fig. 2, the position ofheavily labeled neurons in the juvenilemonkey visual cortex is correlated withthe time of cell origin in the fetus; cellsdestined for deep cortical positions are

generated first, and more superficialones at progressively later times. Thus,most of the neurons of layer VI are

born between E40 and E60, neurons inlayer V between E60 and E70, those inlayer IV between E70 and E80, andthose in layers III and II between E80and El00. These data pertain only to

the time of cell origin and do not define

SCIENCE. VOL. 183

As

when the cells actually attained theirfinal positions.

This study establishes that neuronsgedterated on a day early in develop-ment eventually occupy a relativelynarrow zone of the mature cortex, whiletho'se generated on a later day areeventually distributed over a somewhatwider zone; also, neurons born 8 to10 days apart overlap somewhat in posi-tion along the radial axis (Fig. 2). Onereason for the wide distribution of cellsgenerated simultaneously may be differ-ences in rates of migration. Slower-moving neurons might reach the cortexwhen it had increased several hundredmicrometers in thickness by the addi-tion of neurons that were generatedlater but had migrated faster. Within apopulation of simultaneously labeledcells, the slower-moving neurons wouldpresumably take more superficial posi-tions than the faster-moving ones. In-deed, initial examination of monkeyembryos killed at shorter intervals afterinjection of [3H]dT suggests that somelabeled neurons reach the outer borderof the cortical plate in less than 3 days,whereas others labeled simultaneouslytake 7 or more days to reach a com-parable destination (10). Variations inthe length of the cell generation cyclealso could influence eventual neuronposition in the cortex, as might differ-ences in the detailed aspects of cell in-teraction during migration (11).

Autoradiographic results in the brainof this primate corroborate in generalthe "inside-out" pattern of cell disposi-tion described for rodents (3). Initial ob-servations suggest that cells in othercortical areas in the monkey behavesimilarly, although on slightly differenttime schedules (10). Comparison of thedata in Fig. 2 with studies in mice (12)shows that simultaneously generatedneurons in the monkey eventually be-come confined to relatively narrowstrata of the cortex; that is, the "inside-out" principle is more rigidly followed'in the monkey. This may be the devel-opmental basis for the sharper bounda-ries of cortical layers in the visual cor-tex of adult primates.The monkey neocortex acquires its

full complement of neurons at rela-tively early stages of gestation. The lastcortical neurons are generated close tobirth in mice and rats, and corticalgenesis continues for a few days afterbirth in the hamster (3). In contrast,the last neurons destined for theprimary visual cortex are generatedIXIREBUARY 1974

around El 00 in the monkey, that is,about 2 months before birth. It isprobable that other primates, includingman, also acquire a full complement ofneocortical neurons well before birth.

PASKO RAKICDepartment of Neuropathology,Harvard Medical School, andDepartment of Neuroscience,Children's Hospital MedicalCenter, Boston, Massachusetts 02115

References and Notes

1. D. H. Hubel and T. N. Wiesel, J. Physiol.(Lond.) 195, 215 (1968); Nature (Lond.) 225,41 1970); J. Comp. Neurol. 146, 421 (1972); F.Valverde, Int. J. Neuirosci. 1, 181 (1971); J. S.Lund, J. Comp. Neurol. 147, 455 (1973); S.LeVay, ibid. 150, 53 (1973).

2. W. Vignal, Arch. Physiol. Norm. Pathol. 2,338 (1888); S. Ram6n y Cajal, Cellule 7, 125(1893); A. Kolliker, Handbuch der Gewebelehredes Menschen (Engelmann, Leipzig, 1896); M.Stefanowska, Trav. Lab. Invest. 2, 1 (1898).

We have compared the survival ofDrosophila melanogaster (Oregon-R)males maintained at 100, 200, 250,and 30°C on medium (Carolina InstantMedium, Carolina Biological Supply)prepared with 50 percent D20 withthose maintained on medium preparedwith distilled H2O. The mean life-spanof these Drosophila populations at

0 M

10 ¾. 50 1

\ 10 20 30ii) "t T l°C)

3. J. B. Angevine and R. L. Sidman, Natuire(Lond.) 192, 766 (1961); M. Berry and A. W.Rogers, J. Anat. 99, 691 (1965); S. P. Hicksand C. J. D'Amato, Anat. Rec. 160, 619 (1968);M. Shimada and J. Langman, J. Comp. Neurol.139, 227 (1970).

4. P. Rakic, J. Comp. Neurol. 145, 61 (1972).5. , ibid. 147, 523 (1973).6. R. L. Sidman, in Contemporary Research

Methods in Neuroanatomy, W. J. H. Nautaand S. 0. E. Ebbesson, Eds. (Springer-Verlag,Berlin, 1970), pp. 252-274.

7. K. Brodman, J. Psychol. Neurol. 4, 177 (1905).8. T. Meynert, in Handbuch der Lehre von den

Geweben des Menschen und der Thiere, S.Striker, Ed. (Engelmann, Leipzig, 1871), pp.694-808; W. E. Le Gros Clark, J. Anat. 76,369 (1942).

9. D. E. Schmechel and P. Rakic, Anat. Rec.175, 436 (1973).

10. P. Rakic, unpublished observations.11. R. L. Sidman and P. Rakic, Brain Res. 62, 1

(1973).12. V. S. Caviness, Jr., and R. L. Sidman, J. Comp.

Neurol. 148, 141 (1973).13. Supported by PHS grant NS09081. I am grate-

ful to T. C. Jones and F. Garcia of the NewEngland Regional Primate Research Center,Southboro, Massachusetts, for making avail-able timed pregnancies used in this study.

4 September 1973; revised 15 October 1973 a

20°C was reduced from 85 to 29 dayson a diet prepared wi.th 50 percentD,O. Furthermore, we found that re-ducing the temperature to 10°C causeda decrease in mean survival time from29 to 7 days for populations main-tained on the D20 medium, whereassurvival time was increased from 89to 112 days for populations suppliedwith distilled H.,O medium. Raising thetemperature from 200 to 250C forpopulations supplied with D,O mediumincreased survival from 29 to 36 days.At 30°C mean survival time dropped

Fig. 1. Mean survival time (S) in popu-lations of Drosophila melaniogaster (Ore-gon-R) males maintained from 1 dayof age on Carolina Instant Medium pre-pared with distilled H20 or with 50 per-cent D20 at temperatures (T) of 100, 20°,250, and 30'C. The light-dark cycle was12 hours-12 hours. Flies were reared at200C on standard corn meal-molassesagar. Ten tubes of 20 flies each wereused for each determination of S. (Inset)Difference between mean survival timesfor populations on D20 medium [S(D20)]and H20 medium [S(H20)].

427

Deuterium Oxide Effect on Temperature-Dependent Survivalin Populations of Drosophila melanogaster

Abstract. A comparison of the mean life-spans for populations of Drosophilamelanogaster at 100, 2,00, 250, and 30°C maintained on media prepared withdistilled water and with 50 percent deuterium oxide shows that deuteration de-creases longevity at all four temperatures. The magnitude of the diJference be-tween the mean survival times of populations maintained on deuterated andnondeuterated media is inversely related to temperature between 10° and 30°C.