Anatomical Demonstration of Orientation Columns in Macaque Monkey

DAVID H. HUBEL, TORSTEN N. WIESEL AND MICHAEL P. STRYKER Department ofNeurobiology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT In the macaque monkey striate (primary visual) cortex, the grouping of cells into ocular dominance and orientation columns leads to the pre- diction of highly specific spatial patterns of cellular activity in response to stimu- lation by lines through one or both . In the present paper these patterns have been examined by the 2-deoxyglucose autoradiographic method developed by Sokoloff and his group (Kennedy et al, '76). An anesthetized monkey was given an injection of I4C 2-deoxyglucose and then visually stimulated for 45 minutes with a large array of moving vertical stripes, with both eyes open. The 14Cautora- diographs of striate cortex showed vertical bands of label extending through the full cortical thickness. Layer I was at most only lightly labelled, and layers IV b and VI were the most dense. Layer IV c (the site of terminations of most genicu- late afferents) was labelled uniformly along its length, as expected from the lack of orientation specificity of units recorded in that layer. In the other layers the pattern seen in tangential sections was complex, consisting of swirling stripes with many bifurcations and blind endings, but with occasional more regular re- gions where the stripes were roughly parallel. Interstripe distance was rather constant, at 570 pm. Ocular dominance columns were examined in this same monkey, in the same region, by injecting one with H-proline two weeks before the deoxyglucose experiment, and preparing a second set of autoradi- ographs of the sections after prolonged washing to remove the 14C-deoxyglucose. As seen in tangential sections through layer IV c, these columns had the usual stripe-like form, with a period of 770 pm, but were simpler in their pattern than the orientation stripes, with fewer bifurcations and less swirling. A comparison of the two sets of columns in the same area showed many intersections, but no strict or consistent relationships: angles of intersection showed a distribution that was not obviously different from that expected for any two randomly superimposed sets of lines. Another monkey was stimulated with vertical stripes, but with only one eye open. Deoxyglucose autoradiographs of tangential sections showed regular uni- form rows of label in layer IV c, with all the characteristic features of eye domi- nance columns. In the layers above and below IV c the rows in tangential view were broken up into regularly spaced patches of label, presumably representing aggregations of cells responsive to vertically oriented stimuli. The patches showed no consistent alignment across the ocular dominance rows, and indeed no such tendency would be expected, considering the complexity of the orientation columns. This pattern of labelling is again predicted from and confirms the previ- ous physiological studies.

Two functions of the primary ganized in such a way that cells after the ini- of higher have now been established tial stage respond best to specifically oriented (Hubel and Wiesel, '62, '68). The information straight-line segments, rather than to spots of from the lateral geniculate body is reor- light; and the cortex is for all practical pur-

J. COMP. NEUR. (1978)177: 361-380. 361 362 D. H. HUBEL. T. N. WIESEL AND M. P. STRYKER poses the first site in the retino-geniculo-cor- tions at the column borders. A above or tical pathway at which signals from the two below IV c is presumed to receive convergent eyes converge upon single cells. input, relayed directly or over several syn- Related to and subserving these two func- apses, from several of these monocular regions tions are two independent sets of vertical sub- in IV c, and is consequently likely to be bin- divisions, the orientation columns and the ocularly influenced, but dominated by the eye ocular dominance columns (Hubel and Wiesel, that corresponds to the region in IVc lying '62, '63, '68, '74a). When a micro-electrode below or above along the same radial line, i.e., penetrates through the cortex in a direction in the same column. perpendicular to the surface, recording from The cortex is thus subdivided by two inde- many cells in sequence, the optimal orienta- pendent sets of partitions that are perpen- tion of a stationary or moving short-line stim- dicular to the surface and the layers. These ulus tends to be virtually constant, and the subdivisions are not visible by ordinary histo- same eye remains dominant, though many logical staining methods. Several lines of evi- cells are influenced (usually unequally) by the dence nevertheless suggest that both systems two eyes. In a tangential or oblique penetra- of grouping have the form of parallel vertical- tion the optimal orientation and the ocular ly disposed sheets. For the eye dominance sys- dominance both vary. Orientation changes tem the sheet-like geometry has been made systematically in a clockwise or counter- evident by three independent anatomical clockwise direction, in small steps or perhaps methods: the Nauta/Fink-Heimer/Wiitanen continuously, at rates such that 180' is stain (Hubel and Wiesel, '721, transneuronal covered in about 600 pm or more. Reversals in autoradiography after eye injection (Wiesel et the direction of rotation occur irregularly, al., '74; Hubel et al., '771, and a reduced silver roughly one or two to a centimeter, and occa- stain (LeVay et al., '75). In tangential sections sionally the sequence may be broken by an through layer IV c the ocular dominance col- abrupt shift of up to go", which we term a frac- umns appear as a set of parallel stripes. ture. Ocular dominance meanwhile shifts Though on the whole the stripes are remarka- back and forth, apparently quite independent- bly regular, in places they show bifurcations ly of orientation: first one eye dominates, then and blind endings, and often form loops and the two become roughly equal, and finally the whorls. The evidence that the orientation col- other dominates, a complete cycle occurring umns are arranged in parallel slabs stems roughly every 800 pm. Thus while the individ- from physiological recordings, and is deduced ual subdivisions in the two systems are very from reconstructions of multiple parallel pen- different in size (25-50 pm for the orientation etrations (Hubel and Wiesel, '63, '74a) and columns as opposed to 400 pm for the ocular from the fact that the orientation shifts in dominance) a complete set of either type, left- any single oblique or tangential penetration plus-right eye or a cycle of 180" of orientation, are small and regular. While so far there has occupies about the same distance on the cor- been no anatomical method for demonstrating tex, 0.5-1 mm. One complete set of columns, of the orientation sheets, the recordings, and in either type, has been termed a hypercolumn particular the reversals and fractures, sug- (Hubel and Wiesel, '74b). This is illustrated in gested that they might swirl and branch ex- figure 1. tensively. This description applies to all layers from I1 Figure 1 shows in schematic form a model of to VI, except for IV c, the layer in which the the monkey striate cortex (Hubel and Wiesel, bulk of the geniculo-cortical inputs terminate. '77). The diagram represents two orientation In IV c the cells show no hint of orientation se- hypercolumns and two ocular dominance lectivity, but seem to have circularly sym- hypercolumns. It should be kept in mind that metric center-surround receptive fields re- the orientation columns can be far from flat, sembling the fields of geniculate cells. More- that the choice of vertical orientation to begin over here the cells are virtually all monocular, and end the orientation hypercolumn is so that on proceeding tangenitially along IV c, arbitrary, that nothing is known about the re- crossing from one ocular dominance column to lationship between the two types of columns the next, the electrode passes from a region in - the decision to draw them as orthogonal is which the cells respond only to one eye to a re- again arbitrary - and that whether or not the gion monopolized by the other. There is thus a orientation columns are discrete is still unset- strict alternation of eyes, with abrupt transi- tled (Albus, '75; Hubel and Wiesel, '74a). In ORIENTATION COLUMNS IN MACAQUE MONKEY 363 this figure the orientation slabs represent re- heightened activity leads to an increase in gions of cortex over which optimal orientation glucose consumption. An animal is injected is assumed to be constant, and the shifts in intravenously with a single dose of "C 2-de- orientation from one slab to the next are rep- oxyglucose, which is taken up by nerve cells resented as 10". It is worth stressing, however, and is phosphorylated as if it were normal that the region of cortex activated by a line in glucose, to the 2-deoxyglucose-6-phosphate, a particular orientation would be much wider but not further metabolized. The cell mem- than these slabs, because for most cells the brane is relatively impermeable to this com- range of orientations over which responses are pound, so that the label is effectively trapped evoked is several times greater than 10". inside the cell in concentrations proportional Moreover, such regions of activation would to the integrated uptake of glucose. If the not be precisely defined, since for each cell the brain is quickly frozen and sectioned in a responses vary with orientation according to a frozen state to prevent diffusion of the water- tuning curve, and since these tuning curves soluble label, then regions with increased themselves vary in width from cell to cell. metabolic rates during the period in which the (See, for example, Schiller et al., '76). deoxyglucose was available will be visible on Given these columnar groupings of cells autoradiographs. according to response preferences, it is possi- As one demonstration of the potential of ble to predict the cortical activity patterns this tool in neurobiology, Sokoloffs group has produced by various visual stimuli. Figure 2A used it to demonstrate the ocular dominance represents the activity pattern produced in columns in macaque monkeys by stimulating the upper or lower layers Le., 11-111, IV b, V, one eye only (Kennedy et al., '76). The method or VI) by a set of vertical stimulus stripes has been used in several other systems in the covering a large part of the visual field and by Sharp and his colleagues (Sharp et al., viewed by both eyes. Each shaded strip in the '75, Sharp, '76b), and by Durham and Woolsey figure is of course maximally activated along ('77) to demonstrate whisker barrels in mouse a narrow center line: as discussed above the cortex. We hoped that by stimulating both width of the shaded strips will depend on tun- eyes with lines in one orientation we might ing curve widths, and their borders will not be reveal the corresponding subset of orientation sharp but will shade off just as tuning curves columns and thus obtain anatomical evidence do. for the columnar organization. It would then Figure 2B shows the pattern predicted, for at last be possible to see the 3-dimensional the same layers, when only one eye views the arrangement of the orientation columns, and vertical stripes. And finally 2C shows the pat- compare this with the arrangement of ocular tern for one stimulus orientation (e.g., verti- dominance columns obtained by amino acid cal) and one eye in layer IV c; here the domi- eye injections in the same animal. nance columns corresponding to the stimu- lated eye are activated along their entire METHOD lengths, since cells in IV c respond equally to The present study is based on three experi- all line orientations. Needless to say, a fourth ments done in macaque monkeys about six diagram to illustrate a combination of vertical months old. In each animal we followed in lines, for both eyes, in layer IVc, would most respects the procedure described by require shading throughout. Sokoloff (Kennedy et al., '761, to whom we are Until now these patterns had been inferred indebted for first-hand instruction in the entirely from physiological recordings, except method. The animal, lightly anesthetized with for the case of dominance stripes in layer IV c thiopental and paralyzed with a continuous (fig. 2C), which had been seen anatomically by intravenous infusion of curare and gallamine, the methods already listed. Recently, how- was stimulated (as described below) for 45 ever, a more direct approach has become possi- minutes following a rapid intravenous injec- ble. Over the past few years Sokoloff and his tion of I4C 2-deoxyglucose (New England Nu- group have developed a method by which re- clear, 150 pCi/kg in 1.5 ml/kg of 0.9%saline). cently active regions of nervous tissue can be At 5-minute intervals during the stimulation differentially marked (Sokoloff, '75; Sokoloff period samples of blood were drawn and 14C et al., '77). The technique rests on the fact levels determined to be sure that at the end that nerve cells use glucose as their main they had fallen to a few percent of the initial energy source and on the assumption that level. The animal was then given intravenous- 364 D. H. HUBEL. T. N. WIESEL AND M. P. STRYKER

\ \ @ / / 4 B \ R h Q$ L h R f L

\ / \ \ \ \ Fig. 1 Idealized model of the monkey striate cortex, showing two orientation hypercolumns each covering a full 180",and two ocular dominance hypercolumns. Here the columnar walls are represented as flat, and the two sets are shown intersecting at right angles, but the present paper indicates that neither set of walls is flat, and that the intersections are probably random. R, right eye; L, left eye. (From Hubel and Wiesel, '77: fig. 27)

ly 50 mg/kg of thiopental followed by a lethal the ocular dominance columns. The left eye dose of KC1 (4 ml saturated solution) and de- was occluded with an opaque cover and the capitated. The head was immediately cleaned right eye stimulated with a large brightly of skin, frozen by gradual immersion in Freon- illuminated screen containing white stripes 22 at -125°C over a period of four minutes, 'Ia-'h0 wide and spaced 1s-2"apart, on a black and stored at -80°C. The skull was later re- Fig. 2 Drawings to indicate the patterns of cortical ac- moved and small blocks of brain sectioned at tivity expected from three different visual stimuli. A patch 20 gm in a cryostat at -22" to -26°C. The of monkey striate cortex several millimeters in length and sections were immediately dried on a cover width is viewed face-on. The horizontal lines represent slip heated to 98°C and pressed against X-ray boundaries between orientation columns, the vertical lines, boundaries between ocular dominance columns. H and V film for two to three weeks, after which the stand for horizontal and vertical stimulus orientations; L film was developed. Every fifth section was and R, the left and right eyes. As in figure 1, the column mounted and stained for Nissl substance boundaries are very schematic. Shaded areas indicate the regions in which cells are expected to be activated. (Little is (cresyl violet); in addition some of the sections known about the response properties of cells in layers I or used for autoradiography were later stained IV a,) for Nissl substance. A The pattern expected in layers 11,111, IV b, V and VI when both eyes are stimulated by vertical lines. RESULTS B Activity pattern corresponding to vertical lines Ocular dominance columns stimulating one eye only, in 11, 111, IV b, V, and VI. C Activity in layer IV c, in response to vertical-line In the first monkey we wished to examine stimuli to the left eye only. ORIENTATION COLUMNS IN MACAQUE MONKEY 365

A Vertical lines Both eyes Layers II, Ill, IVb,V. VI

B Vertical lines Left eye Layers II, 111, IVb.V,VI

C Vertical lines Left eye Layer IV c 366 D. H. HUBEL, T. N. WIESEL AND M. P. STRYKER

Fig. 3A, B Deoxyglucose autoradiographs of coronal sections through the right occipital pole of monkey No. 1 to show ocular dominance columns. This animal was stimulated with stripes in all orientations through the right eye only. A is 5 mm in front of the occipital pole, B is 0.5mm in front of A. Down and right in the diagram is superior; up and right is medial. In the upper part of the figure (medial aspect of occipital lobe) the columns are seen in transverse section; two folds below this in the figure (in the superior bank of the calcarine fissure, la- belled S) they are cut very obliquely. C The left portion is from the same section as B, the right is a Nissl-stained section 60 pm distant, matched to show that the highest density of label is in layers IV b and VI. background. The screen, which covered most two levels, one about midway down and one in of the visual field, was held 1 m away (the dis- the deepest part of the cortex. When an adja- tance at which the eye had been focused) and cent Nissl-stained section is matched to the moved in a direction perpendicular to the autoradiograph of B, the two densely labelled stripe orientation at 2-5"/sec. Meanwhile the levels can be seen to correspond to layers IV b screen was rotated slowly and the direction of (the line of Gennari) and VI. In layer IV b movement was changed so as to stimulate in there is some faint higher-than-background all orientations at least once every minute. labelling between columns; this is not nearly Autoradiographs from coronal sections so dense as the label in the columns at that through two regions close to the right oc- level, but it does stand out against the other- cipital pole of this monkey are shown in wise relatively label-free gaps between col- figures 3A and B. B is taken at a level 0.5 mm umns. anterior to A. Bands representing ocular In these sections layer I was very thin, and dominance columns cut in a plane roughly we were not convinced that columns had been perpendicular to the surface are best seen on labelled there. the medial surface (uppermost, in the figures), The distance between bands averaged 760 while more oblique, almost tangential sec- pm, giving 380 pm for the columnar width, a tions show the stripe-like pattern in the supe- figure close to that obtained with other meth- rior bank (S)of the calcarine fissure. The col- ods. umns extend from almost the surface to the These sections closely resemble those pro- white matter, and are most densely labelled at duced in similar experiments by Kennedy et ORIENTATION COLUMNS IN MACAQUE MONKEY 367

Figure 3 368 D. H. HUBEL. T. N. WIESEL AND M. P. STRYKER

Fig. 4 Deoxyglucose autoradiograph of a section through left area 17 (occipital operculum),in monkey No. 2. Section passes perpendicular to the surface. cutting orientation columns transversely. The stimulus consisted of moving vertical stripes; both eyes were open. Note the continuous label in layer IV c, about half way down. In the columns there is a particularly high density of label in layer IV b. and in layer VI just above the white matter al. ('76) (see also Sokoloff 1'751). In some of mapped on the screen, were precisely super- their experiments one eye was enucleated imposed. It was necessary to do this to be sure rather than occluded, and during the stimu- that the eyes would work in synergy for the lation their animals were alert rather cells in area 17 (Hubel and Wiesel, '70) rather than anesthetized. Previous work (Hubel, than compete, as might happen if they were '59; Wurtz, '69) has similarly indicated that out of alignment. The animal was injected as light barbiturate anesthesia does not serious- before with "C 2-deoxyglucose and stimulated ly interfere with specific responses to visual with the moving stripes for 45 minutes, but stimuli in striate cortex. Any general lessen- this time both eyes were open and the stripes ing of background neuronal activity produced were oriented vertically throughout. The re- by the anesthetic may indeed be an advantage cording allowed us to be sure that the animal for these kinds of studies. was in good condition and that our stimuli were actually evoking responses in cortical Orientation columns cells. In the second monkey our main object was An autoradiograph of this monkey's left oc- to reveal the orientation columns in area 17 cipital lobe, from a section perpendicular to and compare them with the eye dominance the exposed outer part of area 17, is shown in columns. To label the dominance columns in figure 4.At first glance the result might seem the same monkey we used the method of similar to that just described for ocular domi- transneuronal autoradiography (Wiesel et al., nance columns: the periodicity of the labelled '74), injecting 2.0 mCi of tritiated proline into regions is not too different and their width in the vitreous of the right eye two weeks before places approaches that of dominance columns. the final experiment. At the time of the ex- One feature, nevertheless, is quite different - periment the animal was anesthetized and set namely the continuous and dense labelling of up for physiological recording from the striate layer IVc, which appears as a conspicuous cortex. Paralysis was induced with an intra- horizontal band placed about half-way down venous infusion of gallamine and curare. The in the cortex, and occupies about one-fifth of eyes were both kept open and focused at 1 m as the thickness. This is, of course, just what is before, and were aligned with a variable prism expected from the lack of orientation speci- so that the projections of the foveas, and also ficity of the units that are recorded in layer the receptive fields of binocular cells as IV c. Each column in addition shows two re- ORIENTATION COLUMNS IN MACAQUE MONKEY 369

Fig. 5 Relationship between cortical layering as seen in Nissl stain, and laminar differences in labelling. De- oxyglucose autoradiograph of tangential section through the right striate cortex of monkey No. 2. The section just grazes layer V and cuts the more superficial layers very obliquely. To the right is a neighboring Nissl- stained section to identify the layers. The main features are the high denaity of label in IV b, the continuous la. be1 in 1V c, and the paucity of label in layer I. (Pattern of orientation columns in this section is obscured by mi- crotome-knife artifacts.) gions of especially high density, one at a level and these differences in density and labelling just above IV c, the other in the very depths, pattern is best seen in oblique or tangential presumably in layer VI. sections, where the layers are more spread out. The relationship between the cortical layers In an example taken from the right occipital 370 D. H. HUBEL, T. N. WIESEL AND M. P. STRYKER

Fig. 6 Deoxyglucose autoradiograph of a tangential section through the left striate cortex of monkey No. 2. Same region as in figure 7. To the left, the same section as was used to make the autoradiograph has been stained for Nissl substance and matched, to identify the layers. Note the high density of label in layers IV b and VI. and the confluence of label in IV c lobe, shown in figure 5, neighboring auto- section through the opposite (left) occipital radiographs and Nissl-stained sections were lobe is shown in figure 6. As in figure 5, it is cut and spliced. The sections are tangential to spliced to a Nissl section, in this case a cresyl- layer V, which appears as an oval, surrounded violet stain of the section from which the in turn by IV c, IV b, 11-111-IV a (these three autoradiograph itself was made. Again one subdivisions cannot be sharply distinguished) can see the high density of label in IV b and and I. The face-on pattern of the labelled re- the confluence of label in IV c; in addition the gions is not particularly well seen in this sec- figure shows a high density of label in layer tion, which was chosen because the layering is VI, and a relatively weak labelling in V. well defined. The columnar pattern seems to Figure 7 shows autoradiographs from tan- extend up so as to include layer I, though the gential sections at four different levels labelling is not dense enough to make this ab- through this region - D is the same as the solutely certain. What this figure shows is (1) right-hand portion of figure 6. These sections the continuous label in IVc, both in its were chosen because the pattern formed by deepest, most densely cell-packed part and in the labelled columns is reasonably clear. Seen its upper, more sparsely populated part; (2) face-on, they form a complex network of inter- the clear correspondence between the densely connected bands. In places the bands run par- labelled portions of the columns and layer IV b allel for short distances, for example in part (roughly equivalent to the Gennari line), and of the upper right quadrant in sections A and (3) the relatively lower density of label in II- C, but more often they branch or end blindly, 111. or form swirls or irregular rings. The spac- An autoradiograph of a deeper tangential ing, nevertheless, is remarkably regular, as ORIENTATION COLUMNS IN MACAQUE MONKEY 371 though there were strong constraints on the tionship between the two, and in particular no frequency with which a given orientation re- marked tendency for them to run parallel or to curs. be orthogonal. We do not consider this ques- By making prints of these four sections on tion settled, however, and plan to examine film and superimposing them it was easy to more macaque brains. The widths of the two show that the patterns closely coincide. Like sets of hypercolumns were determined by the transverse sections of figure 4, this con- tracing the two sets of boundaries, in figures firms the physiological observation that the 8A and B, and for each set selecting a number columns are vertical and extend through the of small, regular regions, determining the full cortical thickness (at least through layers areas of several lengths of columns, and divid- 11-VI), interrupted only by the uniformly la- ing by the lengths. This gave 770 pm for ocu- belled layer IV c. lar dominance hypercolumns, divided equally The overall pattern was reconstructed from (to within 10%) between the two eyes. This is a number of sections parallel to those of figure almost identical to the figure obtained from 7 by cutting out from each the part containing the first monkey, and is similar to that deter- layer VI and matching them. This is shown in mined previously from Nauta, reduced silver, figure 8A. and eye-injection studies (Hubel et al., '77). For the orientation hypercolumns (fig. 8A) the Comparison of orientation and ocular hypercolumn width, obtained by the same dominance column patterns method, was 570 pm. This may be compared to From the block whose sections are shown in a minimum width of 450 pm obtained in phys- figures 6 and 7 every third section was set iological recordings from a maximum slope of aside to examine the ocular dominance col- about 400"/mm in graphs of orientation vs. umns, using the transneuronal 3H label trans- electrode track distance (Hubel and Wiesel, ported from the eye that had been injected. '74a). From these rough figures we conclude The sections were fixed, washed in water for that the orientation and ocular dominance four hours to remove the I4C label, dehydrated hypercolumns are of the same order of mag- and defatted, dipped in photographic emul- nitude, with the orientation hypercolumns sion, exposed in darkness for ten weeks, and slightly smaller. then developed. When viewed in dark-field illumination the sections showed banding in Ocular dominance plus orientation layer IV c typical of ocular dominance col- In a third monkey we combined the pro- umns. The montage of figure 8B was prepared cedures used in the first two deoxyglucose ani- by cutting out layer IV c from several sections mals by stimulating the left eye only, with and assembling them. The stripes form a vertical stripes. The expected distribution of rather irregular pattern, as is usual for re- cells activated by such a stimulus has already gions close to the representation of the been shown schematically in figure 2B, for the horizontal meridian (as this was). On the upper and lower layers and, in figure 2C, for other hand the pattern is simpler than that of layer IV c. The results strongly supported this the orientation columns in the same region expectation. Figure 10A shows a section run- and certainly the two are very different in ning tangential to the right occipital convex- their details. In fact, from inspection of the ity, passing through layers I1 and 111. The la- two, separately, there is no hint of any rela- bel is clearly in patches, which tend to line up tionship between them. In figure 9 we super- in rows that run, in the figure, from lower left imposed tracings of the two sets of columns of to upper right. figure 8, with the dominance columns drawn A much deeper section, B, parallel to A, cuts in thin lines and the orientation columns in the calcarine fissure at right angles over thick. There is in places some suggestion that much of its stem, and again shows how regular the two patterns may be orthogonal; to test and vertical the columns are. The patches are this we measured the angles at each of the 154 shown almost in tangential section just above points of intersection and compared the re- and below the mouth of the fissure, and here sulting distribution of angles with that ex- their arrangement in rows is even more evi- pected if the two sets were randomly related dent. (a sine function). From the result one can only Figure 11 compares tangential sections say that in this region there was no clear rela- from the left hemisphere at various depths, to 372 D H HUBEL. T N WIESEL AND M P STRYKER

Fig. 7 Four deoxyglucose autoradiographs from monkey No. 2, to show the pattern formed by orientation col- umns viewed face-on. Tangential sections through left occipital lobe, exposed surface. Sections A and B and B and C are separated by 250 pm, C and D by 200 pm. D is from the same section as figure 6. Careful inspection shows the similarity of the pattern of columns in upper and lower layers. ORIENTATION COLUMNS IN MACAQUE MONKEY 373

Figure 7 374 D. H. HUBEL. T. N. WIESEL AND M. P. STRYKER

Fig. 8A Reconstruction of orientation columns from monkey No. 2, left occipital lobe, in the same series as that of figure 7, made by cutting and mounting the parts of each section passing through layer VI. B Reconstruction of ocular dominance columns in the same region as A, made from autoradiographs of H-proline sections following injection of the right eye; dark.field photographs, Parts of each autoradiograph passing through layer IV c were cut and mounted. ORIENTATION COLUMNS IN MACAQUE MONKEY 375

5 mm Fig. 9 The vertical-orientation columns, from figure 8A, are traced as thick lines, the left-eye ocular domi- nance columns from figure 8B, as thin lines. The average widths of the hypercolumns are 770 pm for the ocular dominance, 570 wm for the orientation. The two sets are certainly not parallel, but neither are they strictly orthogonal, test the prediction of figure 2C, that an ori- DISCUSSION ented stimulus to one eye would give con- This study partly confirms previous work fluent ocular-dominance stripes in IV c. The and partly supplies new information that sections, three of which are illustrated, were would have been very difficult to obtain taken just behind the lunate sulcus. Each sec- by conventional physiological or anatomical tion was first used for autoradiography and methods. then stained with cresyl violet to identify the The evidence for ocular dominance columns layers with certainty: at each level the cresyl was already massive. In the macaque mon- violet stains are shown in the lower half of the key, microelectrode recordings monitored by figure (posterior, in each of the 6 photographs, electrode track reconstructions and several is up). independent anatomical techniques (Nauta/ The lunate sulcus (L) runs just in front of Fink-Heimer/Wiitanen stain; 3H-proline eye and parallel to the 17-18 border, and from a injection; and reduced silver staining) had all previous study (LeVay at al., '75) it is known demonstrated the subdivision of layer IV c that the ocular dominance stripes are at right into left-eye and right-eye regions, as well as angles to this boundary, running roughly in an revealing the crispness of the segregation. anteroposterior direction. At each level in Until the deoxyglucose method was applied, figure 11 one can see the parallel stripes of la- the evidence that these columns extended bel, corresponding to the left, stimulated eye, through all layers (perhaps excepting layer I) all stopping short right at the 17-18border. In was purely physiological, but was neverthe- the superficial (11, 111, IV a) and deep (V, VI) less compelling. The present study agrees with layers, seen best in A and C, the stripes are the earlier work of the Sokoloff group in pro- broken up into patches, but in both A and B, in viding a confirmation of the extension of the just the regions that pass through IV c, the ocular dominance columns to the upper and patches coalesce to form continuous stripes, as lower layers. Outside layer IV many cells, per- predicted in figure 2. haps about half, are binocular, and the column 376 D. H. HUBEL, T N. WIESEL AND M. P. STRYKER ORIENTATION COLUMNS IN MACAQUE MONKEY 377 378 D. H. HUBEL, T. N. WIESEL AND M. P. STRYKER walls as revealed by the deoxyglucose method fraction of the total deoxyglucose uptake. In would therefore not necessarily be expected to this context it is probably worth noting that in be as sharp as in IV c. Moreover, the apparent the the cells are mainly orientation se- widths of the columns as determined by this lective whereas the geniculate inputs, as in method might be expected to vary from layer the monkey, are not. The cat's orientation col- to layer depending on responsiveness of cells, umns are clearly defined even at the base of the amount of label uptake, and so on. In fact layer IV (Stryker et al., '77). the walls turn out in our autoradiographs to The marked variation in label density from be fairly straight. That the columns in figure layer to layer is at present completely unex- 3 are widest in their deepest parts is perhaps plained. In layer IV b, which is sparsely popu- related to the high density of label in layer VI. lated with cell bodies, the especially high den- The present study provides the first ana- sity of label seems to support the idea that tomical demonstration of the orientation col- neuropil and cell bodies take up about the umns. The morphological evidence that these same amount of deoxyglucose, per unit cross structures are vertically organized and span sectional area (Sharp, '76a). But there is cer- all layers except IV c and possibly I is especial- tainly no clear inverse correlation between la- ly welcome, since the physiological evidence, bel uptake and cell density: layer V is also while strong, was not very direct: It depended sparsely populated, and is lightly labelled; VI on a few penetrations that were so nearly per- is both cell-rich and heavily labelled; I has pendicular that orientation was constant almost no cell bodies, is rich in nerve termi- throughout (Hubel and Wiesel, '681, on the re- nals, but is hardly labelled at all. These lami- construction of multiple parallel penetrations nar variations in uptake are intriguing and (fig. 3 of Hubel and Wiesel, '631, and on the will probably be understood only when the finding that in oblique penetrations the method is increased in resolution by several graphs of orientation plotted against track orders of magnitude. distance were often virtually straight lines The main new finding in this study is obvi- through the full cortical thickness, inter- ously the pattern formed by the sheets of con- rupted only in the part of the penetration stant orientation. That this pattern is con- passing through layer IV c (Hubel and Wiesel, siderably more complex than the orientation- '74a). That the columns in the present deoxy- column pattern was already suspected from glucose study are perpendicular to the surface the high frequency of reversals and fractures is clear not only from their appearance in sec- encountered in oblique penetrations. So far tions perpendicular to the surface (fig. 4) but the pattern appears not only complex, but also also from the similarity of the patterns in irregular and unpredictable, but we would tangential sections taken at different levels have made a similar conclusion for the eye (fig. 7). dominance columns had we only looked at a The lack of even a slight suggestion of orien- few regions such as the foveal (cf. Hubel and tation columns in IV c is itself strong indica- Wiesel, '72: fig. 17; LeVay et al., '75). A larger tion that the columns are orientation col- number of regions must still be examined in umns, since physiologically we see no trace of more animals before one can say whether or orientation preference in that layer. It is gra- not there is any consistent or orderly pattern. tifying to find such a good fit between anat- Our results at least seem to rule out any sim- omy and physiology, especially since the lack ple order, as well as any strict relationship be- of orientation specificity in IV c has not been tween the two sets of columns. Apparently a fact in which one could place extreme confi- random intersection is enough to guarantee dence. The geniculate inputs to this layer are that the two sets intersect frequently and not orientation selective, and given the dif- never remain parallel over long distances, and ficulty in recording large and clearly defined that is probably what is important (Hubel and spikes in this layer there was always the possi- Wiesel, '771. bility that we were recording only from the af- Finally, the constancy of the distance sepa- ferents, as might be so if the cells themselves rating one column representing vertical from did not fire impulses. The present result does the next is striking, just as was the constancy not completely settle the question, however, of spacing of the dominance columns. This since as suggested by Sharp ('76a) uptake by would seem to uphold the notion (Hubel and nerve endings may account for a significant Wiesel, '74b, '77) that contained in each small ORIENTATION COLUMNS IN MACAQUE MONKEY 379

block of cortex, roughly 1 mm X 1 mm, is the ~ 1977 Functional architecture of macaque mon machinery needed to subserve both eyes in all key visual cortex. Proc. Roy. Soc. Lond. B., 298- 1-59. Hubel, D. H., T. N. Wiesel and S. LeVay 1977 Plasticity of orientations. ocular dominance columns in monkey striate cortex. Phil. Trans. Roy. Soc. Lond. B., 278: 377-410. ACKNOWLEDGMENTS Kennedy. C.. M. H. Des Rosiers, 0. Sakurada, M. Shinohara, We are grateful to Birthe Storai, Karen Lar- M. Reivich, H. W. Jehle and L. Sokoloff 1976 Metabolic mapping of the primary visual system of the monkey by son, and Jean Thompson for histological as- means of the autoradiographic I’T-deoxyglucose terh- sistance and to Carolyn Yoshikami for photog- nique. Proc. Natl. Acad. Sci. (U.S.A.).73: 4230-4234. raphy. LeVay, S.,D. H. Hubel and T. N. Wiesel 1975 The pattern of Supported by grants from The Rowland ocular dominance columns in macaque visual cortex revealed by a reduced silver stain. J. Comp. Neur . 259. Foundation, Inc., The Esther and Joseph 559-576. Klingenstein Foundation, Inc., and NIH Schiller, P. H.. B. L. Finlay and S. F. Volman 1976 Quan Grants EY 00605 and EY 00606. Doctor titative studies of single-cell properties in monkey striate Stryker is supported by an NIH training cortex. 11. Orientation specificity and ocular dominance. Grant EY 00082. J. Neurophysiol., 39: 1320.1333. Sharp, F. R. 1976a Relative cerebral glucose uptake of LITERATURE CITED neuronal perikarya and neuropil determined with 2-de- oxyglucose in resting and swimming rat Brain Hes.. I 20: Albus, K. 1975 A quantitative study of the projection 127-139. area of the central and paracentral visual field in area 17 1976b Rotation induced increases of glucose of the cat. 11. The spatial organization of the orientation uptake in rat vestibular nuclei and vestibulocerebellum. domain. Exp. Brain Res., 24: 181-202. Brain Res., 110: 141.151. Durham, D., and T. A. Woolsey 1977 Barrels and columnar Sharp, F. R., J. S. Kauer andG. M. Sheperd 1975 Local sites cortical organization: Evidence from 2-deoxyglucose of activity-related glucose metabolism in rat olfactory (2DG) experiments. Brain Res., 235. bulb during olfactory stimulation Brain Res.. 98 Hubel, D. H. 1959 Single unit activity in striate cortex of 596-600. unrestrained . J. Physiol., 147: 226-238. Hubel, D. H.. and T. N. Wiesel 1962 Receptive fields, bin- Sokoloff. L. 1975 Influence of functional activity on ocular interaction and functional architecture in the local cerebral glucose utilization. In. Brain Work. The Coupling of Function, Metabolism and Blood Flow in the cat’s visual cortex. J. Physiol., 160: 106-154. and N. A. Lassen, eds. Academic 1963 Shape and arrangement of columns in cat’s Brain. Ingvar, D. H Press, New York, pp. 385-388. striate cortex. J. Physiol., 165: 559-568. 1965 Binocular interaction in striate cortex of Sokoloff. L.. M. Reivich, C. Kennedy, M. H. Des Rosiers, C. S. kittens reared with artificial squint. J. Neurophysiol., 28: Patlak, K. D. Pettigrew, 0. Sakurada and M. Shinohara 1041-1059. 1977 The 1’4Cldeoxyglucosemethod for the measurement 1968 Receptive fields and functional architec- of local cerebral glucose utilization: theory, procedure, ture of monkey striate cortex. J. Physiol., 295: 215-243. and normal values in the conscious and anesthetized 1970 Cells sensitive to binocular depth in area albino rat. J. Neurochem., 28: 897-916. 18 of the macaque monkey cortex. Nature, 225: 41-42. Stryker, M. P., D. H. Hubel and T. N. Wiesel 1977 Orienta- 1972 Laminar and columnar distribution of tion columns in the cat’s visual cortex. Neurosci. Abstr.. geniculo-cortical fibers in the macaque monkey. J. Comp. 3: 1852. Neur., 146: 421-450. Wiesel. T. N., D. H. Hubel and D. M. K. Lam 1974 Autoradi- 1974a Sequence regularity and geometry of ori- ographic demonstration of ocular-dominance columns in entation columns in the monkey striate cortex. J. Comp. the monkey striate cortex by means of transneuronal Neur., 158: 267-294. transport. Brain Res., 79: 273-279. 1974b Uniformity of monkey striate cortex: a Wurtz, R. H. 1969 Visual receptive fields of striate cor- parallel relationship between field size, scatter,and mag- tex in awake monkeys. J. Neurophysiol., 32. nification factor. J. Comp. Neur., 158: 295-306. 727-742.