A study of barrels and pyramidal dendritic clusters in the cerebral cortex

Brain Research, 77 (1974) 55-76 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

55

A STUDY OF BARRELS A N D P Y R A M I D A L D E N D R I T I C CLUSTERS IN THE CEREBRAL CORTEX

M A R T I N L. F E L D M A N AND A L A N PETERS

Boston University School of Medicine, Department of Anatomy, Boston, Mass. 02118 (U.S.A.) (Accepted March 27th, 1974)

SUMMARY

Tangential sections from several areas o f rat, cat, macaque, and human neocortex were examined in the light microscope. Two types of neuronal organization barrels and dendritic clusters - - are described. Barrel-shaped aggregations of stellate cell perikarya were examined in layer IV o f the rat parietal cortex. The barrels have their axes oriented perpendicular to the pial surface and are apparent because the concentration o f neurons in their walls is 1.5-2.0 times that in the barrel hollows. Although some ring-shaped neuronal aggregates were observed in the cortices in other species, they could not be found consistently. The dendritic clusters, in rat and cat, consist o f groups o f apical dendrites which radiate vertically from pyramidal cell perikarya. Their form varies with both cortical region and species. It is proposed that clusters are a general feature o f mammalian neocortex. No readily apparent spatial relationship exists between barrels and clusters o f the rat parietal cortex. The dimensions of the dendritic clusters are too small to equate individual clusters with neurophysiologically defined columns. The barrels, in those regions and species in which they have been described, are more likely candidates, although their very limited distribution suggests that, for most functional columns, other morphological correlates exist. It is suggested that, in general, the morphological elements which primarily account for the columns are not perikaryal aggregates but patterns of extrinsic and intrinsic axon terminals. -

-

INTRODUCTION

In the past few years, two forms o f neuronal arrangements have been emphasized in morphological studies of the neocortex. One arrangement is that involving the pyramidal neurons and their apical dendrites. This arrangement first became apparent al by the use o f plastic embedded material examined in the light or electron microscopes, for only by this technique were the dendrites visualized adequately.

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Peters and Walsh 31 in the rat, and later Fleischhauer et al. 7 and Massing and Fleischhauer z2 in the rabbit and cat, demonstrated that the apical dendrites of pyramidal neurons are gathered together into vertically oriented bundles, or clusters, with an intercluster spacing usually measuring between 40 and 50/zm center to center. These studies confirmed the existence of the radial bundles described by von Bonin and Mehler s, in Klfiver Barrera stained preparations of the human and macaque cortex. A different arrangement of neurons has been emphasized by Woolsey and Van der Loos 53 in the cerebral cortex of the mouse. They have shown that in thick, Nissl stained preparations cut tangential to the cortical surface, the cells in layer 1V of the somatosensory cortex are arranged to form darkly stained contiguous rings. The appearance o f rings was presumed to be produced by sectioning barrel-shaped concentrations o f cell bodies, and consequently they have referred to these concentrations as 'barrels'. The axis of the cell sparse hollow of each barrel was perpendicular to the cortical surface, and in sections cut at right angles to this axis, most barrels appeared circular with diameters of about 100 #m. A smaller population of barrels was oval, with a minor axis of about 1O0 # m and a variable major axis, often between 200 and 300/tin. The physiological data obtained from the rat by Welker 45 was explicitly correlated with this anatomical demonstration of barrels. Cortical responses to various somatic stimuli were recorded in the somatosensory (Sml) area of the cortex and discrete non-overlapping fields were found in layer IV. Each field was activated by stimulation of a vibrissa. Cytoarchitecturally, the vibrissa field was shown to contain the barrels and both the relative positions and the number of barrels corresponded closely to the arrangement of the vibrissae on the rat's snout. Hence it was proposed that each barrel is concerned with the sensory input from one vibrissa. Weller 4s has described morphologically similar barrels in layer 1V of the cortex of the deer mouse. In the brush-tailed possum on the other hand, Weller 49 has found the barrels to have no definite hollows so that they are uniformly dense clumps of cells. These dense clumps have also been described by Royce 39 in the armadillo cortex. The present communication concerns three problems which we wished to solve relative to the dendritic clusters o f apical dendrites and the barrels present in layer IV. The first problem was to determine if the dendritic clusters occur generally in the neocortex and if so, whether they have different patterns of organization in various parts of the cortex. The second problem was to explore the question of the generality of barrels in mammalian sensory neocortex. The third problem was to determine the existence of any morphological relationship between the dendritic clusters and the barrels. METHODS

The material examined in this study consisted o f tissue blocks removed from several areas o f normal adult cerebral neocortex in each o f 4 mammals: albino rat, cat, M a c a c a m u l a t t a , and man. The identification o f the cortical areas o f the rat was based upon the studies of

BARRELS AND CLUSTERS IN CEREBRAL CORTEX

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Fig. 1. Tangential 1 p m section, stained with toluidine blue, through layer IV in area 17 of the rat cortex. The apical dendrites of deeper lying pyramidal cells are cut in cross section, and are clustered rather than randomly scattered. One such cluster is indicated by the arrows. The photomicrograph is slightly overexposed to accentuate the clusters. Compare with Fig. 7. b, blood vessel; n, neuronal perikaryon. Calibration line, 50 #m. Fig. 2. As Fig. 1, but in area 41 of the rat cortex. The cluster pattern here (arrows) is less pronounced than in area 17. Compare with Fig. 6. Calibration line, 50/zm.

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Figs. 3-6. Tracings f r o m tangential 1 l+m sections through layer IV in various areas of the rat cortex. Only the transversely cut apical dendrites are drawn. Each small circle represents one apical dendrite. These 4 figures, as well as Figs. 7-10, are all drawn at the same magnification. Fig. 3, cortical area 3: Fig. 4, cortical area 4; Fig. 5, cortical area 2; Fig. 6, cortical area 41. Note that in all of these cortices, as well as in that shown in Fig. 7, also in the rat, there is a marked tendency for apical dendrites to appear in clusters.

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Krieg19,2°. The areas examined were those Krieg designated as 2, 3, 4, 17 and 41, as well as cortex of the frontal pole of the brain. Area 17 corresponds to the primary visual cortex 23 and area 41 to the primary auditory cortex a0,51. The functions of Krieg's areas 2, 3, and 4 have not yet been defined although correlation of the appearance of these areas with the data presented by Welker 45 indicates that at least areas 2 and 3 are included in the somatosensory (SmI) portion of the neocortex. In addition, material was taken from a part of the cortex that will be referred to as the 'barrel field'. This field is located about 20-30 ~o of the distance back from the frontal pole of the cerebral hemisphere and is situated on the lateral and dorsolateral convexity of the hemisphere. The region is defined by the presence of neuronal barrels in layer IV. It includes the rostral portion of Krieg's area 2, and of Welker's Sm145. The identification of cortical areas in the cat was based on studies by Otsuka and Hassler ~s, Hassler and Miihs-Clement10, Woolsey51, Rose and Woolsey8s, and Hubel and Wiesel la. Tissue blocks were removed from area 17, area 3 in the postcruciate gyrus, primary auditory cortex from the rostral half of the region immediately underlying the middle branch of the suprasylvian sulcus, somatosensory cortex immediately rostral to the anterior branch of the suprasylvian sulcus, and cortex of the frontal pole. The determination of the cortical regions in the macaque was based upon the studies of Powell and Mountcastlea4, aS, Ades and Felder~, and Polyakaa. The tissue was removed from the cortex of the frontal pole, area 1 of the somatosensory cortex in the postcentral gyrus 2 mm lateral to the midline, auditory cortex in the middle of the supratemporal plane and primary visual cortex from both the lateral occipital surface posterior to the lunate sulcus and from the superior bank of the calcarine fissure. The human material consisted of cortex removed from the frontal pole, somatosensory and auditory cortex as in the macaque, and primary visual cortex in the superior bank of the calcarine fissure. Fixation of the human brains was carried out by immersion in formalin. The remainder of the brains were fixed by perfusion. Four types of preparation were examined in the light microscope. (1) Plastic sections (1/~m) stained with toluidine blue. This material was taken from rats and cats perfused with glutaraldehyde-formaldehyde solutions. The tissue blocks were then post-fixed with osmium and embedded in Aralditeal. (2) Thick nitrocellulose sections, tangentially oriented and stained with toluidine blue. This tissue, from rat, cat, macaque and human brains, was perfused with either formalin or glutaraldehyde-formaldehyde solutions, embedded in low viscosity nitrocellulose and sectioned at between 40 and 150 #m in a tangential plane, parallel to the pial surface. (3) Selected tangential nitrocellulose sections, 80-100/~m thick, from the barrel fields of rat brains perfused with glutaraldehyde-formaldehyde solutions. After being photographed, the sections were postfixed in osmium, re-embedded in Araldite and sectioned at 1/~m. (4) Serial sections, 15-35 pm thick, of rat, cat and human tissue, embedded in paraffin or celloidin, and stained either by the Nissl or Cajal methods. These sections were oriented in conventional planes.

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Fig. 7. As Figs. 3-6, but in area 17 ol" the rat cortex. Figs. 8-10. As Figs. 3-7, but in v a r i o u s areas of the cat cortex. Fig. 8, cortical area 41 ; Fig. 9, cortic~fl a r e a 17; Fig. 10, cortical area 3. N o t e the tendency for apical dendrites to a p p e a r in clusters.

BARRELS AND CLUSTERSIN CEREBRALCORTEX

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RESULTS Pyramidal cell clusters in rat cortex It has been observed by Peters and Walsh al that the apical dendrites of pyramidal neurons of layer V are not randomly distributed. Instead, they aggregate into relatively discrete clusters which ascend through the more superficial cortical layers. As the pyramidal cell dendritic clusters pass through layer III, and possibly layer II, the apical dendrites o f pyramidal neurons in those layers are added to the clusters. These observations were originally made on area 3 o f the somatosensory cortex o f the rat al. The present report extends these earlier observations and gives an account o f the dendritic clusters as they are visualized in tangential sections in other cortices in the rat and cat. The description is of the appearance of the clusters at the level of layer IV, where the pyramidal cell clustering is most readily apparent. In the rat cortex, micrographs o f areas 17 and 41 as they appear in tangential 1 # m sections through layer IV are shown in Figs. 1 and 2. Tracings of these and other cortical areas, displaying apical dendrites measuring between 2 and 8 #m, are presented in Figs. 3-7. It should be pointed out, however, that it is not possible to ensure that the drawings are taken from exactly equivalent levels of layer IV, for the thickness o f this layer varies in different areas. It is also difficult to equate the distances between the sectioned apical dendrites in the figures and their parent cell bodies. Consequently, perhaps not much weight should be placed upon comparing the relative sizes o f the apical dendrites as they appear in the tracings. As previously shown 31, in area 3 (Fig. 3) the clustering o f the apical dendrites as they traverse layer IV is quite distinct. Each cluster commonly contains 6-8 dendrites, although some may contain more, and an occasional individual dendrite has no immediate neighbors. The most frequent distance separating the centers o f adjacent clusters is about 50 ktm, but the pattern is not perfect and for any particular pair of adjacent clusters the separation may be up to twice as great. In area 4 (Fig. 4) which is located adjacent and medial to area 3, the clusters are less clearly separated and contain more and generally thinner apical dendrites than area 3. In agreement with the earlier observations of Krieg 20, examination o f coronal Nissl preparations through these areas shows that by comparison with area 4 the layer V pyramids in area 3 are larger and less densely packed, and this is one reason for the difference in appearance of tangentially sectioned clusters traversing layer IV in these two areas. Area 2 (Fig. 5), which is located immediately lateral to area 3, also has fewer layer V pyramids than area 4, and this correlates with the fact that area 2 has clusters that contain fewer apical dendrites than those in area 4. The pattern of clusters in area 2 resembles that in area 3 in that the clusters are relatively discrete entities. Thus, comparison among these three adjacent and morphologically distinct areas of the rat neocortex demonstrates that different cytoarchitectonic areas o f the cortex can display different dendritic cluster patterns. More specifically,' the appearance of the dendritic clusters in tangential sections through layer IV is, in part, a reflection o f the size and packing density of the pyramidal cells in layer V. Cluster patterns in the visual (area 17) and auditory (area 41) portions o f the rat

62

M.I..

F I ! L I ) M A N A N D A. P l : l l RS

cortex are shown in Figs. 6 and 7. Typical o f area 41 is the presence o f some relatively large (4-6/zm) apical dendrites either in isolation or in small groups. There is a wide range o f dendritic diameters, however, and presumably this is a reflection of the wide range o f sizes o f layer V pyramids 20 in this part o f the cortex. In the visual cortex, the characteristic features o f the cluster pattern are the discreteness o f the clusters, the small diameter o f the apical dendrites, and the relatively close packing o f the clusters (30-40 ¢tm center to center). Dendritic clusters in cat cortex

The convolutions o f the cerebral cortex in the cat have an appreciable effect on the appearance o f the pyramidal cell dendritic clusters. A t gyral crests the outwardly radiating clusters diverge f r o m one another as they a p p r o a c h the pial surface, while in the depth o f a sulcus they converge. Thus, the apparent intercluster separation depends on the configuration o f the cortex and depth o f the tangential plane being examined. M o s t planar tangential sections o f the cat cortex usually include at least two contiguous cortical layers, and the apical dendrites are sectioned at a gradation o f angles, with only a small n u m b e r exactly in cross section. Dendritic clusters in the cat cortex have been examined in detail in primary visual cortex (area 17), primary auditory cortex (area 41), and postcruciate gyrus (area 3). In general the clusters appear to be composed o f fewer apical dendrites than the clusters in the rat. Furthermore, the average separation between clusters is more variable (Figs. 8-10). In the plane o f layer IV o f the auditory cortex o f the cat (Fig. 8), most clusters are c o m p o s e d o f 2-6 large dendrites between 4 and 6 / z m thick, and the separation between clusters is 50-70/~m. M a n y apical dendrites are isolated and at least some o f the medium sized dendrites in the field are assumed to represent apical dendrites distal to a site o f bifurcationV,2L The clusters o f the primary visual cortex (Fig. 9) closely resemble those in the auditory cortex. The principal differences seem to be that in the clusters o f the visual cortex the apical dendrites are somewhat thinner and more o f them are isolated. The postcruciate gyrus (Fig. 10) has a n u m b e r o f clusters that contain more apical dendrites than those f r o m either the auditory or the visual cortex. The clusters

Fig. 11. Tangential 80/~m section, stained with toluidine blue, through the rat cortex. The light peripheral band, outlining the section, is layer 1. The central region of the section passes through layer IV in the barrel region (see text). Anterior is to the left. The presence of barrels in this section is obvious. In this and succeeding photomicrographs, visualization of barrels is usually facilitated by diffusing the image of the barrels. It is important to recognize that photomicrographs of barrels never reveal them as prominently as they appear when viewed on the slide under low magnification in a dissecting microscope. This is due, in part, to the restricted depth of field of the photomicrographic apparatus. The barrels shown in the present figure are traced out in Fig. 12. Fig. 12. Tracing of the barrels shown in Fig. 1 I. The area enclosed by the dotted line, which appears as a lighter area in the photomicrograph of Fig. 11, is cortical layer V. a, an anterior group of relatively small barrels; p, a more posterior group of relatively large barrels. The arrow indicates an intermediate group of very small barrels. Calibration line, 500 tzm.

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frequently show a rather wide separation between contiguous apical dendrites, with the consequence that adjacent clusters may be almost confluent with each other. In this part of the cortex many apical dendrites are prominent, for some of them are as thick as 10 #m. Barrels in the barrel field of the rat cortex In coronally sectioned and Nissl stained preparations, the portion of' the rat cerebral cortex which is designated as the barrel field (see Methods) appears to be quite thick; the standard 6 cortical laminae are present, although the borders between layers II and III are nebulous. Layer VI is particularly wide and radial strings ot cells are not prominent. As described by Welker 4~, layer 1V appears as a distinct and irregular cell-dense band, 150-200/~m thick, containing many small darkly-staining cells. In Nissl preparations the perikarya of the neurons are generally round and between 8-13/~m in diameter, and have no obvious dendritic roots extending from them. In examining serial tangential sections an important landmark for determining section depth in the cortex is the sharp line of demarcation between the bottom of layer IV and the top of layer V. This transition is particularly marked because the neuronal packing density in layer IV is significantly greater than in layer V, and because layer V neurons often measure 13-15/~m, with an upper limit of about 30/~m. In addition, the perikarya of neurons in layer V have their long axes radially oriented and typically exhibit dendritic roots in Nissl preparations. Most of the remaining observations in this presentation derive from examination of the toluidine blue stained, thick, nitrocellulose-embedded, tangential sections, usually with a dissecting microscope at a total magnification of times 10. Figs. 11 and 12 show the barrels as they are seen in layer IV of the barrel region of the rat cortex. The barrels appear as contiguous rings and, as described by Woolsey and Van der Loos 53, they are of two principal sizes. In the anterior portion of the barrel field the barrel profiles are circular (Fig. 12, a), while in the posterior portion they are larger and oval (Fig. 12, p). The smaller anterior barrels have an average diameter of 190 #m, with a range of between 100 and 230 #m. The larger posterior barrels have an average minor axis of 190/tin (range about 150-250pm) and an average major axis of 390/~m (range about 250-430 pro). Examination of Fig. 11 suggests a population of small barrels with diameters of approximately 75 #m may be present. These are indicated by the arrow in Fig. 12. Such small barrels have thinner walls than either of the two principal groups of barrels described previously and are more difficult to discern. Figs. 13-15 illustrate barrels of the principal variety at higher magnifications. It can be seen from these figures that a barrel profile is produced by a ring-shaped aggregation of small, darkly staining cells, in the middle is a light central area which in the terminology of Woolsey and Van der Loos a3 is called the hollow, and this is separated from an adjacent hollow by a wall about 4 cells thick. Where barrels abut they may appear to share a common wall, but occasionally (see Fig. 13) the wall appears to be composed o f two sides, one side belonging to each barrel, separated by a septum which is almost free of Nissl stained perikarya. The wall-septum-wall

BARRELS AND CLUSTERSIN CEREBRAL CORTEX

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Fig. 13. Tangential 80/~m section, stained with toluidine blue, through the rat barrel region (see text). The pial surface is to the right. The lower left portion of the figure passes through cortical layer IV. Numerous barrels are present in this region. Two barrels are indicated by arrows. These barrels are shown at higher magnification in Figs. 14 and 15. Calibration line, 250/~m. Fig. 14. Higher magnification of the barrel indicated by the left arrow in Fig. 13. Fig. 15. Higher magnification of the barrel indicated by the right arrow in Fig. 13.

66

M. l_. FELl)MAN AND A. Plil IRS

appearance presumably depends critically on plane of section. Analysis of serial tangential sections, enabling reconstruction of individual barrels in 3 dimensions, leads to the conclusion that a barrel wall is formed by a cuff- or barrel-shaped sheet of cells and that the height of each barrel is about 200 ktm. This indicates that in the rat the barrels probably extend through the full thickness of cortical layer IV. From the position of the barrels in layer IV and the size and shape of the component neurons, we assume that the neurons forming the barrel walls are stellate cells. In the cortices we have examined, no cell-dense patches (without hollows), described as 'barrels' in the cortex of the brush-tailed possum 49 and armadillo ag, have been observed. As described under Methods, 5 selected 100/~m nitrocellulose-embedded sections containing barrels were examined and photographed, and then resectioned at a thickness of 1 #m for further analysis. Examination of these tangential 1/tm sections through portions of individual barrels permitted estimation of the differential distribution of neuronal and neuroglial perikarya and of blood vessel profiles in the barrel walls and hollows. This estimation was carried out by making tracings of the positions of these elements as they appeared in the 1 Mm sections and then superimposing on the drawing the outlines of the barrel walls and hollows as they appeared in the photographs of the initial 100 # m nitrocellulose sections. These composite drawings were then overlain with a transparent sheet marked with grid squares, each square representing 370 sq./~m of section area, and counts made of the numbers of different elements contained in squares lying within the boundaries of either barrel walls or hollows. The average numbers of the neuronal and neuroglial perikarya and blood

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blood vessels in barrel walls and hollows. This analysis was carried out on tangential 1 ,urn toluidine blue stained sections through layer IV in a regmn containing known barrels. The data indicate that it is the increased packing density of neuronal perikarya in the barrel wall that is primary responsible for the appearance of a barrel in a thick tangential section. See text for additional details.

BARRELS AND CLUSTERSIN CEREBRALCORTEX

67

vessel profiles present in the walls and hollows of the barrels in a representative 1 # m section are given in Fig. 16. The data in this figure show that while blood vessels are not differentially distributed, cell perikarya are more densely packed in the barrel walls than in the hollows. This increase in cell density in the walls is not due to neuroglial cells, which are evenly distributed, but to neurons. On the basis of the analysis of 1 # m thick sections the average neuronal packing density in barrel walls is about 1.5 times greater than that in hollows. In a parallel analysis carried out by direct examination of an 80/zm thick nitrocellulose section the packing density of cells in the walls was estimated to be twice that in the hollows. An incidental observation made during the examination of 1 #m sections through known barrels is that the presence of strings of three or more apparently contiguous neurons seems to be diagnostic of a barrel wall.

Cluster-barrel relationships in the rat In addition to the analysis of neurons, neuroglial cells, and blood vessels in

Fig. 17. Tracing of the positions of the clustered apical dendrites (small circles) and barrel walls (interrupted lines). The outlines of the barrel walls were traced from an 80/~m tangential section through layer IV in the rat barrel region. This section was then re-sectioned at 1 ~tm and the dendrites drawn in. No particular relationship between the position of the clusters and the barrel walls is evident, b, blood vessel. See text for additional details.

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M. L. FELDMAN AND A. P|III-RS

Fig. 18. A: photomicrograph of 80/~m toluidine blue stained tangential section, cat somatosensory cortex. The area enclosed by the rectangle is shown at higher magnification and with diffusion in B. Calibration line, 500/~m. B: the area indicated in A printed through a diffusing screen using high contrast materials. The arrows indicate several cellular rings.

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the 1 #m sections, the positions of clusters of the apical dendrites of layer V pyramidal cells passing through layer IV were also analyzed with respect to locations of the barrels. As shown in Fig. 16, as well as in the data from the other thick sections resectioned at 1 #m, the dendrites contributing to the clusters, whether they are considered as individual entities as in Fig. 16, or considered in terms of the locations of clusters, are equally disposed in the walls and hollows of the barrels. A typical example of one of these analyses is shown in Fig. 17. We conclude, therefore, that there is no relationship between the spatial disposition of the barrels formed by stellate cell perikarya of layer IV and the clusters formed by the apical dendrites of layer V pyramidal neurons. Cell groups in other cortices

Our examination of tangential sections through other cortical regions of the rat brain and through the cortices of the cat, macaque and man has revealed no cell groupings which appear with the same consistency and clarity as those in the rat barrel field. However, some ring-shaped cellular aggregates could be seen. They differ from the barrels described above in that they are difficult to detect, possess walls that are often only one or two cells thick, and although they occur preferentially in layer IV, they are occasionally found in layer II. In addition, these configurations often occur as isolated rings of cells rather than as part of a field of contiguous rings, and generally the rings cannot be traced from one thick section to another. The shapes of these rings of cells as seen in tangential sections are round or oval, although in some instances, as in the human frontal and auditory cortex, they have more variable outlines. It seems that these configurations are not produced by the chance juxtaposition of strings of cells, since we have seen some individual rings which extend through the depth of thick nitrocellulose sections. Examples of such ring-like configurations of neurons are shown in Figs. 18 and 19. The diameters of the rings vary between 50 and 300/~m, but, as pointed out above, the positions are so variable that is it impossible to specify any location in which they can be consistently observed. At present, therefore, we consider it appropriate to simply mention the existence of these ring-like configurations of cells, examples of which we have seen in most of the different cortices examined. DISCUSSION

The principal conclusions suggested by the data of the present study are: first, that clusters of pyramidal ceils are a general feature o f mammalian neocortex, and that clusters in cytoarchitectonically different areas may vary in morphology; second, that barrels are not a general feature of mammalian neocortex: and third, that there is no clear anatomical relationship between the position of the clusters and the position of the barrels. Clusters

The present results comparing clusters formed by the apical dendrites of pyramidal cells in different neocortical areas of the rat and cat expand our earlier account 31

70

M. L. I~ELDMAN A N D A. PEI'/~:R,";

Fig. 19. A: photomicrograph of 35 pm cresyl violet stained tangential section, human primary visual cortex. The area enclosed by the rectangle is shown at higher magnification and with diffusion in B. Calibration line, 500/~m. B: the area indicated in A printed as Fig. 18B. The arrows indicate several cellular rings.

o f this aspect o f cortical o r g a n i z a t i o n in area 3 o f the rat cortex. In other studies, Fleischhauer e t al. 7 a n d Massing a n d Fleischhauer 22 have described the dendritic clusters in the parietal cortex o f the rabbit a n d cat, while von B o n i n a n d Mehler 5

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have given a survey of clusters in different areas of human and macaque brains. Von Bonin and Mehler ~ find the clusters to be about 80/zm apart and closest together in the visual cortex, while in the rabbit, Fleischhauer et al. 7 give the separation between clusters as about 40-45 #m. These values compare with the separation of about 50 #m in the rat and 50-70 #m in the cat. Such figures are only approximate, for the dendritic clusters do not form a regular pattern. The separation between neighboring, large clusters is often much greater. The values are also dependent upon the degree of curvature of the cortical area being examined. It is clear that the morphology of the clusters may vary between cytoarchitecturally different cortical areas. In the parietal cortex of the rat, for example, the clusters in area 4 are relatively large and tend to be almost confluent with each other, while in areas 2 and 3 the individual clusters contain comparatively fewer dendrites and are more discretely localized. Essentially, the size and number of the apical dendrites reflect the size and packing density of the pyramidal neurons in layer V. The clustering of apical dendrites has now been described in the rat, cat, rabbit, macaque and human, and is also present in the opossum (Walsh and Peters, unpublished observations), so this form of vertical organization seems to be generally present in mammalian neocortex. No doubt the frequently observed arrangement of neuronal perikarya in radial strings 21 is related to the presence of the dendritic clusters. Similarly, the axons of the neurons forming the dendritic clusters may contribute to the fascicles of myelinated axons radiating through the deeper laminae of the cortex 17. Caja136 considered these radiate fascicles to contain fibers of projection that originate from pyramidal neurons, and Nauta and Bucher 26 found that lesions in the grey matter of the rat striate cortex produce degeneration of the radiate fascicles. As discussed previouslyal, it seems that the size of the clusters and the 30-80 /zm intercluster separation are too small to allow these entities to be considered as the anatomical correlates of the neurophysiologically defined functional columns which generally are reported to be 100-500/zm in width. However, in their discussion of the dendritic clusters in the rabbit parietal cortex, Fleischhauer et al. 7 conclude that the dendritic clusters do represent the morphological substrate of a particular kind of vertical column. They point out that to explain penicillin spikes it is necessary to postulate the existence of a functional unit for discharges, i.e., a generator, most likely consisting of a few tens of apical dendrites that are synchronized in their discharge. On the basis of studies by Petsche et al. 32, they suggest that a grid of homologous, densely packed and vertically arranged units must be assumed, and from electrophysiological findings in the rabbit they postulate a separation of about 50/~m between units. This distance is approximately the same as that separating the dendritic clusters. Barrels

With respect to the barrels in the rat barrel field, our observations make it apparent that their walls are obvious because the concentration of neurons in them is 1.5-2.0 times that present in the hollows. A similar finding has recently been re-

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ported by Pasternak and Woolsey ')9 for barrels in the mouse cortex, in which they ' find a barrel side to have 1.42-1.97 times more cells than the hollow. Other than in material from the rat barrel field we have not been able to recognize a clearcut, consistently identifiable barrel field in any of our cortical material. In a number o f areas, isolated rings of cells have been found in tangentially oriented sections passing through layers IV and II, and although we have the impression that these are more than chance arrangements of neurons, we have been unable to produce any data which would allow support of a concept that groups of neurons arranged in definite tangentially oriented groups are a general feature of the m a m m a lian neocortex. This is also suggested by the data of Weller 49, who examined the cortex of 2l marsupial species, but found 'barrels' with the form of irregular dense clumps of cells, lacking hollows, in only 5. Similarly, Killackey's recent findings based on thalamocortical degeneration suggest that barrels may be absent in opossum, hedgehog, and Eastern grey squirrel is. It is necessary, however, to be cautious about interpreting reports of failure to find barrels, for inappropriate section plane and thickness could easily account for the non-appearance of barrels. In this context it is interesting that in Woolsey's 52 initial account of barrels in the mouse similar entities were not observed in serial sections o f the rat brain. Apart from our observation of isolated and scattered neuronal rings in layer IV in a variety of cortices - - an observation of indeterminate significance at the present time - - two clearly different structures have been described as 'barrels'. These are: (1) the orderly, hollow structures observed in mouse SI cortex by Woolsey and Van der Loos 53, in rat Sml cortex by Welker 45 and the present authors, and in the cortex of the deer mouse by Weller 4s, and (2) the irregular cellular clumps, without hollows, observed in marsupial parietal cortex by Weller 49 and in armadillo postsupraorbital cortex by Royce 39. At present, only the hollow barrels have been investigated from the standpoint of functional significance. Taken together, these latter investigations 44,4a, 4s,sa build a strong case for the proposition put forward by Woolsey and Van der Loos 53 that each barrel is concerned with information originating from one facial sinus hair. This specificity is reinforced by the finding that in rat SmII cortex, lying adjacent to the SmI (barrel) field, neither sinus hair representation nor barrels are found 46. With respect to the irregular cellular clumps in the parietal region of the brush-tailed possum, the field in which the clumps are found coincides with the somatic sensory representation o f the entire body 49. The cellular clumps in the armadillo cortex are located in the somatic m o t o r and sensory area for the forelimb and trunk 39. From a functional standpoint, then, the clumps appear to be more generalized structures than the hollow-type barrels. Morphologically, they differ from the hollow barrels in their relative irregularity and, o f course, in their lack of cell-sparse hollows. Since Killackey a8 has recently observed dense nests of thalamocortical fibers that appear to fit within the hollows of rat barrels, one may speculate that the form o f the thalamic input to the two varieties of barrel differs. We do not yet have a complete explanation o f the limited distribution of barrels in mammalian cortex. Woolsey and Van der Loos 5~ have called attention to the possible relationship between the hollow barrels and the punctate nature o f the vibrissae

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considered as discrete sensory receptors, and have suggested that the prominence o f the barrels may reflect the relative significance of the vibrissae system in certain rodents. Subsequent work has tended to be consistent with each of these hypotheses. The terminal nests of thalamocortical afferents in rat somatosensory cortex is, and the barrels with which they seem to be associated, can be viewed as one attempt to maintain the discrete nature of sensory information originating from punctate receptors. And the demonstration that barrels are preserved even in the severely disrupted cortex of micrencephalic rats 47 suggests the fundamental nature o f this sensory system in the rat. With respect to species differences, it is interesting that a thalamic lesion which produces discrete groupings of degeneration in rat somatosensory cortex produces a uniform lamina of degeneration in the cortex o f the eastern grey squirrel, the hedgehog, and the opossum, all species which have vibrissae is.

Relationship to functional columns The size and distribution of the hollow barrels tempt speculation that these structures are the correlates, in layer IV, of the neurophysiological columns described initially by Mountcastle z4 and subsequently by a number of other investigators. Lines of evidence which favor this view have been summarized by Woolsey and Van der Loos ~3. Proof of the equivalence of barrels and columns is still lacking. However, it is important to stress the fact that even if the barrels in rodent cortex are found to be congruent with functional columns, this would not mean that barrels are the general structural substrate for all functional columns. If they were, then barrels would presumably be visible in all of the cortices in which a columnar arrangement has been demonstrated neurophysiologically. Neither the present study nor the literature supports the existence of clearcut barrels in these locations, such as cat auditory 1,9,11,27, visual 12,1a, somatic sensory24, ~5, and motor4, 50 cortex, and monkey visual14,15, a4 and motor 3 cortex. Two entities which do satisfy the requirements of a generalized distribution are the clusters of pyramidal cells and their dendrites, described here, and the radial strings o f cells described by I.orente de N6 zl, among others. The dendritic clusters, which are spaced 30-80/~m apart, are too small to be the direct correlate o f the functional columns, which are generally reported to be 100-500 # m wide. However, the dendritic clusters do provide a substrate of vertically oriented units that could be a basis for the similarity in response properties of neurons encountered successively in normally oriented electrode penetrations. If the dendritic clusters are components o f the columns, then a column must include a number of pyramidal cell clusters, and these are presumably linked to each other and to other neurons in the column by the pattern o f axons in the cerebral cortex. The vertical linking of neurons into units may be achieved through vertically oriented intracortical axons and axon collaterals6,21,a6,4z, 43, while the thalamocortical axons 8,15,16,18,21,4°,41,43 must be considered to be of primary importance in defining the sizes and shapes o f the columns. Such axonal components derived from both intrinsic and extrinsic sources could combine to define columnar fields, each possessing some degree o f functional independence. However, the hypothesis that the functional

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columns are primarily defined by the distribution of axons implies that no particul~lr pattern of neuronal perikarya conforming to the boundaries of the columns need exist. Indeed the results of the present and of other studies on the cerebral cortex indicate that such patterns are somewhat exceptional. Even when such a neuronal pattern, for example a barrel, does exist it is possible that the pattern is formed as a response to axonal proliferation. Developmental studies show that barrels are not present in newborns37, 4s, but that they may form in relation to the somatotopically organized axons which arrive in the cortex and displace perikarya laterally to form the barrels 37. Presumably these proliferating axons are the ones demonstrated in their mature pattern by Killackey is in the rat. Killackey has shown that lesions of the ventral posterior nucleus of the thalamus of the rat lead to the formation of discrete groups o f degenerating axons and their terminals in layer IV of the parietal cortex. Such a distribution of the axon terminals can probably be attributed to the highly circumscribed nature o f the individual mystacial vibrissae and the preservation of this discreteness at thalamic and cortical levels. In contrast to these discrete patches of thalamic axon terminals in the rat are the results obtained from lesions in the thalami of the Eastern grey squirrel, the hedgehog, and the opossum is. These animals have vibrissae but the pattern of degeneration in the cortex is a uniform lamina, so that the receptive fields corresponding to the vibrissae of these animals seem to have no morphological arrangements of neurons which can be readily identified. Another example of columns defined on the basis of the axonal input are the stripe-like fields o f terminal degeneration, corresponding to eye-preference columns, produced by Hubel and Wiese115,1~ after making small lesions confined to a single layer of the lateral geniculate nucleus of the monkey. No arrangements of neurons can be identified which correspond to the areas of axonal degeneration. Thus, on the basis of the evidence presently available, we conclude the following. Barrels may be a morphological manifestation o f the functional columns corresponding to the mystacial vibrissae of the rat's snout, but such arrangements of neurons in the cortex are unusual. Pyramidal cell clusters must pass through functional columns because they occur throughout the neocortex. The columns are probably defined by the extrinsic axonal input to that area of cortex, with the vertical linking of neurons effected by the distribution patterns of intrinsic axons. ACKNOWLEDGEMENTS

For their valuable assistance during the course of this project, the authors wish to thank Charles Ribak, Lawrence McCarthy, and Salvatore Lunetta. They also wish to acknowledge the co-operation of Dr. Thomas Kemper in making available to us human material in the collection of the Elmer E. Southard Unit Research Laboratory. We are also grateful to Dr. D. Pandya for furnishing us with macaque and human material. This work was supported by U.S. Public Health Service Research Grant NS07016, from the National Institute of Neurological Diseases and Stroke.

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