Journal o f Neurocytology 5 , 5 0 9 - 5 2 9 (1976)

Aspects of cortical organization related to the geometry of neurons with intra-cortical axons F. V A L V E R D E

SecciSn de Neuroanatomz'a Corrzparada,lnstituto Cajal, Madrid, Spain Received 12 February 1976; revised 13 April and 14 May 1976; accepted 21 May 1976

Summary Using the Golgi method, cells with intra-cortical axons in the visual cortex of young mice were classified according to defined geometrical axonal shapes. This study principally describes a computer technique and its application to the study of neuronal morphology. Neurons were converted in a sequence of three-coordinate points which were stored in digital form on magnetic tape. From the stored data the total real length in space of dendrites and axons was obtained and the results compared in two groups of mice raised under different conditions. Preliminary observations show short axonal lengths in mice raised in darkness. Using Eulerian coordinate transformations, reconstructions of individual neurons and of groups of several neurons and fibres were obtained by generating displays of different views after rotation around the horizontal axis. Reconstructed pictures were compared with their corresponding original drawings in order to describe particular aspects of cortical organization. Introduction The basic plan of the structural organization of the visual cortex was established by the early Golgi studies of Cajal (1911, 1921), O'Leary (1941) and Polyak (1957). In recent years, a renewed interest in the Golg[ method originated from two main trends of fundamental investigation: firstly, from a search for structural correlates related to the use of modern techniques, such as electron microscopy, anterograde tracing of pathways, label injection and neurophysiology, and, secondly, from the need for the establishment of a perfectly defined scheme of neuronal morphology and connectivity, in order to understand and clearly define the changes in cortical cells brought about by environmental manipulations. Along this second line, we began a series of studies designed to investigate the extent to which neuronal structures and cortical organization were affected by sensory deprivation (Valverde, 1967, 1968, 1971a; Ruiz-Marcos and Valverde, 1970). These studies provided details of the distribution of dendritic spines, dendritic organization, the form and distribution of specific afferents and their presumed inter-relationships. 9 1975 Chapman and Hail Ltd. Printed in Great Britain

510

F. V A L V E R D E

T h e p r e s e n t s t u d y r e p o r t s f u r t h e r o b s e r v a t i o n s on t h e q u a l i t a t i v e , q u a n t i t a t i v e a n d t h r e e - d i m e n s i o n a l c h a r a c t e r i s t i c s o f cell t y p e s a n d n e u r o n a l circuits in t h e visual c o r t e x o f t h e m o u s e . In this w o r k , c o r t i c a l n e u r o n s h a v e b e e n d i v i d e d i n t o t w o g r o u p s : cells w i t h a x o n s e n t e r i n g t h e w h i t e m a t t e r ( p r o j e c t i n g n e u r o n s ) , and cells whose axons do not enter the white matter (neurons with intra-cortical axons).

Material and methods

Source, histology and recording of material The present observations were made on the brains of a large collection of a closed colony of black mice derived from an inbred stock (C57BL/6J). The brains were stained by the Golgi method, performed either by immersion-fixation, or by perfusion-fixation with osmium-dichromate solution, followed by a triple impregnation cycle, as described elsewhere (Valverde, 1970). 38 brains, from animals ranging in age from 1 0 - 1 9 days, were selected for this study, sectioned in the frontal plane at 1 5 0 - 2 0 0 / J m , and mounted serially. 58 cells with intra-cortical axons, and 36 pyramidal cells from the visual cortex were drawn by camera lucida, at a final magnification of x 1140, using x 40 (NA 0.65) and x 63 (NA 0.90) dry objectives with working distances of 0.7 and 0.09 mm respectively. Most selected cells appeared entirely included within a single Golgi section. When this was not the case, it was necessary to follow the axons or dendrites into adjacent sections. Cells with processes extending into adjacent sections were drawn on sheets of transparent paper and the ramifications of the whole neuron were obtained by the superposition of the individual drawings. Reconstruction of blocks containing several cells were obtained from three to seven adjoining sections. The identification of equivalent processes from one section to another was achieved by superposition of tomographic drawings made of the two confronting planes in every pair of adjoining sections.

Computer technique for data storage The series of individual drawings of neurons, or fibres, were transformed into a set of points by reading x, y and z coordinate values along axonal and dendritic branches. These values were stored on magnetic tape for permanent filing and further processing. Observations were compiled by reading z-ordinate values using the fine focus knob of the microscope (Zeiss Ultraphot II) supplemented with a plastic disc 7 cm in diameter. One complete turn of the fine focus knob resulted in 155/.tm of vertical displacement. A number of different Fig. 1. Reading procedure for data storage and for the calculation of the angle of rotation. (A) The depth from the surface of the section in/lm is recorded for each point including origins, bifurcations, sample points and terminals. Subsequent reading of the x- and y-values results in a set of three-coordinate values and codes which are stored on magnetic tape. During the reading procedure, codes 3 and 4 (bifurcations and terminals) cause a respective increase or decrease by 1 in the level of bifurcation: this latter parameter is kept in a separate accumulator. When a dendrite or an axon has been read, the level of bifurcation should be set at 0 for codes 1, 5 and 6 (next origin, end of dendritic reading or end of neuron tracking respectively); otherwise an error message is printed. The dashed paths indicate the sequence followed during the reading procedure. (B) This scheme shows a side view of a block of five consecutive sections (1-5). In the drawing the plane of sectioning, or the observer's plane OB, is not oriented perpendicular to the tangential plane, or the projection plane OA, at the surface of the brain. In order to obtain projections of recorded structures on the OA plane, the angle 0 is calculated from measures (a-b, c-d) obtained from apical dendrites o f surrounding pyramidal cells. The measure e represents the thickness of each section, and e' its projection on the OA plane (see text).

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circular scales in that range could be fitted onto the disc so that correlative z-values for successive serial sections were obtained. Beginning at a point which corresponded to the centre of the cell body, z-values of the following selected points were brought into sharp focus and recorded on the drawing (Fig. 1 A); namely the centre of the cell body, the origin of each dendrite, the origin of the axon, all bifurcation points, all terminal points, and a number of selected sample points along segments of the processes. Cortical afferent fibres were treated in the same way. The drawing of each cell was later positioned on a l a n e drawing board to record the corresponding x- and y-ordinate values of each of the z-ordinate points recorded previously. The board was equipped with a precision technigraph (Laster Junior Florett from Nestler) consisting of a horizontal fixed arm 120 cm in length (the x-axis), and a vertical sliding arm 100 cm in length (the y-axis). Both arms bore scales graduated in micrometres at the same final magnification as the drawing. A free-moving, hand-held, cursor attached to the goniometer head was slid along the cell processes to obtain x- and y-ordinate values simultaneously. The topological identifiers used were: 0 for the coordinate values of the centre of the body, 1 for origins of dendrites and axons, 2 for sample points, 3 for bifurcations, 4 for end points, 5 for end of dendritic arbor reading, and 6 for end of axonal reading and end of neuron tracking. The values of the three coordinates of each point, plus a topological identifier, were typed on the key board of a microcomputer (Compucorp model 425 G, using a model 3000 Processor which has a maximum memory size addressable of 16 384 8-bit bytes). Each coordinate point, and its corresponding topological identification code (typed at the right of the decimal point), was stored in a register (8 bytes or 16 BCD digits in length) and addressed sequentially into the data storage section of the computer which can hold up to 512 registers, which is a sufficient capacity to accommodate all the dendritic and axonal data points of the types of neuron analysed in this study. In this procedure a program was used to record, check errors and store the set of all values onto a magnetic tape with cassette drive unit (Compucorp model 492). Neurons were always processed in a specific sequence. Beginning at the centre of the body, whose coordinate values served to position it in space, dendrites were tracked distally from their origin at the cell body. When a bifurcation occurred, the tracking continued along the right branch; if a second bifurcation was reached the tracking proceeded again along the right branch and so on. When a terminal point was reached, the tracking continued from the next point after the last recorded bifurcation, or the point of origin of another dendrite, or the axon, if the previous branch had been completed (Fig. 1 A). If n t is the number of terminal points, and n b the number of bifurcations, when the tracking of a particular dendrite, or an axon, had been completed a subroutine checked that: nt=nb +1. This equation must be satisfied, otherwise the computer printed an error message. Simple instructions permit the deletion of any given point, or series of stored points. The time required to perform the tracking of one neuron consisting of 4 0 0 - 5 0 0 three-coordinate data points is about 45 min. The volume of cortical tissue that can be covered with this system is

Fig. 2. Original camera lucida drawing ( A ) a n d model (B--D) representations of a cell with an intra-cortical axon (class I) recorded from three adjacent Golgi sections. (A) The cell was drawn in a plane in which the O - Y axis is parallel to its principal axonal branch a. (B) Reconstruction in the same plane made from data points stored on magnetic tape. (C) Reconstruction obtained after rotation of 120 ~ around the horizontal O - X axis. (D) Representation of all bifurcation points of the axon in the same plane as in C. Bifurcation points are coded according to an arbitrary scale represented along the O - Y axis in B (see text). The centre of the b o d y is represented by small black circles in B and C and by a dot with a curved arrow in D. A single bifurcation pointp(x,y,z) can be identified in all representations.

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unlimited, because the three scales can be made and installed for the desired extension. The accuracy is • 1/am, and no more than a 2% variation was found in computations of total axonal lengths (see later) performed by three different operators. Once the data from neurons had been stored on magnetic tape (one 90 metre conventional cassette stored about 150 neurons each one consisting of 3 0 0 - 5 0 0 points) they could be analysed in various ways. A recall program could retrieve all coordinate points in the same order as the tracking sequence and it was possible, by connecting all points with straight paths, to display a model of the neuron equivalent to the original camera lucida drawing (Figs. 2, A and B). The displays could be drawn from the data outprints on paper tape, or automatically reproduced via an X-Y plotter (Compucorp model 493) interfaced to the microcomputer.

Real dendritic and axonal lengths This analysis was carried out on 14 cells with intra-cortical axons of which 6 were from control animals and 8 from animals raised in complete darkness from birth to 19 days post partum. All dendritic and axonal branches of the cells in these groups were traced to all their terminal points. Dendritic and axonal lengths were calculated from the distance, in real space, between consecutive stored points using the Pythagorean theorem for three dimensions:

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The computer calculated, for each cell, running totals for every dendrite, total dendritic length, total axonal length and the axonal-dendritic quotient. From this calculation, it will be seen that the values obtained are an approximation to the true length because the chord length (distance between two consecutive points) is shorter than the length of the arc it subtends. If the distance between consecutive points is made smaller it is possible to obtain computed lengths as close as is desired to the true length, but only by recording from a very large number of points. An approximation factor has been calculated as follows: The first collateral axonal branch originating from axon I f of cell f in Fig. 4 was read every 2 ~tm interval of shaft length giving a set of 590 triplets of data points which, with their corresponding codes, were stored on magnetic tape. The computer was programmed to print out; firstly the total length, obtained by summing the distances between all recorded points; subsequent totals were obtained by computing the distances between every other points, between every third point, and so on, until the total sum lengths of the distances between points representing only bifurcations and terminals were finally obtained. Considering the first computed value as being very approximate to the true total length, all the subsequent values differ from the first in stepwise quantities that are nearly proportional to the difference between all recorded points and the number of points used to calculate the subsequent value. On the basis of these results we have generalized that the computed lengths from stored data differ by about 14% from the total length, or in other words, the figures obtained after computing dendritic and axonal lengths should be multiplied by the factor 1.16 to obtain the best approximation to the true length. Fig. 3. Axons of cells with intra-cortical axons arranged according to the depth of cell bodies below the pial surface. The first row ( 1 - 7 ) comprises axons ramifying above the cell body (class I axons). The second row ( 8 - 1 5 ) shows ascending axons with a vertical main stem emitting collaterals at different levels (class II axons). The first three examples in the third row ( 1 6 - 1 8 ) represent axons with recurving and long descending collaterals ramifying above and below their cell bodies (class III axons). 1 9 - 2 1 represent types with diversely oriented axonal collaterals with non-specific patterns (class IV axons). 22 and 23 are the axons of pyramid-like cells with intra-cortical axons forming recurrent arcs. 24 is an intra-cortical axon of a typical pyramidal cell with collaterals distributed in layers IV and V. (Golgi method. Camera lucida drawings. Mice 1 0 - 1 9 days of age).

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F. V A L V E R D E

Reconstruction of cells after rotation With a neuron, or group of neurons, transformed into an orderly set of three-coordinate points, it is possible to generate projections on different planes using standard Eulerian coordinate transformations. In this work, individual neurons and groups of related neurons in the same, or in several adjacent Golgi sections, were rotated in space in order to obtain individual, or whole block projections, from different angles of view. Fig. 2 A shows a camera lucida drawing of one cell with an intra-cortical axon (corresponding to axon No. 3 in Fig. 3) whose body is located at the junction between layers III and IV. This neuron was drawn from three consecutive sections. Fig. 2 B shows a model of this neuron obtained from data stored on magnetic, tape. Fig. 2 C shows the reconstruction when the cell is rotated 120 ~ around the horizontal O - X axis. With this procedure we wanted to obtain projections on planes tangential to the pial surface at the point of intersection of a line perpendicular to the surface of the brain passing through the body of the neuron. For obvious reasons, the plane of sectioning in Golgi material never coincides with the radial axis passing through a given neuron. Therefore, an angle ( 0 ) h a s to be added to, or substracted from 90 ~ The angle of rotation (~0) is defined as = 90 ~ + 0 depending on whether the plane of section forms acute, or obtuse angles, with the tangent to the surface of the brain in relation to a fixed sagitta[ orientation. The angle 0 (Fig. 1 B) is obtained by averaging the values of the angles that neighbouring apical dendrites form with the surface of the section: 0 = arctan

(ab - cd)/ac.

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Since we have framed all structures into a volume of nssue in which the O - Y axis always lies perpendicular to the axis of rotation, the x-values remain the same, while the new j - v a l u e s are either ignored (since this axis is now perpendicular to the observer) or are used to generate colour coded tracings in the X - Y p!otter for separation of different depths. The display of neurons after rotation shows nevr that were not apparent in frontal views, b u t the impression of depth along the y-axis is lost. A complementary program abstracts all bifurcation points from the whole neuron, giving their distribution around the centre of the body. Rotated bifurcation points can now be grouped into a number of subsets according to their depth from the pial surface. The thickness of the visual cortex was divided into eight levels numbered 1 - 8 from medullary white to pial surface. Bifurcation points are coded by giving them the same number as the segment in which they were found. The orientation of separated bifurcation points are clearly displayed by this technique (Fig. 2 D).

Fig. 5. Reconstruction of all cells represented in Fig. 4 after rotation of 92 ~ around the horizontal O - X axis. The drawing represents a projection onto a plane tangential to the surface of the brain, of three consecutive sections ( 1 0 - 1 2 ) . The dendrites of cell f have not been represented. The axon of the latter (in red) ramifies preferentially among pyramidal cell dendrites. Note truncated branches anterior to cell g in section 11, which could not be followed in section 10.

Fig. 6. A group of cells recorded from two adjacent sections. Cell g has moderately spinous dendrites radiating in all directions and an ascending axon (in red) emitting collaterals in layers IV and V, and terminal branches spreading in the zone of superficial pyramidal cells. Cells a - f and h appear to be related (see text). (Golgi method. Camera lucida drawing. Normal mouse 11 days of age).

Neurons with intra-cortical axons

517

Observations CLASSIFICATION OF NON-PYRAMIDAL CORRELATION

INTRA-CORTICAL AXONS AND

WITH THEIR CELLS OF ORIGIN

Axons of non-pyramidal cells having an intra-cortical distribution in the visual cortex of the mouse were grouped into five classes. Three of these classes represented about 80% of recorded intra-cortical axons and were classified on the basis of the spread of collateral branches above the level of their cell bodies, on the existence of a main, vertically ascending axonaI stem, and on the distribution of a number of recurving collateral branches, descending below the level of their cell bodies. The fourth class comprised axons which could not be classified according to the above criteria. The fifth class included axons regarded as transitional types with characteristics similar to axons of pyramidal cells with intra-cortical axons. 24, apparently fully impregnated, intra-cortical axons were arranged in three rows according to the depth of their cell bodies below the pial surface (Fig. 3). They represent examples of all classes of intra-cortical axon encountered in the present material. Class I axons (Fig. 3, Nos. 1 - 7 ) The bifurcations and collateral branches of the axon are all superficial to the cell body. All collaterals are distributed in cylindrical or ovoidal volumes. Cells with class I axons have been found in layers I I - I I I (Rose's, 1929 stratification) and the vertical extensions of all collateral branches is never larger than 300-400/am, so that deeply situated cells such as Nos. 5--7 (Fig. 3) have the majority of their collaterals below layer IV. In the visual cortex of the mouse, the most commonly found cell type with an intra-cortical class I axon is a large cell with dendrites radiating in all directions. These cells were called large stellate cells in a previous study (Valverde, 1968). The shape of the dendritic and axonal fields of these cells resembles some short axoned stellate cells described by Cajal (1911, 1921) in his layer of stellate cells, and the star cells with extended axonaI arbors described by O'Leary (1941) in the visual cortex of the cat. They appear more similar to the giant short axon cells described by Lorente de N6 (1922) in layer IV of the mouse's neocortex. In the visual cortex of the monkey, Lund (1973) has described types of stellate cells with dendrites having few spines and whose axons resemble the class I axons of Fig. 9. Reconstruction of a block of five consecutive Golgi sections ( 7 - 1 1 ) containing 29 ceils, rotated 107 ~ around the horizontal O - X axis. The drawing represents a projection onto a plane which is tangential to the surface of the brain. This reconstruction includes all the cells of Fig. 8. The cells were originally recorded on magnetic tape from frontal sections, having previously fixed a common origin in a three-coordinate system. The axons of pyramidal cells were not represented. Specific afferent fibres appear in blue, and the axons of cells with intra-cortical axons are red. An observer looking through a window with an angle of view given by arrows x and y would see the perspective drawing as in Fig. 8.

518

F. VALVERDE

the present work. However, on the basis of her classification, the comparison is difficult, as she has included, under the general term stellate neurons with spine-free or sparsely spined dendrites, such types as Martinotti cells and Cajal double bush dendritic cells, which others consider as very specific neuron types.

Class H axons (Fig. 3, Nos. 8--15) A vertically ascending, main axonal stem forms the axis of the axonal arborization. These axons have been found in layers II-III, in the lower part of IV and in V. When the cell body is located in the upper part of layer I I - I I I the main axonal stem ascends for a short distance and turns through a right angle to course horizontally for various distances within layer I (Fig. 3, Nos. 8 and 9). Collateral branches, originating from the main stem, may be in the form of long and thin descending fibres. When the cell body is located more deeply, the collateral branches appear segregated into two groups. The first group always originates within the first 150 ~m segment along the main stem, and consists of a number of descending branches traversing layer IV, and sometimes layers V and VI. The second group of collaterals originates at upper levels where they can be followed for long horizontal distances. Cells having class II axons represent the second most frequently found type observed in the visual cortex of the mouse. Neurons with class II axons located in lower levels of layer IV, and in layers V and VI, seem to correspond to a cell type described by Martinotti (1889), and later described by Cajal (1911) as Martinotti cells or as ascending axon cells. We have observed the dendrites of cells with ascending axons located in the lower part of layer IV completely enveloped by the terminal fibres of specific afferents, in a way suggesting synaptic contacts (Ruiz-Marcos and Valverde, 1970). Class lII axons (Fig. 3, Nos. 16--18) They originate from cells located in layer II-III. The main axonal stem ascends for a short distance but soon breaks u p into numerous slender hanging branches which may reach layer V. Axons of this group clearly differ from those of class I, since the collaterals and bifurcations abound below the level of the cell body. The three-dimensional shape of these axonal arbors, observed in rotational views, is laminar. They possess densely arranged descending collaterals which frequently

Fig. 4. A group of layer V pyramidal cells surrounding a cell (f) with an intra-cortical axon. The latter ascends emitting numerous collaterals in variable directions which pass among the dendrites of the surrounding pyramidal cells. This composite drawing was made from three consecutive frontal sections and redrawn at different magnifications in order to obtain a perspective impression. The pyramidal cells n and o are within the first section (anterior), whereas pyramidal ceils b, c and m were drawn from the rear section. The axons of pyramidal cells are not illustrated. All cells in this drawing were recorded on magnetic tape. (Golgi method. Camera lucida drawing. Normal mouse 16 days of age).

520

F. V A L V E R D E

adopt the form of braids, or a bush of fibres, enveloping the basal dendrites of superficial pyramidal cells. Axons with similar fields are well illustrated in the study of Lorente de N6 (1922) in the mouse, but could not be correlated with any cell type described by Cajal (1921) in the visual cortex of the cat. This type of axon resembles the axons of types 2 and 3 described by Jones (1975) in the primate somatosensory cortex. Class I V a x o n s (Fig. 3, Nos. 1 9 - 2 1 ) We have included in this class axons with collaterals displaying no consistent direction and non-specific geometrical patterns. Such axons may share some of the features of other axon classes. They are present in the lower parts of layer I I - I I I and in layer IV. Class V axons (Fig. 3, Nos. 22 and 23) They originate from cells located in layer II-III. The main axonal stem descends for variable distances not exceeding 200 ~m, forming one, or more, acute bends from which long ascending and descending branches originate. This type of axon has been observed to originate from neurons having an apical dendrite-like shaft bearing few spines and with basal dendrites orientated in various directions. The shape of this axon does not differ, except in the lengths of collaterals, from axons of true pyramidal cells having an intra-cortical distribution (Fig. 3, No. 24). Class V axons were not frequently found in the visual cortex of the mouse, but are characteristic of the visual cortex of the cat (Cajal, 1911, 1921) and can be found in both somatosensory (Jones, 1975) and visual (Vatverde, I971b) cortices of the monkey. This classification of intra-cortical axons into five different classes permits an approach to the study of comparable parameters between axons having a similar morphology. It has not yet been possible to obtain a sufficient number of examples of identical axons to make comparisons between the same type, but the following study represents preliminary comparisons between intra-cortical axons in two groups of mice raised in different conditions. THE EFFECTS

OF DARK REARING

ON D E N D R I T I C

AND AXONAL

LENGTHS

Results of calculations on the 14 cells with intra-cortical axons considered in this analysis are summarized in Table I. The values given were obtained after using the correction factor described above. Values for individual dendrites were not entered as these varied considerably from one dendrite to another. In the data listed in Table I, we have introduced the A / D quotient which is obtained by dividing the total axonal length by the total dendritic length. This quotient has been found to range between 1.2 and 3.8 with a mean value around 2.5 in the visual cortex of normally reared mice. It is a useful estimate of the total length of an axon in relation to the sum-length of all dendrites of the same cell. It indicates that a neuron in control material with an A / D quotient of below 1 has not been completely recorded.

Neurons with intra-cortical axons

521

Table I. C o m p u t e d three-dimensional total dendritic and axonal lengths

Cell No. 36 43 44 46 47 48

3 5 7 8 9 10 12 15

Total dendritic length in t.tm (TDL)

Total axonal leng tb in t.tm (TAL)

Condition

A/D quotient

16 I 308 19 I 620 19 III 180 16 I 470 15 I 600 15 III 260 Mean values +95% confidence limits based on the t-distribution . . . . . . . .

3615 2109 2620 1634 2008 2767

7525 4721 3811 5459 5555 8220

Control Control Control Control Control Control

2.081 2.238 1.454 3.339 2.766 2.970

19 II 526 19 III 607 19 III 648 19 III 567 19 III 486 19 II 648 19 II 607 19 I 749 Mean values +-95% c o n f i d e n c e limits based on the t-distribution . . . . . . . .

3932 1489 1546 851 1682 2893 1207 2184

Age in days

Class of axon

Deptb from surface in IJm (body)

2458 -+ 698 . . . 5 8 8 1 + 1670 . . . Mean A/D ...2.474 3001 3162 1508

969 1669 806

899 1334

Darkness Darkness Darkness Darkness Darkness Darkness Darkness Darkness

0.763 2.123 0.975 1.138

0.992 0.278 0.744 0.610

1973 -+ 822 . . . 1668 -+ 755 . . . . Mean A / D . . . 0 . 9 5 2

A/D q u o t i e n t = TAL/TDL C o m p a r i s o n of TDL values b e t w e e n control and dark-reared groups: t = 1.008, p < 0.4 Comparison of TAL values b e t w e e n control and dark-reared groups: t = 6.034, p < 0.001

In the analysis of data in Table I, two important results should be considered. The mean value of total dendritic length in dark raised mice and in control animals shows no significant difference. The mean value of the total axonal length in dark raised mice is lower than that for control animals, the difference being highly significant. A diminution in the AID quotient reflects these differences. THREE-DIMENSIONAL

ORGANIZATION

OF THE. VISUAL

CORTEX

Fig. 4 is a camera lucida drawing of a group of layer V pyramidal cells surrounding cell f, whose axon corresponds to No. 4 in Fig. 3. The drawing has been made from three adjoining Golgi sections. Axon if arises from a thick dendrite ascending Vertically for about 100/am and breaking up into complex collaterals distributed within layer IV. The dendrites are moderately covered with thick spine-like protrusions. The axonal branches of this cell interlace with dendrites of pyramidal ceils. All neuronal structures in this figure were rotated 92 ~ around the horizontal O - X axis as is shown in Fig. 5. In this view the radial disposition of pyramidal cells about cell f is obvious. The axon of cell f (in red) spreads out in three main

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directions coinciding with the orientation of surrounding bands of pyramidal cells. Fig. 6 is a camera lucida drawing made from two adjacent Golgi sections showing details of vertical inter-connections between a group of neurons. The axon of cell g corresponds to axon No. 15 in Fig. 3 and is located in layer V; the dendrites of this cell extend through the zone in which axonal collaterals of layer V pyramidal cells abound. Ascending branches of the axon (in red) reach layer I. Along its path, the axon emits a number of relatively short horizontal collaterals and very long ascending, and descending, branches. Note that most branches originate along the first 200/am of the axon. Terminal branches ending in layer I probably contact the apical dendrites of pyramidal cells such as a-f. The first group of collateral branches of the axon of cell g extends around basal dendrites and the first collaterals of the apical dendrites of layer V pyramidal cells. At superficial cortical levels there are pyramidal cells such as e and f whose axons do not leave the cortex. The collateral branches of their axons (Fig. 6, le and lf) were observed to approach closely the dendrites of cell g and the dendrites of the deep pyramidal cell h. The horizontal axon collaterals (2h) of the latter enter the domain of the dendrites of cell g. The group of cells described in Fig. 6 was not seen in relation to specific afferents but none the less might be interpreted as a continuation of the basic circuit of Fig. 2 in Valverde and Ruiz-Marcos (1969). Thus, specific cortical afferents spreading in restricted portions of layers III and IV would be able to extend the activity of their inputs into complex neuronal circuits organised as neuronal chains subserved by cells with ascending intra-cortical class II axons. In Fig. 7 three different cells A, B and C with intra-cortical axons that have been rotated through the angles indicated are shown. The three axons correspond to Nos. 16, 18 and 6 respectively in Fig. 3. The axon and dendrites of cell A are disposed in an ovoid with a major axis slightly oblique in relation to the frontal plane. The axon and dendrites of cell B appear to be contained in a circular profile with some long branches running parallel to the frontal place. Cell C has an irregular orientation, the dendrites being polarized toward the left side whereas the axon appears to be compressed in an arc to the right of the cell body. There appear to be two basic patterns of organization of intra-cortical axons: ascending class II axons with associated pyramidal cells (Fig. 6) form systems extending through the thickness of the cortex in a vertical direction, whereas cells with intra-cortical axons of classes I and III may be responsible for communication with the neighbouring dendrites of pyramidal cells in restricted transversely oriented

Fig. 7. (A--C). Reconstruction of individual cells with intra-cortical axons rotated around the horizontal O - X axis to obtain projections on planes tangential to the surface of the brain. The axon of cell A corresponds to No. 16 of Fig. 3, B to No. 18 of Fig. 3, and C to No. 6 of Fig. 3. The dendrites are grey, the axon black. The top legend line in each reconstruction gives the brain and section number, the second line the angle of rotation and the third line the age of the mouse and the depth from the pial surface. (From Golgi preparations).

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I

M227 RT108~PSL 40'6 19days18014m

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F. V A L V E R D E

discoids slabs (Figs. 5 and 7). Both systems seem to be arranged in concentric irregular shells around specific cortical afferents. Fig. 8 shows a group of cells and fibres in the frontal plane reconstructed from separate camera lucida drawings. This reconstruction was obtained from five consecutive sections from a 19 day mouse raised in darkness from birth. It represents a parallelepipedic volume of cortical tissue measuring 750/am along the Z-axis (perpendicular to the observer's plane) and with front-wall dimensions of 650/am x 800/am. All the neurons and fibres were individually tracked and entered on the computer file with reference to a c o m m o n three-coordinate system. The reconstruction after rotation by 107 ~ around the horizontal O - X axis is shown in Fig. 9. In Fig. 8, two cells with intra-cortical axons (g, h) and the pyramidal cell f show interweaving of axons and dendrites into a vertical braid (3). The axon l h does not emit collateral branches until a level which is slightly above layer IV and not in the area shown. The axon lg emits descending collaterals (2g, 3g and 4g) which form complex twists around the cell body and dendrites of pyramidal cell f. In the background cells b, d, e, n and o with intra-cortical axons, and pyramidal cell c lie in the same plane. Axons ld and le give off descending collaterals which run mainly in vertical directions. Axons lb and i n break up into tightly bound, long braids, made up of thin collaterals. Cell n is of particular interest because the dendrites form curving trajectories completely polarized towards the white matter. It was not unusual to find similarly polarized dendrites in the visual cortex of older enucleated mice (Valverde, 1968). Axon braids such as l b and I n have not as yet been observed in normal mice though they are a c o m m o n feature in other species (Cajal's cellule double bouquet with a horse tail like axon). Cells b to e, n and o are sandwiched between specific afferent fibres 1 and 2 at the front and back of the perspective. Fibre 1 enters the figure from the left; fibre 2 enters from the background to the right of the figure. The criteria for identification of geniculo-striate fibres have been presented elsewhere (Ruiz-Marcos and Valverde, 1970). T h e y occupy elongated bands, or slabs, disposed in the transverse plane and divide into long, slightly oblique - almost horizontal - and vertical branches that may reach the upper part of layer II-III. No particular direction appears to be preferred, for when viewed vertically (Fig. 9) all trajectories are equally represented. Fig. 9 shows all the cells in the sections of the reconstruction arranged around two afferent fibres as if the latter were centres of wide irregular cylindrical formations with diameters in the range 4 0 0 - 5 0 0 / a m .

Fig. 8. Composite perspective drawing made from five consecutive frontal sections showing a large group of cells (a-o) and two specific afferent fibres (1,2) in the visual cortex of a mouse 19 days of age raised in darkness since birth. Cells f, g and h were drawn on the first perspective plane. Succeeding planes contain neurons and fibres drawn at the scales represented in the drawing. (Golgi method. Camera lucida drawing).

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Discussion

The technique of computer reconstruction Several systems for three-dimensional reconstruction of neuronal morphology from Golgi preparations obtained by computer methods have been recently developed (Glaser and Van der Loos, 1965; Garvey et al., 1973; Wann et al., 1973; Marfi-Padilla and Stibitz, 1974; Llin~s and Hillman, 1975). These systems were used mainly for the study of some quantitative aspects of dendrites and to present different views of neurons rotated in space (e.g. Marin-Padilla and Stibitz, 1974). Thus far, only Marin-Padilla and Stibitz (1974) and Jones (1975), using the system developed by Wann et al. (1973), have obtained interesting results on morphological characteristics of axonal fields. In the somatosensory cortex of the monkey Jones (1975) has made extensive use of computer tracings to describe shapes and axonal orientations in an attempt to find a correlation with the possible columnar arrangement in primary cortical sensory areas. In the present study the reconstruction of intra-cortical axons rotated around the horizontal O - X axis has revealed different spatial organizations. Class I axons of cells situated above layer IV are distributed in an overall spherical or cylindrical shape, whereas those of ceils classified as class II axons with ascending vertical stems are always contained in very narrow vertical cylinders of cortical tissue. When the main axonal stem of class 11 axons, or its terminal branches, enter layer I they run in planes nearly perpendicular to the antero-posterior axis of the brain. Finally, intra-cortical class III axons tend to occupy thin, flat or curved, slabs amidst diversely oriented bands of pyramidal cells. The technique of computer reconstruction has been found to be extremely useful for the study of neurons impregnated by the Golgi method. With this technique it is possible to reconstrlact groups of ceils from different spatial angles and to correlate them with their corresponding original drawings in order to describe particular aspects of cortical organization. The classification o f intra-cortical axons Several types of intra-cortical axon described in the present work share features with the axons of different cell types described by other authors, in various species. The correlation between these ~xon and cell types and those identified in other studies has been made under Observations in the description of the particular classes. It should be mentioned that most authors classify small sized neurons, whose axons arborize in a very limited volume, or do not extend beyond the domain of their dendritic trees, as Golgi 2nd type cells. These are typically found in higher mammals and seem to conform to an homogeneous cell type, probably corresponding to the small stellates, grain or granular cells of Cajal (19 t 1, 1921), Lorente de N6 (1922) and Polyak (1957), 'clewed' cells of Va~verde (197 lb), stClate neurons with beaded axons of Lurid (1973) and midget, or web-like, cells of the Russian authors (Poliakov, 1961; Shkolnik-Yarros, 1965; Beritoff, 1965). No such cells appeared stained in young mice.

Neurons with intra-cortical axons

527

The five classes of intraocortical axons recognized in this study belong to different cell types. They transmit impulses to different levels, to different cells and most probably, the dendrites of their cells of origin receive different impulses. However, this classification into a limited number of defined geometrical shapes has facilitated the study of comparable parameters (e.g., length of axons) and the qualitative investigation of their spatial relationships.

The effects of dark rearing on dendritic and axonal patterns. The analysis of total dendritic and axonal lengths could not be completed satisfactorily. Even lower mammals, such as the subject of the present study, contain a large variety of cell types. However taking into account these restrictions, our results show that retarded or arrested growth of axonal branches in cells with intracortical axons may occur in mice raised in darkness, with no corresponding variation in total dendritic length.

Anatomical features specifically related to the concept of columnar organi2ation Vertical chains o f related neurons have been described in the present work. These chains are composed of ascending class II axons and the complexes formed by ascending class I axons around basal and apical dendrites of pyramidal cells (Figs. 6 and 8). In reconstructions rotated around the horizontal O - X axis, specific afferents were seen to alternate with flat or curved vertically inter-connected bands of neurons. Three complex groups of neurons containing specific afferents were reconstructed three-dimensionally and rotated, and one of these reconstructions is illustrated in Fig. 9. In this view the group of neurons surrounding the centrally placed fibre labelled 1 (sections 9-11) gives a fairly good idea of what might be the organization of a cylinder of cortical tissue with a diameter in the range of the postulated functional columns. In the previous studies on Golgi preparations, specific afferents were seen to contact relatively smooth dendrites of cells with class I intra-cortical axons (Valverde and Ruiz-Marcos, 1969), the dendrites of cells with ascending class II axons and basal dendrites of pyramidal cells of layer II-III (Ruiz-Marcos and Valverde, 1970). These observations were in part corroborated by the electron microscopic studies of Garey and Powell (1971) in the cat and monkey and of Peters et al. (1976) in the rat. These authors observed degenerating geniculo-striate afferents ending upon spines and on smooth dendrites of neurons they recognize as stellate cells. The terminations of specific afferents upon these cells might represent the first link in the thalamo-cortical pathway as was suggested previously (Valverde and Ruiz-Marcos, 1969). Surrounding this elementary system, the apical dendrites of layer V pyramidal cells, cell groups with ascending and descending intra-cortical axons and collateral axons of superficial pyramidal cells may be sequentially related into a model of cortical organization.

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Acknowledgements The author gratefully aknowledges the skillful technical assistance of Mrs Inma Alia and Miss Laura L6pez. This work was supported by the Spanish Government under a grant from Fondo Nacional para el Desarrollo de la Investigaci6n Cientffica. References BERITOFF, J. S. (1965) Neural Mechanisms of Higher Vertebrate Behavior (translated from the Russian and edited by LIBERSON, W. T.) London: J. & A. Churchill Ltd. CAJAL, S. R. (1911) Ilistologie du Systkme Nerveux de l'Homme et des Vertkbr~s. Vol. II. Paris: A. Maloine. Reimpress: Madrid: Instituto Cajal, 1955. CAJAL, S . R . (1921) Textura de la corteza visual del gato. Trabajos del Laboratorio de Investigaciones Biolbgicas de la Universidad de Madrid 19, 1 1 3 - 4 4 . GAREY, L. J. and POWELL, T. P. S. (1971) An experimental study of the termination of the lateral geniculo-cortical pathway in the cat and monkey. Proceedings of tbe Royal Society of London, Series B 179, 41--63. GARVEY, C.F., YOUNG, J. H., Jr., COLEMAN, P.D. and SIMON, W. (1973) Automated three-dimensional dendrite tracking system. Electroencepbalography and Clinical Neurophysiology 35, 1 9 9 - 2 0 4 . GLASER, E. M. and VAN der LOOS, H. (1965) A semi-automatic computer-microscope for the analysis of neuronal morphology. IEEE Transactions on Bio-medicaI Engineering 12, 22--31. JONES, E.G. (1975) Varieties and distribution of non-pyramidal cells in the somatic sensory cortex of the squirrel monkey. Journal of Comparative Neurolog:)/160, 2 0 5 - 6 8 . LLINA.S, R. and HILLMAN, D. E. (1975) A multipurpose tridimensional reconstruction computer system for neuroanatomy. In Golgi Centennial Symposium: Perspectives in Neurobiology (edited by SANTINI, M.), pp. 71--79. New York: Raven Press. LORENTE DE NO, R. (1922) La eorteza cerebral det rat6n (Primera contribuci6n. - La corteza acfistica). Trabajos del Laboratorio de Investigaciones Biolhgicas 20, 41--78. LUND, J. S. (1973) Organization of neurons in the visual cortex, Area 17, of the m o n k e y (Macaca mulatta). Journal of Co rnparative Neurology 147, 4 5 5 - 9 6 . MARIN-PADILLA, M. and STIBITZ, G. R. (1974) Three-dimensional reconstruction of the basket cell of the human motor cortex. Brain Research 70, 5 1 1 - 1 4 . MARTINOTTI, C. (1889) Contributo allo studio della eorteecia cerebrale, ed all'origine centrale dei nervi. Annali di Freniatria e Scienze Affini 1, 3 1 4 - 8 0 . O'LEARY, J. L. (1941) Structure of the area striata of the cat. Journal of Comparative Neurology 75, 1 3 1 - 6 4 . PETERS, A., FELDMAN, M. and SALDANHA, J. (t976) The projection of the lateral geniculate nucleus to area 17 of the rat cerebral cortex, lI. Terminations upon neuronal perikarya and dendritic shafts. Journal of Neurocytology 5, 8 5 - 1 0 7 . POLIAKOV, G. 1. (1961) Some results of research into the development of the neuronal structure of the cortical ends of the analyzers in man. Journal of Comparative Neurology 117, 1 9 7 - 2 1 2 . POLYAK, S. (1957) The Vertebrate Visual System (edited by KLOVER, H.) Chicago: The University of Chicago Press. ROSE, M. (1929) Cytoarchitektonischer Atlas der Grosshirnrinde der Maus. Journal /hr Psycbotogie und Neurologie 40, 1--51. RUIZ-MARCOS, A. and VALVERDE, F. ( 1 9 7 0 ) D y n a m i c architecture of the visual cortex. Brain Research 19, 25--39. SHKOLN1K-YARROS, E.G. (1965) Neurons and Interneuronal Connections. The Visual Analyzer. (Russian edition). Leningrad: Academy of Medical Sciences.

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VALVERDE, F. (1967) Apical dendritic spines of the visual cortex and light deprivation in the mouse. Experimental Brain Research 3, 3 3 7 - 5 2 . VALVERDE, F. (1968) Structural changes in the area striata of the mouse after enucleation. Experimental Brain Research 5,274--92. VALVERDE, F. (1970) The Golgi method. A tool for comparative structural analyses. In Contemporary Research Methods in Neuroanatomy (edited by NAUTA, W . J . H . and EBBESSON, S. O. E.), pp. 12--31. Berlin, Heidelberg, New York: Springer. VALVERDE, F. (1971a) Rate and extent of recovery from dark rearingin the visual cortex of the mouse. Brain Research 33, 1 - 1 1 . VALVERDE, F. (1971b) Short axon neuronal subsystems in the visual cortex of the monkey. International Journal of Neuroscience 1, 1 8 1 - 9 7 . VALVERDE, F. and RUIZ-MARCOS, A. (1969) Dendritic spines in the visual cortex of the mouse.. Introduction to a mathematical model. Experimental Brain Research 8, 2 6 9 - 8 3 . WANN, D . F . , WOOLSEY, T . A . , DIERKER, M.L. and COWAN, W.M. (1973) An on-line digital-computer system for the semiautomatic analysis of Golgi-impregnated neurons. IEEE Transactions on Bio-medical Engineering 20, 233--47.