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Specific Thalamo-cortical Afferents and their Presumptive Targets in the Visual Cortex. A Golgi Study
Specific Tha1amo- cort ical Afferent s and their Presumptive Targets in the Visual Cortex A Golgi Study A. FAIREN and F. VALVERDE
Seccidn d e Neuroanatomia Comparada, Instituto Chjal, CSIC, Velazquez 144, Madrid 6 (Spain)
INTRODUCTION In the present paper, two questions relating t o the circuitry of the visual cortex are discussed on the basis of Golgi observations. The first is: What types of neurons with intracortical axons can be contacted by geniculo-cortical fibres? the second: How are these fibres three-dimensionally arranged in layer IV of the primary visual cortex? Clearly, although the answers that the Golgi method offers to the first question are not conclusive, they nevertheless provide the necessary background for the interpretation of experimental data. To this end, a classification of presumptive recipient non-pyramidal cells is introduced, which takes into account dendritic, as well as axonal, characteristics. Some preliminary results on the geometrical arrangement of geniculo-cortical fibres are also discussed and some possible consequences of geometry on connectivity are considered. The observations were made on a large collection of brains of mice, cats and monkeys stained during the last 12 years by the rapid Golgi method, as described elsewhere (Valverde, 1970, 1978). Details of interest were recorded on camera lucida drawings or photographed to be later assembled as mosaic reconstructions. Selected examples of individual cortical afferent fibres were tracked for storage on magnetic tape, to be later displayed o n an X-Y plotter in various spatial orientations, using a micro-computer based system (Valverde, 1976,1978). RECEPTIVE CELLS IN THE INTERNAL GRANULAR LAYER: AN ESSAY ON CLASSIFICATION Despite considerable efforts during recent years, identification of the diverse types of neurones receiving thalamocortical synapses is not yet complete. Recent approaches like anterograde degeneration combined with a new Golgi-EM technique (Fairin et al., 1977; Peters et al., 1977) or intracellular labelling of physiologically identified cells (Kelly and Van Essen, 1974; Christensen and Ebner, 1978) offer new possibilities. For obvious reasons, however, these techniques have t o deal with a rather limited number of cells. To facilitate
420 interpretation of the experimental data, then, a detailed catalogue of the types of presumptive recipient cells is wanting. In spite of many recent attempts t o define neuronal types in the sensory areas of a number of species (Garey, 1971; Shkol’nik-Yarros, 1971; Valverde, 1971, 1976; Lund, 1973; Szentigothai, 1973; Jones, 1975b; Parnavelas et al., 1977; Feldman and Peters, 1978; Tombol, 1978) some clarification is needed, for the results are not completely concordant, owing t o differences in cataloguing criteria, and are not applicable to all animal species. For this reason, a classification is proposed of non-pyramidal cell types in the primary visual cortex, specially of those present in layer IV. These cells possess, with a few exceptions, intracortical axons. The main criteria used are morphology of dendritic surfaces and axonal arborization patterns. In no way is it intended t o cover all cell types exhaustively, but rather to characterize some clear groups which are well represented in our material, and to follow possible homologies between different animal species. Two major categories are distinguished on the basis of the presence or absence of dendritic spines, because this is relevant in interpreting EM results on anterograde degeneration.
In area 17 of rats, cats and monkeys, 14-15% of the degenerating terminals resulting from lateral geniculate lesions contact dendritic shafts; 2- 3% contact neuronal somata and the remainder, 83-84%, dendritic spines (Garey and Powell, 1971; Peters and Feldman, 1976). These cell somata and dendritic shafts have been interpreted as belonging t o stellate cells (Colonnier and Rossignol, 1969; Garey and Powell, 1971; Peters et al., 1976) of the type which either lacks spines or is poor in them (sparsely spined stellate cells). This has been confirmed in Golgielectron microscopic (EM) studies of the rat visual cortex (Peters, 1978). These cells, however, do not constitute a uniform group: Peters et al. (1976) have Pointed out, on EM grounds, that two different cell types might be involved and Feldman and Peters (1978), basing their observations on dendritic morphology in Golgi material, have described various types of spineless neurones. On the other hand, the spines which receive geniculo-cortical afferents belong to dendrites of small diameter (Garey and Powell, 1971; Peters and Feldman, 1976,1977), which show different morphologies, indicating that they belong to different cell types (Peters and Feldman, 1977). Golgi-EM studies in both rat visual and mouse somatosensory cortex have indicated that they are in fact diverse parts of pyramidal cells (Peters et al., 1977; Peters, 1978; Somogyi, 1978; White, 1978). Stellate cells with spiny dendrites are also involved (Peters et al., 1977; Peters, 1978; White, 1978). These results indeed agree with those obtained through dye injections in the cat visual cortex (Kelly and Van Essen, 1974). Interestingly enough, many of these connections have previously been suggested by Golgi studies (Valverde, 1968, 1971; Valverde and Ruiz-Marcos, 1969; Ruiz-Marcos and Valverde, 1970).
Besides the presence or absence of dendritic spines, neuronal types are considered as having either generalized or specialized axonal arborization patterns. Generalized axons are defined by exclusion, for their lack of specific preterminal arborizations, being common in rats (Feldman and Peters, 1978; Peters and Fairen, 1978) and mice.
Neurones with smooth and sparsely spined dendrites and generalized intracortical axons In the mouse, spineless or sparsely spined neurones have axons that can be included in classes I, I1 and 111 of the Valverde (1976) classification. Class I and I11 axons bear certain similarities, whereas those of class I1 constitute a quite different entity. Another group recently described in rats as bipolar cells (Feldman and Peters, 1978) does not show well impregnated axonal plexuses in our mouse material and is not dealt with here, but, undoubtedly, its frankly interesting features deserve further study. (a) Cells with recuwing axonal arcades (classes I and HI). Axons of classes I and 111 belong to cells stellate in form, whose dendrites are smooth or bear a very small number of spines. They are present in all cortical layers of the mouse area 17, with the exception of layer I. An example of a layer 11-111 cell bearing a class 111 axon is shown in Fig. 1,b.
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Fig. 1. Frontal section through the visual cortex of the mouse, Varieties of cells with intracortical axons (b, e) and pyramidal cells (a, c, d , f) can be observed. The figure shows the contrasting difference between the extension of the axonal plexus of cell b (labelled l b ) and the ascending axon (labelled le) of cell e. In the latter, the possible sites of synaptic contacts with dendrites of pyramidal cells are labelled s. Golgi method, camera lucida drawing. Mouse, 21 days old.
422 Typically, the axon emerges from the upper pole of the cell body or from the base of a dendrite, ascends vertically and after the first bifurcation forms thick recurving arcades giving rise t o horizontal or oblique branches. These form a plexus of variable density, within or in the vicinity of the dendritic field. On occasion (Fig. 1,b) there is a dense local plexus forming a curtain around the central portion of the neurone, while some branches form looser plexuses which in this case invade layer I. Class I axons ramify exclusively above the cell body, whereas class 111 do so above and below; these distinct axonal patterns result in different connection possibilities. An interesting fact, also observed in rats (Peters and Fairen, 1978) is that the axon is not exclusively confined t o the vicinity of the cell body, for a descending branch may sometimes be followed over considerable distances. In a recent study, the structural characteristics of spine-free stellate cells were studied in rat (Peters and Fairen, 1978). Some cells (numbered 1, 2, 3 and 6) are entirely comparable to those bearing class I/III axons in the mouse. By their cytological features they correspond to one of the cell types shown to receive geniculocortical input (Peters et al., 1976). After an electron microscopic analysis of their axonal plexuses it became feasible to define the role played by these cells in the intracortical circuits. Entirely comparable neurones are present in monkey (Lund, 1973) visual and somatosensory (Jones, 1975b, type 2) cortices and have been described as large stellate cells in several electron microscopic studies (see Tigges et al., 1977, for a review). ( b ) Cells with ascending axons (classII). Neurones possessing class I1 axonal arborizations (Valverde, 1976) are rather frequently observed in mice. They are stellate cells whose dendrites range from smooth (spine-free) to sparsely spinous (Fig. 1,e). In some instances, even in adult animals, the dendrites are so richly endowed with spines that their classification as sparsely spinous would seem inadequate, although the number of spines never reaches the level typical of true spiny stellate cells (Figs. 2-5); however, it is clear that only quantification can lead t o conclusive criteria. The axon (Fig. 1,le) emerges from the upper pole of the cell body and forms a straight ascending stem which reaches layer I, where it arborizes. According to the localization of the cell bodies, two subtypes may be recognized. Neurones located in layer 11-111 send the ascending axon towards layer I, where it bends and follows horizontal courses for variable distances. Collaterals originate from the main stem throughout its course in the form of long, thin descending branches, but thick arcades are never present. On the other hand, cells located in lower layer IV or in layer V originate a more conspicuous ascending axon; a main group of thin, descending collaterals originates from the initial portion of the axonal trunk and forms a relatively dense local arborization (Fig. 1). A second group of axonal branches derives from the upper part of the axonal stem and distributes within layer I. These deeply located cells correspond t o the type described by Cajal (191 1) as Martinotti cells. On the basis of Golgi observations, Ruiz-Marcos and Valverde (1970) considered these cells t o be recipient of thalamo-cortical synapses. No data on the types of terminals formed by these neurones are available. Golgi observations in mice suggest that the axon might synapse with apical dendritic shafts (s-s on cells f and d), but also other postsynaptic elements may well be involved.
Neurones with specialized axonal arborizations Several neurone types of the non-spiny variety have been described whose axonal terminal branches adopt very specific arborization patterns. These include pericellular baskets (Cajal,
423 191 1 ; Marin-Padilla, 1969; Szentagothai, 1973; Tombol, 1978), horse-tail arborizations (Cajal, 1899, 191 1; Colonnier, 1966; Szentagothai, 1973; Jones, 1975b), vertical axonal bundles provided with grape-like knobs (Valverde, 1978), and candlesticks (Szentagothai and Arbib, 1974). In a broader sense, clewed cells (Valverde, 1971; Jones, 1975b) which show very elaborate, strictly local arborizations might also be included in this group. Although the versatility of the Golgi procedure can create false quantitative impressions, the information gathered from the study of our own Golgi material seems to support the idea that cells with specialized axons are more frequently present in higher species. If this were true, it could be that in lower species, generalized types assume the same roles in cortical circuitry as specialized ones do in higher species. In rat, cells with generalized axons have shown the same connections as those presumed, on the basis of Golgi studies, to have at least two different types of neurones with well organized axonal formations (Peters and Fairln, 1978). However, generalized and specialized axons have been found t o coexist in the cerebral cortex of a given animal species. It is evident that more precise data on connectivity patterns of specialized neurones are needed t o clarify this problem. To date, there is a single instance (Somogyi, 1977; Szentagothai, 1978) in which a type of neurone, in rat visual cortex, has been shown t o produce a unique type of synaptic contact, in relation to axon initial segments of pyramidal cells. Work in progress in this laboratory shows, in cat visual cortex, the presence of such a type of specific synaptic relationship. Some types of neurones with specialized axons are not well enough represented in our Golgi material and consequently, our review is restricted t o 3 categories only: clewed cells, chandelier cells and neurones with vertical axonal bundles. ( a ) Clewed cells. In layer IVc of monkey area 17, Valverde (197 1) described short axon cells which were named “clewed cells” on account of the complex, twisted disposition of their dendritic and axonal branches. An example is shown in Fig. 3g. They are small cells with a low number of smooth, beaded dendrites. The axon (Ig) may issue from any point around the cell body and soon resolves into numerous collaterals which are densely interwoven with the dendrites of the same cell, forming a plexus strictly local in nature. On the basis of Golgi observations, it was suggested that clewed cells were contacted by specific afferents (Valverde, 1971). This cell type corresponds to Jones’ (1975b) type 5; a comparable granular cell is present in monkey area 18 (Valverde, 1978) but here, axonal plexuses are not so dense and the dendritic fields are vertically elongated. In layer IV of cat area 17, a type of short axon cell is observed (Fig. 2,e) which bears resemblance t o primate clewed cells. In cat, the cells are larger and the axonal arborizations looser. Clewed cells are not present in rodents but, interestingly, some class I11 axonal arborizations are reminiscent of clewed cell axons. ( b ) Chandelier cells. These cells, described by Szentigothai and Arbib (1974), Szentigothai (1975) and Tomb61 (1978) are similar to type 4 cells of Jones (1975b). They are considered ubiquitous cells in the cortex but t o our knowledge, no examples of such cells have been described in monkey visual cortex. In area 17 of cats we have recently found some examples which agree in almost every detail with previous descriptions (Fig. 2,a). Usually, dendrites are sparsely spinous. The terminal axonal formations (candlesticks) are apparent (2a). One major point of discrepancy is that, almost invariably, a thick axonal trunk (la) leaves the principal area of axonal arborization and descends t o lower layers where it arborizes again, though never so richly as in superficial levels, or even enters the white matter.
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No data showing that chandelier cells could be targets of the geniculo-cortical input are available. In cat visual cortex they are frequently present in layer IV but no evidence for these connections has been found so far. ( c ) Cells with vertical axonal bundles and grape-like terminal knobs. These cells have been described by Valverde (1978) in layer I1 and upper 111 of area 18 in monkeys, but no examples have been found in area 17. They are considered here only because their axons bear certain similarities with class I/III axons in mice (see Figs. 3,a and 5,b, in Valverde, 1978). The dendrites of these neurones are smooth and beaded. The axon, originating from the upper or lower pole of the cell body, forms arcades from which several long, thin collaterals arise. These descend to lower levels, emitting characteristic grape-like terminal knobs, specially abundant in layer IV, and group together constituting compact bundles. Non-pyramidal neurones with spinous dendrites In cats and monkeys, spinous non-pyramidal neurones are frequent in all subdivisions of layer IV. They are multipolar cells whose dendrites are almost as densely studded with spines as pyramidal cells (see Jones, 1975b). However, a typical apical dendrite is lacking. Density of spines is, then, a criterion which serves to differentiate true spiny stellate cells from other neuronal types whose dendrites bear some spines. Although the term spiny stellate cell is not entirely adequate, it is retained here for the sake of simplicity. It is clear that they do not constitute a uniform group (Figs. 2-5); subgroups can be defined according to cell size, dendritic orientation and axonal distribution. In all cases axons can be considered generalized, as defined above, for they lack specific terminal arborizations. However, their overall distributions follow rather definite patterns, which can also help to fully define spiny stellate cells as a special neurone category. In layer IV of cat visual cortex (Fig. 2,b, c, d) cell bodies are large (up t o 18 pm in diameter) and sometimes show cilia (Fig. 4A). Dendrites are richly supplied with drumsticklike spines and span a very large field. The axon (Figs. 21b, lc, and Fig. 4A1) originates from the lower pole and soon gives rise t o collaterals, either horizontal or recurrent. Horizontal collaterals (Figs. 22b, 3b, 2c, 2d and Fig. 4A4) are thick and extend for long distances; less frequently, they ascend and ramify poorly in upper layers (Fig. 23b). Recurrent collaterals form loops (Fig. 4A2, 3) and ascend obliquely or in some cases, vertically (Figs. 2 and 4 4 ) . It has been possible t o observe these ascending branches in intimate contact with the spines of apical dendrites (Figs. 2s on ap2 and Fig. 4% ) in a way suggesting functional contacts. The main descending branch of the axon (Figs. 24, and 4%) descends vertically and sometimes enters the white matter. These spiny stellate cells clearly correspond to the giant stellate cells described by Cajal (1899, 191 1, 1921) and should be considered presumptive targets of thalamic afferent fibres (Szen tigothai, 1975). In monkey area 17, spiny stellate cells vary in morphology according t o the sublayer where they are located. In layer IVa (which receives input from the parvocellular geniculate laminae), stellate cells (Fig. 3,b, c)aremedium sized and in some cases their dendrites adopt horizontal courses. The axon ( l b , lc) leaves the lower pole of the perikaryon and descends vertically for variable distances, sending off recurrent collaterals which ascend more or less vertically (2b). Layer IVb contains a group of clearly multipolar stellate cells (Fig. 3 ,d, e), which resemble
Fig. 2 (see following pagc). Frontal section through the visual cortex of the cat. The figure is a composite drawing recording varietics of cells from several adjacent sections and one specific afferent fibre (in red) which has been traced in 3 consecutivc sections. Large stellate cells with markedly spinous dendrites are labelled b , c, d . One cell (c) with smooth dendrites and localized axonal field (le) and one “chandelier” cell (a) with characteristic terminal formations (221) complcte a pattern of cell varieties most frequently Ftaincd in kittens, Golgi method, camera lucida drawing. Cat, 1 month old.
Fig. 3 (see following right-hand pagc). Frontal section through the visual cortex of the monkey. The drawing is a composite illustration of varieties of cells rccorded in adjacent sections corrcsponding to the central part of the thickness of the cortex and showing also specific afferent fibres ( I , 2, in red). Medium Fized conventional pyramidal cclls can be observed in layer 111 (a) as well as in the middle of sublayer IVb (f). Small stellate cells with spinous dendrites (h, i, j) and recurrent axons (in blue) ascend t o develop localized axonal plexuses in sublayer IVa. Here another variety of stellate cell with spinous dendrites (b, c) have dendrites running horizontally probably in relation to the disposition of specific afferent cortical terminal fibres of sublayer IVa. This level might also be affected by recurrent axon terminals (in yellow) of small pyramidal cells (k, 1, m ) located in the upper part of layer V. Two stellate cells with spinous dendrites (d, e) and one clewed cell (8) can also be obscrved. Goki method, camera lucida drawing. Adult monkey (Macaca rhesus).
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Fig. 4A: mosaic photomicrographic reconstruction of one large stellate cell with spinous dendrites in layer IV of the visual cortex of the cat. The main axonal trunk (1J splits into several collateral branches (2-5) coursing in various directions. Inset B shows a series of parallel contacts (b) of a fibre f which is a continuation outside the field of the collateral 4 in A, along a portion of one apical dendrite. Note spine heads (arrowheads) at the end of long stalks attached to the dendrite. Golgi method. Cat, 1 month old.
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Fig. 5A: mosaic photomicrographic reconstruction of one small stellate cell with spinous dendrites in sublayer IVc of the visual cortex of the monkey. The main, descending axonal trunk ( I ) forms several loops (2) which emit recurrent ( 3 , 4 ) and descending (5) collaterals. In comparison with the cell in Fig. 4, this cell shows dendrites with fewer, but larger long-stalked spines. Both reconstructions were made to the same final magnification to facilitate comparison. Inset B shows one portion of a dendrite (d) belonging to a similar stellate cell enveloped by a fibre (f) which is a collateral of one specific afferent fibre. Notice spines marked by arrowheads and enlargements of fibre f, notably b, which might be sites of synaptic contact, Go@ method, adult monkey (Macaca rhesus).
43 1 the large stellate cells of Cajal. They are clearly smaller than in the cat, however (compare with Fig. 2 ) , and cannot receive direct thalamic input, except if located at the bordersof 1%; those located in lower IVb (e) do not differ from spiny stellate cells in upper layer IVc. The lower part of layer IVc, which also receives parvocellular geniculate input, contains a very typical group of spiny stellate cells (Figs, 3,h, i, j and 5A). The cell bodies are some 12 pm in diameter, while dendrites are less densely covered with spines and some are long stalked. As in other spiny stellate cells, the axon goes down initially but soon forms recurving loops (Fig. 5A,, 2) which are followed by vertically ascending collaterals (Figs. 33,.,, li, lj and Fig. 5 , 4), strictly columnar in distribution, that reach sublayer IVa where they form terminal arboriz'ations (4h, 2i) or follow horizontal courses (5h). Other collaterals are horizontal (2h) and, sometimes, the main axonal trunk continues as a descending fibre (Fig. 55). These layer IVc stellate cells are likely to receive fibres of geniculate origin. In Fig. 5B, a fibre (f) identified as a specific afferent apparently makes contact with a dendritic spine of one such stellate cell. In layer V there is a group of pyramidal cells with recurrent axons (Cajal, 191 1, 1921) which have a quite similar pattern of axonal distribution (Fig. 31, m); they have also been described in monkey area 18 (Valverde, 1978). In seeking for cell types in mice that might be considered homologous of the spiny stellate cells, it became evident that they were not as abundant as in higher species or, alternatively, that they were not so easily stainable by the Golgi method. However, typical examples are present and a very clear one has been illustrated previously (Valverde, 1968, Fig. 7). The axonal distribution of these stellate cells follows the pattern described as class V by Valverak (1976). This arborization pattern does not differ from the one shown by some pyramidal cells with intracortical axons in mouse, and by star pyramids (Lorente de N6, 1949; Jones, 1975b). GEOMETRY OF THE SPECIFIC AFFERENT FIBRES Presumed specific, geniculo-cortical fibres have been studied in layer IV. In young mice, as well as in cat and monkey, the criteria for identification of geniculo-cortical fibres in Golgi preparations are based entirely on consideration of the following points: (a) the parent branch can be traced to the white matter, (b) the intracortical trajectories correspond t o those shown by degeneration studies, and (cj the level of termination and extension of their final branches agrees with degeneration and autoradiography results (cf., Valverde, 197 1 ; Lund, 1973; Jones, 1975a).
The mouse In young mice, specific afferents can be recognized as the thickest fibres entering the visual cortex from the w h t e matter. As they ascend in the cortex, they follow oblique ascending, wavy courses (see Fig. 2 in Valverde and Ruiz-Marcos, 1969, and Fig. 1 in RuizMarcos and Valverde, 1970). In layer V, or sometimes even below in layer VI, they may divide dichotomously, giving rise t o secondary fibres generally unequal in thickness. In layer IV, these secondary fibres give origin t o preterminal branches provided with both en passant and terminal dilations of presumably synaptic character (cf., Peters and Fairen, 1978). The fibres form a plexus which distributes mainly in layer IV but also in the deepest part of layer Ill; isolated fibres can be followed up to layer I . In Golgi preparations, pre-
43 2 terminal fibres occur in elongated clouds possessing an oblique major axis 300-400 pm in length (Ruiz-Marcos and Valverde, 1970). Within the domain of these clouds, repeated branching at acute angles is observed. In their spatial distribution, preterminal fibres tend to follow oblique courses, as has previously been shown (Cajal, 1911; Lorente de NO, 1949) and sometimes they are vertical. Except for these characteristics, it seems that mice show rather unspecific patterns of terminal distribution when compared t o cats or monkeys. Successful staining of the specific afferent fibres can only be attained in young mice and, therefore, i t is not infrequent t o observe enlarged growing tips. During postnatal development the thick fibres myelinate and thus become refractory to Golgi staining. However, in adult animals it is frequent to observe plexuses of fine horizontal or oblique fibres in layers IV and lower 111, corresponding in part to specific axons. Fig. 6A shows, in a frontal view, a computer reconstructed afferent cortical fibre as it enters the visual cortex from the w h t e matter. This fibre was digitized from a 220 p m t h c k Golgi section of a mouse 10 days old. Although the fibre shows a principal bifurcation in a lower level corresponding to the bottom of layer VI, most branches originate at the level of layer IV and lower 11-111. Some long collaterals ascend unbranched to higher levels. Fig. 7A shows the same fibre after computer rotation of 90" around the horizontal OX axis; the fibre thus appears as if it were seen from the surface of the brain. No definite preferential orientation is observed, so that any possible direction of its collaterals seems t o be equally probable (cf., Fig. 9 in Valverde, 1976).
The cat Golgi impregnations of specific fibres have been particularly successful in 1-month-old kittens; by this age, most neurones exhibit adult features even though myelination is not complete. As has been shown by LeVay et al. (1978), segregation of ocular dominance columns is completed between the 33rd and 39th postnatal days. Fig. 2 is a composite drawing synthetising information from several adjacent Golgistained sections. In red, a reputed geniculo-cortical fibre is depicted; it has been traced in 3 consecutive sections and can be considered quite complete. It stems from a thick fibre (1) which has been partially impregnated, as if it were an unmyelinated segment of a myelinated fibre. The thick parent fibre follows an oblique, almost horizontal course in the bottom of layer IV. Whenever it has been possible to trace the specific fibres down to the white matter, specially in younger animals, it has been found that the parent fibres are thick (up to 3 pm) and sometimes divide into diverging fibres before entering the cortex, in such a way that the span of a single afferent fibre may be quite large. Fibre 1 in Fig. 2 gives rise t o a thick ascending branch which soon subdivides dichotomously into slightly thinner secondary branches, which follow long, wavy trajectories inside layer IV. Preterminal branches originate from the secondary branches and, on occasions, even from the primary trunk. They follow oblique ascending or descending, but not vertical, courses and originate, by repeated subdivisions, interlacing bouquet-like plexuses which are denser than the ones present in mice. The fine fibres show synaptic enlargements along their lengths and short appendages ending in dilations also occur. It is worth noting, however, that the thick secondary branches (which presumably acquire myelin sheaths later in development) also give rise to synaptic contacts (unpublished Golgi-EM observations). This fact agrees with the observation by Carey and Powell (1971) of degenerating geniculo-cortical axons making synapses immediately after the loss of a myelin sheath. Fig. 6B shows a computer display of the same specific fibre shown in Fig. 2, in the same
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C Fig. 6 . Computer display of specific afferent fibres in the visual cortex. A: mouse, 10 days old. B: cat, 1 month old. C: adult monkey. From Gold material.
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Fig. 7 . Computer display of the same fibres shown in Fig. 6 after rotation of 90" on the horizontal OX axis.
435 position. The fibre is here represented as seen in a frontal section. As the figure shows, the total tangential spread of the fibre is in the order of 700 pm. A dense preterminal plexus is visible in the centre of the picture and a less profuse one appears on the left. After a 90" computer rotation on the OX axis (Fig. 7B), the tangential distribution of the fibre becomes visible, as if it were seen perpendicularly from the pial surface. Horizontal lines indicate the limits of the 3 Golgi-stained sections used t o reconstruct the fibre. One of the salient features revealed by the rotation is that the dense plexus shown in the centre of Fig. 6B is resolved into distinct plexuses coming out from main collaterals extending in a slightly oblique anteroposterior direction. Another interesting feature observed in computer rotations like this is that the plexuses of preterminal fibres occur as discrete entities, roughly 100-150pm in diameter, separated by clear fields of the same size. From Golgi observations, it is impossible t o know whether or not other incoming fibres overlap and then obscure such a pattern, but in support of the suggestion that the pattern does exist, there is the observation that, after geniculate lesions, degeneration occurs in clusters with a periodicity of about 100 pm (Carey and Powell, 1971). The monkey As seen in Golgi preparations of the visual cortex in adult monkeys, presumed geniculocortical fibres entering from the white matter, ascend vertically through layers VI and V. Immediately above the well marked boundary between IVc and V, the parent fibres begin to arborize by sending out at right angles a number of collateral branches which extend for variable distances in sublayer IVc. These collaterals are provided with numerous tightly packed short-side appendages and dilations giving a most characteristic image in well impregnated Golgi preparations (Fig. 3, red fibre 1). These collateral branches pierce through groups of stained small stellate cells with spinous dendrites and clewed cells (cf., Fig. 6 in Valverde, 1971), where it is not infrequent t o observe close approximations t o spinous dendritic segments in a way suggesting functional relationships (Fig. 5B). There is a second and thmner stratum of terminal fibres in sublayer IVa. Here parent fibres (Fig. 3, red fibre 2 ) can be traced ascending vertically from the white matter t o end in this plexus of sublayer IVa; collaterals from the same parent fibre are also seen in sublayer IVc (Fig. 3, fibre 3), so that dual innervation of both levels from an identical origin can be presumed to exist. Fig. 6C is a computer display of the terminal arborization of a single afferent fibre entering sublayer IVc in which all short-sided appendages and terminal dilations were recorded. The computed total number of terminal end bulbs in this fibre is 366; these are distributed in a volume of tissue of 32.7 X lo6 pm3. The high concentration of presumed synaptic sites in a given space is notorious. This fibre can be seen in Fig. 7C after 90" rotation around the horizontal OX axis. In this view it seems evident that the plexus of terminal fibres extends in a more or less elongated field with predominant transverse orientation, though no definite patterns are visible. Genera1 considerations Despite some common characteristics, which served for identification purposes, the patterns of distribution of specific fibres in the 3 animal species studied are rather diverse, probably in correlation with the progressive complication of the mammalian brain during evolution. In mouse, the distribution of preterminal fibres on the tangential plane appears t o be
436 unspecific and does not seem t o bear any correspondence with the cellular composition of layer IV, and in the rat visual cortex, Peters and Feldman (1976) found that degenerating axon terminals were no more densely grouped around apical dendritic clusters (Peters and Walsh, 1972) than elsewhere. Afferent fibres in cat (Fig. 7B) form distinct groupings of preterminal fibres. Whether this pattern as seen in Golgi preparations reflects a discreteness in the distribution of terminals is not clear. However, Carey and Powell (1971) found that such discreteness bears no relation t o any morphological feature in layer IV. The situation in the motor cortex appears to be different. In monkey (Sloper, 1973), a denser aggregation of degenerating terminals of thalamic origin occurs around apical dendrites. It seems that this different grouping of terminals reflects the fact that in the motor cortex, thalamo-cortical synapses on apical dendritic spines are apparently frequent; they have moreover been reported in EM degeneration studies (Strick and Sterling, 1974). In the primary visual cortex no such synapses have been seen by using this technique, suggesting that they do not exist or perhaps are less frequent. In Golgi preparations of cat and monkey visual cortex, the majority of preterminal fibres follow horizontal or oblique courses (Fig. 6B, C). The probability for these fibres t o establish synaptic contacts with apical dendritic spines is therefore less than if they followed vertical trajectories (cf., Szentigothai, 1975). If this were true, preterminal thalamo-cortical fibres in the motor cortex would be expected to course vertically. From the data shown by Jones (1975a, Fig. 15), it is clear that this is indeed the case. In mouse, preterminal fibres distribute in a more perpendicular fashion, much as they do in monkey motor cortex; our mouse material, however, is probably immature. Unfortunately, no EM degeneration data are available on mouse to decide whether or not apical dendrites are involved. In the closely related rat, a case of such contact has been found by the Golgi-EM technique (Peters, 1978), but the distribution of geniculo-cortical fibres in rat visual cortex is still unknown. In fact, one major unsolved question remains behind these considerations: are axonal arborizations specific, in terms of the postsynaptic elements they contact? To approach this problem, a much more detailed knowledge of the connectivity of diverse types of axons, extrinsic or intrinsic, must be attained. SUMMARY The Golgi method is used to study the forms of non-pyramidal cells in the primary visual cortex in 3 animal species; neuronal types that could be recipients of the geniculo-cortical input are sought. These neurones are classed according t o dendritic and axonal characteristics, into several distinct groups: spine-free (or sparsely spinous) neurones with either generalized or specialized axons, and spiny neurones always bearing axons of the generalized type. Specific afferent fibres are identified in Golgi preparations and their geometrical properties analysed by means of computer rotations. The morphology differs in the animal species studied, but some basic properties are maintained. Except for laminar distribution,no definite patterns of arborization are found in mouse and monkey, whereas, in cat, afferent fibres form discrete preterminal arborizations. ACKNOWLEDGEMENTS We wish to thank Miss Laura Lbpez for her excellent technical assistance. The work was
437 supported in part by a grant from Fundacion E. Rodriguez Pascual to A.F. and by Fondo Nacional para el Desarrollo de la Investigacibn Cientifica.
REFERENCES Cajal, S. R a m h y (1899) Comparative study of the sensory areas of the human cortex, In: Centennial Volume, Clark University, Worcester, Mass., pp. 31 1-382. Cajal, S. Ram6n Y (191 1) Histologie du Systtme Nerveux de I’Homme et des Vertdbrds, Tome II, Maloine, Paris (Reimpress., Consejo Superior de Investigaciones Cientificas, Instituto Cajal, Madrid, 1955). Cajal, S. Ram6n y (1921) Textura de la corteza visual del gato. Trab. Lab. Invest. biol. Univ. Madrid, 19: 113-144. Christensen, B. N. and Ebner, F. F. (1978) The synaptic architecture of neurons in opossum somatic sensory-motor cortex: a combined anatomical and physiological study. J. Neurocytol., 7 : 61 -70. Colonnier, M. (1966) The structural design of the neocortex. In: Brain and Conscious Experience, J. C. Eccles (Ed.), Springer, Berlin, pp. 1-23, Colonnier, M. and Rossignol, S. (1969) Discussion: heterogeneity of the cerebral cortex. In: Basic Mechanisms o f the Epilepsies, H. H. Jasper, A. A. Ward and A. Pope (Eds.), Little, Brown and Co., Boston, Mass., pp. 29-40. Fair&, A., Peters, A. and Saldanha, J. (1977) A new procedure for examining Gold impregnated neurons by light and electron microscopy. J. Neurocytol., 6: 311-337. Feldman, M. L. and Peters, A, (1978) The forms of non-pyramidal neurons in the visual cortex of the rat. J. comp. Neurol., 179: 761-794. Garey, L. J. (1971) A light and electron microscopic study of the visual cortex of the cat and monkey, Proc. roy. Soc. B, 179: 21-40. Garey, L. J. and Powell, T. P. S. (1971) An experimental study of the termination of the lateral geniculocortical pathway in the cat and monkey.Proc. roy. Soc. B, 179: 41 -63. Jones, E. G. (1975a) Lamination and differential distribution of thalamic afferents within the sensorymotor cortex of the squirrel monkey. J. comp. Neurol., 160: 167-204. Jones, E. G. (1975b) Varieties and distribution of non-pyramidal cells in the somatic sensory cortex of the squirrel monkey. J. comp. Neurol., 160: 205-268. Kelly, J. P. and Van Essen, D. C. (1974) Cell structure and function in the visual cortex of the cat. J. Physiol. [Lond.], 238: 515-547. LeVay, S., Stryker, M. P. and Shatz, C. J. (1978) Ocular dominance columns and their development in layer N of the cat’s visual cortex: a quantitative study. J. comp. Neurol., 179: 223-244. Lorente de N6, R. (1949) Cerebral cortex: architecture, intracortical connections, motor projections. In: Physiologyof theNeruousSystem, J. F. Fulton (Ed.),Oxford University Press, London,pp.288-330, Lund, J. S. (1973) Organization of neurons in the visual cortex, area 17, of the monkey (Macuca mulatta). J. comp. Neurol., 147: 455-496, Marin-Padilla, M. (1969) Origin of the pericellular baskets of the pyramidal cells of the human motor cortex: a Golgi study. Brain Res., 14: 633-646. Parnavelas, J. G., Lieberman, A. R. and Webster, K. E. (1977) Organization of neurons in the visual cortex, area 17, of the rat. J. Anat. (Lond.), 124: 305-322. Peters, A. (1978) Personal communication. Peters, A . and Fairdn, A. (1978) Smooth and sparsely-spined stellate cells in the visual cortex of the rat: a study using a combined Golgielectron microscope technique.J. comp. Neurol., 181 : 129-172. Peters, A. and Feldman, M. L. (1976) The projection of the lateral geniculate nucleus to area 17 of the rat cerebral cortex. I. General description. J. Neurocytol., 5 : 63-84. Peters, A. and Feldman, M. L. (1977) The projection of the lateral geniculate nucleus to area 17 of the rat cerebral cortex. N.Terminations upon spiny dendrites. J. Neurocytol., 6: 669-689. Peters, A. and Walsh, T. M. (1972) A study of the organization of apical dendrites in the somatic sensory cortex of the rat. J. comp. Neurol., 144: 253-268. Peters, A,, Feldman, M. and Saldanha, J. (1976) The projection of the lateral geniculate nucleus to area 17 of the rat cerebral cortex. 11. Terminations upon neuronal perikarya and dendritic shafts. J. Neurocytol., 5 : 85-107. Peters, A,, White, E. L. and F a i r h , A. (1977) Synapses between identified neuronal elements. An electron
microscopic demonstration of degenerating axon terminals synapsing with Golgi impregnated neurons. Neurosci. Lett., 6: 171-175. Ruiz-Marcos, A. and Valverde, F. (1970) Dynamic architecture of the visual cortex. Brain Res., 19: 25-39. Shkol‘nik-Yarros, E. G . (1971) Neurons and Interneuronal Connections of the Central Visual System. Plenum Press, New York. Sloper, J . J. (1973) An electron microscope study of the termination of afferent connections to the primate motor cortex. J. Neurocytol., 2: 361-368. Somogyi, P. (1977) A specific “axo-axonal” interneuron in the visual cortex of the rat. Brain Res., 136: 345-350. Somogyi, P. (1978) The study of Golgi stained cells and of experimental degeneration under the electron microscope: a direct method for the identification in the visual cortex of three successive links in a neuron chain, Neuroscience, 3: 167-180. Strick, P. L. and Sterling, P. (1974) Synaptic termination of afferents from the ventrolateral nucleus of the thalamus in the cat motor cortex. A light and electron microscope study. J. comp. Neurol., 153: 77-106. Szentigothai, J . (1973) Synaptology of thevisual cortex. In: Handbook ofSensory Physiology. Vol. VI1/3, Central Processing of Visual Information, Part B, Visual Centers in the Brain, R. Jung (Ed.), Springer, Berlin, pp. 269-324. Szentigothai, J . (1975) The “module-concept” in cerebral cortex architecture. Brain Res., 95: 475-496. Szentigothai, J . (1978) The neuron network of the cerebral cortex: a functional interpretation. Proc. roy. SOC.B, 201: 219-248. Szentigothai, I . and Arbib, M , A . (1974) Conceptual models of neural organization. Neurosci. Res. Progr. Bull., 12: 307-510. Tigges, M.,Bos, J., Tigges, J . and Bridges, E. (1977) Ultrastructural characteristics of layer lV neuropil in area 17 of monkeys. Cell Tiss. Res., 182: 39-59. Tombol, T. (1978) Comparative data on the Golgi architecture of interneurons of different cortical areas in cat and rabbit. In: Architectonics of the Cerebral Cortex, M. A. B. Brazier and H. Petsche (Eds.), Raven Press, New York, pp. 59-76. Valverde, F. (1968) Structural changes in the area striata of the mouse after enucleation. Exp. Brain Res., 5 : 274-292. Valverde, F. (1970) The Golgi method. A tool for comparative structural analyses. In: Contemporary Research Methods in Neuroanatomy, W. J . H. Nauta and S . 0. E. Ebbesson (Eds.), Springer, Berlin, Pp. 12-31. Valverde, F. (1971) Short axon neuronal subsystems in the visual cortex of the monkey. Int. J. Neurosci., 1: 181-197. Valverde, F. (1976) Aspects of cortical organization related to the geometry of neurons with intra-cortical axons. J. Neurocytol., 5 : 509-529. Valverde, F. (1978) The organization of area 1 8 in the monkey. Anat. Embryol., 154: 305-334. Valverde, F. and Ruiz-Marcos, A. (1969) Dendritic spines in the visual cortex of the mouse: introduction to a mathematical model. Exp. Brain Res., 8: 269-283. White, E. L. (1978) Identified neurons in mouse SmI cortex which are postsynaptic to thalamocortical axon terminals: a combined Golgielcctron microscopic and degeneration study. J. comp. Neurol., 181: 627-662.
Seccidn d e Neuroanatomia Comparada, Instituto Chjal, CSIC, Velazquez 144, Madrid 6 (Spain)
INTRODUCTION In the present paper, two questions relating t o the circuitry of the visual cortex are discussed on the basis of Golgi observations. The first is: What types of neurons with intracortical axons can be contacted by geniculo-cortical fibres? the second: How are these fibres three-dimensionally arranged in layer IV of the primary visual cortex? Clearly, although the answers that the Golgi method offers to the first question are not conclusive, they nevertheless provide the necessary background for the interpretation of experimental data. To this end, a classification of presumptive recipient non-pyramidal cells is introduced, which takes into account dendritic, as well as axonal, characteristics. Some preliminary results on the geometrical arrangement of geniculo-cortical fibres are also discussed and some possible consequences of geometry on connectivity are considered. The observations were made on a large collection of brains of mice, cats and monkeys stained during the last 12 years by the rapid Golgi method, as described elsewhere (Valverde, 1970, 1978). Details of interest were recorded on camera lucida drawings or photographed to be later assembled as mosaic reconstructions. Selected examples of individual cortical afferent fibres were tracked for storage on magnetic tape, to be later displayed o n an X-Y plotter in various spatial orientations, using a micro-computer based system (Valverde, 1976,1978). RECEPTIVE CELLS IN THE INTERNAL GRANULAR LAYER: AN ESSAY ON CLASSIFICATION Despite considerable efforts during recent years, identification of the diverse types of neurones receiving thalamocortical synapses is not yet complete. Recent approaches like anterograde degeneration combined with a new Golgi-EM technique (Fairin et al., 1977; Peters et al., 1977) or intracellular labelling of physiologically identified cells (Kelly and Van Essen, 1974; Christensen and Ebner, 1978) offer new possibilities. For obvious reasons, however, these techniques have t o deal with a rather limited number of cells. To facilitate
420 interpretation of the experimental data, then, a detailed catalogue of the types of presumptive recipient cells is wanting. In spite of many recent attempts t o define neuronal types in the sensory areas of a number of species (Garey, 1971; Shkol’nik-Yarros, 1971; Valverde, 1971, 1976; Lund, 1973; Szentigothai, 1973; Jones, 1975b; Parnavelas et al., 1977; Feldman and Peters, 1978; Tombol, 1978) some clarification is needed, for the results are not completely concordant, owing t o differences in cataloguing criteria, and are not applicable to all animal species. For this reason, a classification is proposed of non-pyramidal cell types in the primary visual cortex, specially of those present in layer IV. These cells possess, with a few exceptions, intracortical axons. The main criteria used are morphology of dendritic surfaces and axonal arborization patterns. In no way is it intended t o cover all cell types exhaustively, but rather to characterize some clear groups which are well represented in our material, and to follow possible homologies between different animal species. Two major categories are distinguished on the basis of the presence or absence of dendritic spines, because this is relevant in interpreting EM results on anterograde degeneration.
In area 17 of rats, cats and monkeys, 14-15% of the degenerating terminals resulting from lateral geniculate lesions contact dendritic shafts; 2- 3% contact neuronal somata and the remainder, 83-84%, dendritic spines (Garey and Powell, 1971; Peters and Feldman, 1976). These cell somata and dendritic shafts have been interpreted as belonging t o stellate cells (Colonnier and Rossignol, 1969; Garey and Powell, 1971; Peters et al., 1976) of the type which either lacks spines or is poor in them (sparsely spined stellate cells). This has been confirmed in Golgielectron microscopic (EM) studies of the rat visual cortex (Peters, 1978). These cells, however, do not constitute a uniform group: Peters et al. (1976) have Pointed out, on EM grounds, that two different cell types might be involved and Feldman and Peters (1978), basing their observations on dendritic morphology in Golgi material, have described various types of spineless neurones. On the other hand, the spines which receive geniculo-cortical afferents belong to dendrites of small diameter (Garey and Powell, 1971; Peters and Feldman, 1976,1977), which show different morphologies, indicating that they belong to different cell types (Peters and Feldman, 1977). Golgi-EM studies in both rat visual and mouse somatosensory cortex have indicated that they are in fact diverse parts of pyramidal cells (Peters et al., 1977; Peters, 1978; Somogyi, 1978; White, 1978). Stellate cells with spiny dendrites are also involved (Peters et al., 1977; Peters, 1978; White, 1978). These results indeed agree with those obtained through dye injections in the cat visual cortex (Kelly and Van Essen, 1974). Interestingly enough, many of these connections have previously been suggested by Golgi studies (Valverde, 1968, 1971; Valverde and Ruiz-Marcos, 1969; Ruiz-Marcos and Valverde, 1970).
Besides the presence or absence of dendritic spines, neuronal types are considered as having either generalized or specialized axonal arborization patterns. Generalized axons are defined by exclusion, for their lack of specific preterminal arborizations, being common in rats (Feldman and Peters, 1978; Peters and Fairen, 1978) and mice.
Neurones with smooth and sparsely spined dendrites and generalized intracortical axons In the mouse, spineless or sparsely spined neurones have axons that can be included in classes I, I1 and 111 of the Valverde (1976) classification. Class I and I11 axons bear certain similarities, whereas those of class I1 constitute a quite different entity. Another group recently described in rats as bipolar cells (Feldman and Peters, 1978) does not show well impregnated axonal plexuses in our mouse material and is not dealt with here, but, undoubtedly, its frankly interesting features deserve further study. (a) Cells with recuwing axonal arcades (classes I and HI). Axons of classes I and 111 belong to cells stellate in form, whose dendrites are smooth or bear a very small number of spines. They are present in all cortical layers of the mouse area 17, with the exception of layer I. An example of a layer 11-111 cell bearing a class 111 axon is shown in Fig. 1,b.
42 1
Fig. 1. Frontal section through the visual cortex of the mouse, Varieties of cells with intracortical axons (b, e) and pyramidal cells (a, c, d , f) can be observed. The figure shows the contrasting difference between the extension of the axonal plexus of cell b (labelled l b ) and the ascending axon (labelled le) of cell e. In the latter, the possible sites of synaptic contacts with dendrites of pyramidal cells are labelled s. Golgi method, camera lucida drawing. Mouse, 21 days old.
422 Typically, the axon emerges from the upper pole of the cell body or from the base of a dendrite, ascends vertically and after the first bifurcation forms thick recurving arcades giving rise t o horizontal or oblique branches. These form a plexus of variable density, within or in the vicinity of the dendritic field. On occasion (Fig. 1,b) there is a dense local plexus forming a curtain around the central portion of the neurone, while some branches form looser plexuses which in this case invade layer I. Class I axons ramify exclusively above the cell body, whereas class 111 do so above and below; these distinct axonal patterns result in different connection possibilities. An interesting fact, also observed in rats (Peters and Fairen, 1978) is that the axon is not exclusively confined t o the vicinity of the cell body, for a descending branch may sometimes be followed over considerable distances. In a recent study, the structural characteristics of spine-free stellate cells were studied in rat (Peters and Fairen, 1978). Some cells (numbered 1, 2, 3 and 6) are entirely comparable to those bearing class I/III axons in the mouse. By their cytological features they correspond to one of the cell types shown to receive geniculocortical input (Peters et al., 1976). After an electron microscopic analysis of their axonal plexuses it became feasible to define the role played by these cells in the intracortical circuits. Entirely comparable neurones are present in monkey (Lund, 1973) visual and somatosensory (Jones, 1975b, type 2) cortices and have been described as large stellate cells in several electron microscopic studies (see Tigges et al., 1977, for a review). ( b ) Cells with ascending axons (classII). Neurones possessing class I1 axonal arborizations (Valverde, 1976) are rather frequently observed in mice. They are stellate cells whose dendrites range from smooth (spine-free) to sparsely spinous (Fig. 1,e). In some instances, even in adult animals, the dendrites are so richly endowed with spines that their classification as sparsely spinous would seem inadequate, although the number of spines never reaches the level typical of true spiny stellate cells (Figs. 2-5); however, it is clear that only quantification can lead t o conclusive criteria. The axon (Fig. 1,le) emerges from the upper pole of the cell body and forms a straight ascending stem which reaches layer I, where it arborizes. According to the localization of the cell bodies, two subtypes may be recognized. Neurones located in layer 11-111 send the ascending axon towards layer I, where it bends and follows horizontal courses for variable distances. Collaterals originate from the main stem throughout its course in the form of long, thin descending branches, but thick arcades are never present. On the other hand, cells located in lower layer IV or in layer V originate a more conspicuous ascending axon; a main group of thin, descending collaterals originates from the initial portion of the axonal trunk and forms a relatively dense local arborization (Fig. 1). A second group of axonal branches derives from the upper part of the axonal stem and distributes within layer I. These deeply located cells correspond t o the type described by Cajal (191 1) as Martinotti cells. On the basis of Golgi observations, Ruiz-Marcos and Valverde (1970) considered these cells t o be recipient of thalamo-cortical synapses. No data on the types of terminals formed by these neurones are available. Golgi observations in mice suggest that the axon might synapse with apical dendritic shafts (s-s on cells f and d), but also other postsynaptic elements may well be involved.
Neurones with specialized axonal arborizations Several neurone types of the non-spiny variety have been described whose axonal terminal branches adopt very specific arborization patterns. These include pericellular baskets (Cajal,
423 191 1 ; Marin-Padilla, 1969; Szentagothai, 1973; Tombol, 1978), horse-tail arborizations (Cajal, 1899, 191 1; Colonnier, 1966; Szentagothai, 1973; Jones, 1975b), vertical axonal bundles provided with grape-like knobs (Valverde, 1978), and candlesticks (Szentagothai and Arbib, 1974). In a broader sense, clewed cells (Valverde, 1971; Jones, 1975b) which show very elaborate, strictly local arborizations might also be included in this group. Although the versatility of the Golgi procedure can create false quantitative impressions, the information gathered from the study of our own Golgi material seems to support the idea that cells with specialized axons are more frequently present in higher species. If this were true, it could be that in lower species, generalized types assume the same roles in cortical circuitry as specialized ones do in higher species. In rat, cells with generalized axons have shown the same connections as those presumed, on the basis of Golgi studies, to have at least two different types of neurones with well organized axonal formations (Peters and Fairln, 1978). However, generalized and specialized axons have been found t o coexist in the cerebral cortex of a given animal species. It is evident that more precise data on connectivity patterns of specialized neurones are needed t o clarify this problem. To date, there is a single instance (Somogyi, 1977; Szentagothai, 1978) in which a type of neurone, in rat visual cortex, has been shown t o produce a unique type of synaptic contact, in relation to axon initial segments of pyramidal cells. Work in progress in this laboratory shows, in cat visual cortex, the presence of such a type of specific synaptic relationship. Some types of neurones with specialized axons are not well enough represented in our Golgi material and consequently, our review is restricted t o 3 categories only: clewed cells, chandelier cells and neurones with vertical axonal bundles. ( a ) Clewed cells. In layer IVc of monkey area 17, Valverde (197 1) described short axon cells which were named “clewed cells” on account of the complex, twisted disposition of their dendritic and axonal branches. An example is shown in Fig. 3g. They are small cells with a low number of smooth, beaded dendrites. The axon (Ig) may issue from any point around the cell body and soon resolves into numerous collaterals which are densely interwoven with the dendrites of the same cell, forming a plexus strictly local in nature. On the basis of Golgi observations, it was suggested that clewed cells were contacted by specific afferents (Valverde, 1971). This cell type corresponds to Jones’ (1975b) type 5; a comparable granular cell is present in monkey area 18 (Valverde, 1978) but here, axonal plexuses are not so dense and the dendritic fields are vertically elongated. In layer IV of cat area 17, a type of short axon cell is observed (Fig. 2,e) which bears resemblance t o primate clewed cells. In cat, the cells are larger and the axonal arborizations looser. Clewed cells are not present in rodents but, interestingly, some class I11 axonal arborizations are reminiscent of clewed cell axons. ( b ) Chandelier cells. These cells, described by Szentigothai and Arbib (1974), Szentigothai (1975) and Tomb61 (1978) are similar to type 4 cells of Jones (1975b). They are considered ubiquitous cells in the cortex but t o our knowledge, no examples of such cells have been described in monkey visual cortex. In area 17 of cats we have recently found some examples which agree in almost every detail with previous descriptions (Fig. 2,a). Usually, dendrites are sparsely spinous. The terminal axonal formations (candlesticks) are apparent (2a). One major point of discrepancy is that, almost invariably, a thick axonal trunk (la) leaves the principal area of axonal arborization and descends t o lower layers where it arborizes again, though never so richly as in superficial levels, or even enters the white matter.
424
No data showing that chandelier cells could be targets of the geniculo-cortical input are available. In cat visual cortex they are frequently present in layer IV but no evidence for these connections has been found so far. ( c ) Cells with vertical axonal bundles and grape-like terminal knobs. These cells have been described by Valverde (1978) in layer I1 and upper 111 of area 18 in monkeys, but no examples have been found in area 17. They are considered here only because their axons bear certain similarities with class I/III axons in mice (see Figs. 3,a and 5,b, in Valverde, 1978). The dendrites of these neurones are smooth and beaded. The axon, originating from the upper or lower pole of the cell body, forms arcades from which several long, thin collaterals arise. These descend to lower levels, emitting characteristic grape-like terminal knobs, specially abundant in layer IV, and group together constituting compact bundles. Non-pyramidal neurones with spinous dendrites In cats and monkeys, spinous non-pyramidal neurones are frequent in all subdivisions of layer IV. They are multipolar cells whose dendrites are almost as densely studded with spines as pyramidal cells (see Jones, 1975b). However, a typical apical dendrite is lacking. Density of spines is, then, a criterion which serves to differentiate true spiny stellate cells from other neuronal types whose dendrites bear some spines. Although the term spiny stellate cell is not entirely adequate, it is retained here for the sake of simplicity. It is clear that they do not constitute a uniform group (Figs. 2-5); subgroups can be defined according to cell size, dendritic orientation and axonal distribution. In all cases axons can be considered generalized, as defined above, for they lack specific terminal arborizations. However, their overall distributions follow rather definite patterns, which can also help to fully define spiny stellate cells as a special neurone category. In layer IV of cat visual cortex (Fig. 2,b, c, d) cell bodies are large (up t o 18 pm in diameter) and sometimes show cilia (Fig. 4A). Dendrites are richly supplied with drumsticklike spines and span a very large field. The axon (Figs. 21b, lc, and Fig. 4A1) originates from the lower pole and soon gives rise t o collaterals, either horizontal or recurrent. Horizontal collaterals (Figs. 22b, 3b, 2c, 2d and Fig. 4A4) are thick and extend for long distances; less frequently, they ascend and ramify poorly in upper layers (Fig. 23b). Recurrent collaterals form loops (Fig. 4A2, 3) and ascend obliquely or in some cases, vertically (Figs. 2 and 4 4 ) . It has been possible t o observe these ascending branches in intimate contact with the spines of apical dendrites (Figs. 2s on ap2 and Fig. 4% ) in a way suggesting functional contacts. The main descending branch of the axon (Figs. 24, and 4%) descends vertically and sometimes enters the white matter. These spiny stellate cells clearly correspond to the giant stellate cells described by Cajal (1899, 191 1, 1921) and should be considered presumptive targets of thalamic afferent fibres (Szen tigothai, 1975). In monkey area 17, spiny stellate cells vary in morphology according t o the sublayer where they are located. In layer IVa (which receives input from the parvocellular geniculate laminae), stellate cells (Fig. 3,b, c)aremedium sized and in some cases their dendrites adopt horizontal courses. The axon ( l b , lc) leaves the lower pole of the perikaryon and descends vertically for variable distances, sending off recurrent collaterals which ascend more or less vertically (2b). Layer IVb contains a group of clearly multipolar stellate cells (Fig. 3 ,d, e), which resemble
Fig. 2 (see following pagc). Frontal section through the visual cortex of the cat. The figure is a composite drawing recording varietics of cells from several adjacent sections and one specific afferent fibre (in red) which has been traced in 3 consecutivc sections. Large stellate cells with markedly spinous dendrites are labelled b , c, d . One cell (c) with smooth dendrites and localized axonal field (le) and one “chandelier” cell (a) with characteristic terminal formations (221) complcte a pattern of cell varieties most frequently Ftaincd in kittens, Golgi method, camera lucida drawing. Cat, 1 month old.
Fig. 3 (see following right-hand pagc). Frontal section through the visual cortex of the monkey. The drawing is a composite illustration of varieties of cells rccorded in adjacent sections corrcsponding to the central part of the thickness of the cortex and showing also specific afferent fibres ( I , 2, in red). Medium Fized conventional pyramidal cclls can be observed in layer 111 (a) as well as in the middle of sublayer IVb (f). Small stellate cells with spinous dendrites (h, i, j) and recurrent axons (in blue) ascend t o develop localized axonal plexuses in sublayer IVa. Here another variety of stellate cell with spinous dendrites (b, c) have dendrites running horizontally probably in relation to the disposition of specific afferent cortical terminal fibres of sublayer IVa. This level might also be affected by recurrent axon terminals (in yellow) of small pyramidal cells (k, 1, m ) located in the upper part of layer V. Two stellate cells with spinous dendrites (d, e) and one clewed cell (8) can also be obscrved. Goki method, camera lucida drawing. Adult monkey (Macaca rhesus).
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Fig. 4A: mosaic photomicrographic reconstruction of one large stellate cell with spinous dendrites in layer IV of the visual cortex of the cat. The main axonal trunk (1J splits into several collateral branches (2-5) coursing in various directions. Inset B shows a series of parallel contacts (b) of a fibre f which is a continuation outside the field of the collateral 4 in A, along a portion of one apical dendrite. Note spine heads (arrowheads) at the end of long stalks attached to the dendrite. Golgi method. Cat, 1 month old.
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Fig. 5A: mosaic photomicrographic reconstruction of one small stellate cell with spinous dendrites in sublayer IVc of the visual cortex of the monkey. The main, descending axonal trunk ( I ) forms several loops (2) which emit recurrent ( 3 , 4 ) and descending (5) collaterals. In comparison with the cell in Fig. 4, this cell shows dendrites with fewer, but larger long-stalked spines. Both reconstructions were made to the same final magnification to facilitate comparison. Inset B shows one portion of a dendrite (d) belonging to a similar stellate cell enveloped by a fibre (f) which is a collateral of one specific afferent fibre. Notice spines marked by arrowheads and enlargements of fibre f, notably b, which might be sites of synaptic contact, Go@ method, adult monkey (Macaca rhesus).
43 1 the large stellate cells of Cajal. They are clearly smaller than in the cat, however (compare with Fig. 2 ) , and cannot receive direct thalamic input, except if located at the bordersof 1%; those located in lower IVb (e) do not differ from spiny stellate cells in upper layer IVc. The lower part of layer IVc, which also receives parvocellular geniculate input, contains a very typical group of spiny stellate cells (Figs, 3,h, i, j and 5A). The cell bodies are some 12 pm in diameter, while dendrites are less densely covered with spines and some are long stalked. As in other spiny stellate cells, the axon goes down initially but soon forms recurving loops (Fig. 5A,, 2) which are followed by vertically ascending collaterals (Figs. 33,.,, li, lj and Fig. 5 , 4), strictly columnar in distribution, that reach sublayer IVa where they form terminal arboriz'ations (4h, 2i) or follow horizontal courses (5h). Other collaterals are horizontal (2h) and, sometimes, the main axonal trunk continues as a descending fibre (Fig. 55). These layer IVc stellate cells are likely to receive fibres of geniculate origin. In Fig. 5B, a fibre (f) identified as a specific afferent apparently makes contact with a dendritic spine of one such stellate cell. In layer V there is a group of pyramidal cells with recurrent axons (Cajal, 191 1, 1921) which have a quite similar pattern of axonal distribution (Fig. 31, m); they have also been described in monkey area 18 (Valverde, 1978). In seeking for cell types in mice that might be considered homologous of the spiny stellate cells, it became evident that they were not as abundant as in higher species or, alternatively, that they were not so easily stainable by the Golgi method. However, typical examples are present and a very clear one has been illustrated previously (Valverde, 1968, Fig. 7). The axonal distribution of these stellate cells follows the pattern described as class V by Valverak (1976). This arborization pattern does not differ from the one shown by some pyramidal cells with intracortical axons in mouse, and by star pyramids (Lorente de N6, 1949; Jones, 1975b). GEOMETRY OF THE SPECIFIC AFFERENT FIBRES Presumed specific, geniculo-cortical fibres have been studied in layer IV. In young mice, as well as in cat and monkey, the criteria for identification of geniculo-cortical fibres in Golgi preparations are based entirely on consideration of the following points: (a) the parent branch can be traced to the white matter, (b) the intracortical trajectories correspond t o those shown by degeneration studies, and (cj the level of termination and extension of their final branches agrees with degeneration and autoradiography results (cf., Valverde, 197 1 ; Lund, 1973; Jones, 1975a).
The mouse In young mice, specific afferents can be recognized as the thickest fibres entering the visual cortex from the w h t e matter. As they ascend in the cortex, they follow oblique ascending, wavy courses (see Fig. 2 in Valverde and Ruiz-Marcos, 1969, and Fig. 1 in RuizMarcos and Valverde, 1970). In layer V, or sometimes even below in layer VI, they may divide dichotomously, giving rise t o secondary fibres generally unequal in thickness. In layer IV, these secondary fibres give origin t o preterminal branches provided with both en passant and terminal dilations of presumably synaptic character (cf., Peters and Fairen, 1978). The fibres form a plexus which distributes mainly in layer IV but also in the deepest part of layer Ill; isolated fibres can be followed up to layer I . In Golgi preparations, pre-
43 2 terminal fibres occur in elongated clouds possessing an oblique major axis 300-400 pm in length (Ruiz-Marcos and Valverde, 1970). Within the domain of these clouds, repeated branching at acute angles is observed. In their spatial distribution, preterminal fibres tend to follow oblique courses, as has previously been shown (Cajal, 1911; Lorente de NO, 1949) and sometimes they are vertical. Except for these characteristics, it seems that mice show rather unspecific patterns of terminal distribution when compared t o cats or monkeys. Successful staining of the specific afferent fibres can only be attained in young mice and, therefore, i t is not infrequent t o observe enlarged growing tips. During postnatal development the thick fibres myelinate and thus become refractory to Golgi staining. However, in adult animals it is frequent to observe plexuses of fine horizontal or oblique fibres in layers IV and lower 111, corresponding in part to specific axons. Fig. 6A shows, in a frontal view, a computer reconstructed afferent cortical fibre as it enters the visual cortex from the w h t e matter. This fibre was digitized from a 220 p m t h c k Golgi section of a mouse 10 days old. Although the fibre shows a principal bifurcation in a lower level corresponding to the bottom of layer VI, most branches originate at the level of layer IV and lower 11-111. Some long collaterals ascend unbranched to higher levels. Fig. 7A shows the same fibre after computer rotation of 90" around the horizontal OX axis; the fibre thus appears as if it were seen from the surface of the brain. No definite preferential orientation is observed, so that any possible direction of its collaterals seems t o be equally probable (cf., Fig. 9 in Valverde, 1976).
The cat Golgi impregnations of specific fibres have been particularly successful in 1-month-old kittens; by this age, most neurones exhibit adult features even though myelination is not complete. As has been shown by LeVay et al. (1978), segregation of ocular dominance columns is completed between the 33rd and 39th postnatal days. Fig. 2 is a composite drawing synthetising information from several adjacent Golgistained sections. In red, a reputed geniculo-cortical fibre is depicted; it has been traced in 3 consecutive sections and can be considered quite complete. It stems from a thick fibre (1) which has been partially impregnated, as if it were an unmyelinated segment of a myelinated fibre. The thick parent fibre follows an oblique, almost horizontal course in the bottom of layer IV. Whenever it has been possible to trace the specific fibres down to the white matter, specially in younger animals, it has been found that the parent fibres are thick (up to 3 pm) and sometimes divide into diverging fibres before entering the cortex, in such a way that the span of a single afferent fibre may be quite large. Fibre 1 in Fig. 2 gives rise t o a thick ascending branch which soon subdivides dichotomously into slightly thinner secondary branches, which follow long, wavy trajectories inside layer IV. Preterminal branches originate from the secondary branches and, on occasions, even from the primary trunk. They follow oblique ascending or descending, but not vertical, courses and originate, by repeated subdivisions, interlacing bouquet-like plexuses which are denser than the ones present in mice. The fine fibres show synaptic enlargements along their lengths and short appendages ending in dilations also occur. It is worth noting, however, that the thick secondary branches (which presumably acquire myelin sheaths later in development) also give rise to synaptic contacts (unpublished Golgi-EM observations). This fact agrees with the observation by Carey and Powell (1971) of degenerating geniculo-cortical axons making synapses immediately after the loss of a myelin sheath. Fig. 6B shows a computer display of the same specific fibre shown in Fig. 2, in the same
433
1
200 prn
A
C Fig. 6 . Computer display of specific afferent fibres in the visual cortex. A: mouse, 10 days old. B: cat, 1 month old. C: adult monkey. From Gold material.
434
A
J
C
Fig. 7 . Computer display of the same fibres shown in Fig. 6 after rotation of 90" on the horizontal OX axis.
435 position. The fibre is here represented as seen in a frontal section. As the figure shows, the total tangential spread of the fibre is in the order of 700 pm. A dense preterminal plexus is visible in the centre of the picture and a less profuse one appears on the left. After a 90" computer rotation on the OX axis (Fig. 7B), the tangential distribution of the fibre becomes visible, as if it were seen perpendicularly from the pial surface. Horizontal lines indicate the limits of the 3 Golgi-stained sections used t o reconstruct the fibre. One of the salient features revealed by the rotation is that the dense plexus shown in the centre of Fig. 6B is resolved into distinct plexuses coming out from main collaterals extending in a slightly oblique anteroposterior direction. Another interesting feature observed in computer rotations like this is that the plexuses of preterminal fibres occur as discrete entities, roughly 100-150pm in diameter, separated by clear fields of the same size. From Golgi observations, it is impossible t o know whether or not other incoming fibres overlap and then obscure such a pattern, but in support of the suggestion that the pattern does exist, there is the observation that, after geniculate lesions, degeneration occurs in clusters with a periodicity of about 100 pm (Carey and Powell, 1971). The monkey As seen in Golgi preparations of the visual cortex in adult monkeys, presumed geniculocortical fibres entering from the white matter, ascend vertically through layers VI and V. Immediately above the well marked boundary between IVc and V, the parent fibres begin to arborize by sending out at right angles a number of collateral branches which extend for variable distances in sublayer IVc. These collaterals are provided with numerous tightly packed short-side appendages and dilations giving a most characteristic image in well impregnated Golgi preparations (Fig. 3, red fibre 1). These collateral branches pierce through groups of stained small stellate cells with spinous dendrites and clewed cells (cf., Fig. 6 in Valverde, 1971), where it is not infrequent t o observe close approximations t o spinous dendritic segments in a way suggesting functional relationships (Fig. 5B). There is a second and thmner stratum of terminal fibres in sublayer IVa. Here parent fibres (Fig. 3, red fibre 2 ) can be traced ascending vertically from the white matter t o end in this plexus of sublayer IVa; collaterals from the same parent fibre are also seen in sublayer IVc (Fig. 3, fibre 3), so that dual innervation of both levels from an identical origin can be presumed to exist. Fig. 6C is a computer display of the terminal arborization of a single afferent fibre entering sublayer IVc in which all short-sided appendages and terminal dilations were recorded. The computed total number of terminal end bulbs in this fibre is 366; these are distributed in a volume of tissue of 32.7 X lo6 pm3. The high concentration of presumed synaptic sites in a given space is notorious. This fibre can be seen in Fig. 7C after 90" rotation around the horizontal OX axis. In this view it seems evident that the plexus of terminal fibres extends in a more or less elongated field with predominant transverse orientation, though no definite patterns are visible. Genera1 considerations Despite some common characteristics, which served for identification purposes, the patterns of distribution of specific fibres in the 3 animal species studied are rather diverse, probably in correlation with the progressive complication of the mammalian brain during evolution. In mouse, the distribution of preterminal fibres on the tangential plane appears t o be
436 unspecific and does not seem t o bear any correspondence with the cellular composition of layer IV, and in the rat visual cortex, Peters and Feldman (1976) found that degenerating axon terminals were no more densely grouped around apical dendritic clusters (Peters and Walsh, 1972) than elsewhere. Afferent fibres in cat (Fig. 7B) form distinct groupings of preterminal fibres. Whether this pattern as seen in Golgi preparations reflects a discreteness in the distribution of terminals is not clear. However, Carey and Powell (1971) found that such discreteness bears no relation t o any morphological feature in layer IV. The situation in the motor cortex appears to be different. In monkey (Sloper, 1973), a denser aggregation of degenerating terminals of thalamic origin occurs around apical dendrites. It seems that this different grouping of terminals reflects the fact that in the motor cortex, thalamo-cortical synapses on apical dendritic spines are apparently frequent; they have moreover been reported in EM degeneration studies (Strick and Sterling, 1974). In the primary visual cortex no such synapses have been seen by using this technique, suggesting that they do not exist or perhaps are less frequent. In Golgi preparations of cat and monkey visual cortex, the majority of preterminal fibres follow horizontal or oblique courses (Fig. 6B, C). The probability for these fibres t o establish synaptic contacts with apical dendritic spines is therefore less than if they followed vertical trajectories (cf., Szentigothai, 1975). If this were true, preterminal thalamo-cortical fibres in the motor cortex would be expected to course vertically. From the data shown by Jones (1975a, Fig. 15), it is clear that this is indeed the case. In mouse, preterminal fibres distribute in a more perpendicular fashion, much as they do in monkey motor cortex; our mouse material, however, is probably immature. Unfortunately, no EM degeneration data are available on mouse to decide whether or not apical dendrites are involved. In the closely related rat, a case of such contact has been found by the Golgi-EM technique (Peters, 1978), but the distribution of geniculo-cortical fibres in rat visual cortex is still unknown. In fact, one major unsolved question remains behind these considerations: are axonal arborizations specific, in terms of the postsynaptic elements they contact? To approach this problem, a much more detailed knowledge of the connectivity of diverse types of axons, extrinsic or intrinsic, must be attained. SUMMARY The Golgi method is used to study the forms of non-pyramidal cells in the primary visual cortex in 3 animal species; neuronal types that could be recipients of the geniculo-cortical input are sought. These neurones are classed according t o dendritic and axonal characteristics, into several distinct groups: spine-free (or sparsely spinous) neurones with either generalized or specialized axons, and spiny neurones always bearing axons of the generalized type. Specific afferent fibres are identified in Golgi preparations and their geometrical properties analysed by means of computer rotations. The morphology differs in the animal species studied, but some basic properties are maintained. Except for laminar distribution,no definite patterns of arborization are found in mouse and monkey, whereas, in cat, afferent fibres form discrete preterminal arborizations. ACKNOWLEDGEMENTS We wish to thank Miss Laura Lbpez for her excellent technical assistance. The work was
437 supported in part by a grant from Fundacion E. Rodriguez Pascual to A.F. and by Fondo Nacional para el Desarrollo de la Investigacibn Cientifica.
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