I. the Lissencephalic Brain of Rodentia, Lagomorpha, Insectivora and Chiroptera I

J. Anat. (1986), 145, pp. 217-234 217 With 10 figures Printed in Great Britain A Golgi study of the sixth layer of the cerebral cortex. I. The lissencephalic brain of Rodentia, Lagomorpha, Insectivora and Chiroptera I. FERRER, I. FABREGUES AND E. CONDOM Unidad de Neuropatologia, Departamento de Anatomla Patologica, Hospital Principes de Espania, Hospitalet de Llobregat, Barcelona, Spain (Accepted 8 August 1985)

INTRODUCTION Over the past century considerable information has been obtained, using the Golgi technique, on the structure of the cerebral cortex in various mammalian species. Using a similar approach, the present paper has investigated neuronal structure and cellular organisation in the sixth layer of the cerebral cortex in several representative mammalian species. Previous investigations furnished much insight into the distinctive types of neuron within the sixth layer in mouse (Lorente de No, 1922, 1933,1949; Meller, Breiphol & Glees, 1969), rat (Parnavelas, Lieberman & Webster, 1977; Feldman & Peters, 1978; Peters & Proskauer, 1980), rabbit (O'Leary & Bishop, 1938; Tombol, 1978 a, 1984; McMullen& Glaser, 1982), hedgehog (Valverde & Popez-Mascaraque, 1981), cat (O'Leary, 1941; Tombol, 1976, 1978a, 1984; Peters & Regidor, 1981), dog (Tunturi, 1971; Miodonski, 1974), monkey (Jones, 1975; Valverde, 1978; Tombol, 1978b, 1984) and man (Ramon y Cajal, 1911). The first part of the present study includes species with lissencephalic brains: Rodentia (rat, mouse, hamster and vole), Lagomorpha (rabbit), Chiroptera (dwarf bat) and Insectivora (European hedgehog). Some of these mammals are more pri- mitive than others having evolved earlier (McKenna, 1975; Kowalski, 1976); most are terrestrial, but mammals living in an aerial ecological environment are also considered. The second part of the study (Ferrer, Fabregues & Condom, 1986) deals with species with gyrencephalic brains and includes the Carnivora (cat and dog), Artio- dactyla (cow and sheep) and Primates (man). It is the aim of this study to provide further morphological data for a comparative anatomy of layer VI of the cerebral cortex.

MATERIAL AND METHODS The material was obtained from adult and 15-20 days old Sprague-Dawley rats (Rattus norvegicus); adult and 15-20 days old mice (Mus musculus); adult hamsters (Cricetus cricetus); adult voles (Microtus agrestis); adult and 20-22 days old rabbits (Oryctologus cuniculus); 20-25 cm common hedgehogs (Erinaceus europaeus) and 40-5 mm dwarf bats (Pipistrellus pipistrellus). The animals were killed under ether anaesthesia and the brains removed immediately. Blocks 3 mm thick were cut from the sensorimotor cortex and placed in a solution 8 ANA 145 218 I. FERRER, I. FABREGUES AND E. CONDOM of 3 % potassium bichromate and 0 5-1 % osmium tetroxide (20/6; vol/vol) for 2-7 days, rinsed in a solution of 0 75 % silver nitrate and placed in a fresh solution of 0 75 % silver nitrate for 24-48 hours. The tissue samples were then dehydrated in ethanol, embedded in paraffin and sections, 50-75 ,um thick, were cut. To allow for the somewhat uncertain Golgi reaction and ensure maximal staining, multiple assays were performed with varying concentrations of potassium bichromate-osmium tetroxide, and occasional double impregnations were carried out. Two hundred neurons selected from approximately 600 sections were traced in detail using a drawing tube mounted in a Leitz microscope. Additional brain samples from the same species were fixed with 2 % para- formaldehyde-I % glutaraldehyde, embedded in paraffin and sectioned at a thickness of 1O,m. The sections were stained with either haematoxylin and eosin, Nissl, Sudan black or Gros-Bielschowsky stains.

RESULTS General considerations The sixth layer occupied from 20 to 24 % of the sensory neocortex in the species studied. In routine histological sections, the superficial region (lamina VIa) contained symmetrically shaped large neuronal cell bodies while the deeper region (lamina VIb) contained cells with smaller asymmetrical soma and horizontal neurons. In the rodent and the rabbit, layer VI was well defined, whereas in the hedgehog and the bat the upper boundary with layer V and the lower boundary with the white matter were indistinct. In the last two species, the neurons in the deeper region were organised in rows separated by myelinated bundles of white matter. The rat as a modelfor the different neuronal types in layer VI ofthe neocortex In Golgi sections, the total thickness of the motor cortex of the rat was 1700 #sm, the molecular lamina accounting for approximately 200 ,um, laminae II and III for 500 Itm, lamina IV for 100 ,um, lamina V for 470 ,um and lamina VI for 430 ,um. In earlier studies (Ferrer & Martinez-Matos, 1981), it was found that during the second to the third postnatal week the neuronal morphology of the neocortex of the rat could be studied in detail because the different neuronal types were well developed and because the course ofthe axon could easily be followed over long distances since myelination of the intracortical fibres had only just started. For these reasons the following description is based on findings in 15-21 days old rats. Pyramidal neurons were found throughout layer VI; lamina VIa contained large and medium pyramidal neurons while small and medium pyramidal neurons pre- dominated in lamina VIb. Cell bodies were triangular, polygonal or globular. Apical dendrites were vertically aligned, and terminated in one or two distal branches (Fig. 1). The apical dendrites of the large pyramidal neurons were traced for more than 800 #um and entered into the supragranular layers. The apical dendrites of the medium pyramidal neurons measured approximately 400 ,um in length and extended across the lower regions of layer V. The apical dendrites oftheflattenedpyramidal neurons had a short trajectory before dividing into oblique terminal branches within layer VI itself. Although arborisation of the basilar branches of the pyramidal neurons was Neurons ofcerebral cortex. I 219

Fig. 1. Pyramidal neurons in layer VI of the cerebral cortex in a 15 days old rat. Soma are triangular, polygonal or globular (G), with apical dendrites ofvariable length. The axon descends towards the white matter, but ascending collaterals emerge from some of the cells before the white matter is entered (ax). IP, inverted pyramidal neuron; F, fusiform neuron. Bar, 100 4tm. generally symmetrical, some neurons exhibited a large asymmetrical basilar dendrite which often entered the white matter. These cells were similar to the atypical or triangular pyramidal cells of other species (Ramon y Cajal, 1911). The axons of the pyramidal neurons always penetrated the white matter to a considerable depth. Many of the cells also exhibited horizontal or oblique collateral branches and ascending collaterals reaching the upper layers of the cortex. However, these ascending collaterals could only rarely be traced for more than 500 ,sm. A pyramidal cell characteristically found in layer VI was tentatively classified as a multiapical pyramidal neuron because numerous apical dendrites emerged from its polygonal soma and entered the middle regions of the neocortex (Fig. 2); these multiple apical dendrites further arborised into several ascending or oblique narrow branches; as a result, the apical dendritic arborisation of these cells frequently extended to a width of 250-300,um. The axons of multiapical pyramidal neurons 8-2 220 I. FERRER, I. FABREGUES AND E. CONDOM

Fig. 2. Multiapical pyramidal neurons show multiple primary apical dendrites and several secondary ascending and oblique secondary branches. The axon penetrates the white matter but an ascending collateral extends towards the upper layers. 15 days old rat. Bar, 100 4m. penetrated deeply into the white matter but, just before the white matter was reached, a collateral branch emerged and curved at a right angle towards the surface for a length of 700-800,um, showing several oblique, lateral branches originating along its trajectory. In addition to the ascending collateral, several horizontal or oblique axonal branches extended across the median and deep regions of the sixth layer. Inverted pyramids were commonly found in the upper region of lamina VIa. Cell bodies were triangular and exhibited a stout descending dendrite, approximately 200-250,m in length, which frequently entered the white matter. The basilar dendrites were similar to those of classical, 'correctly' oriented pyramidal neurons. The course of the axon as described by van der Loos (1965) descended and always entered deeply into the white matter (Fig. 1). The axon left from either the cell body or the region proximal to the descending dendrite. On occasion, the axon originated at the upper edge of the soma but reversed its direction through a characteristic loop towards the white matter. Fusiform neurons also were found in the upper region of lamin VIa. They had fusiform vertical soma from which a stout, vertically oriented dendrite, approximately 400 mm long, originated at each pole. The upper dendrite penetrated layer V and the lower one the subcortical white matter (Fig. 1). The axon always descended deeply into the white matter, giving off few collateral branches. Neurons ofcerebral cortex. I 221

0=0-

c / Fig. 3(A-C). Horizontal neurons (A), horizontal pyramidal cells (B) and fan shaped neurons (C) in layer VI in a 21 days old rat. Note the horizontal course of the axon in horizontal neurons and the descending course of the axon towards the white matter with its emerging horizontal lateral branch in the fan shaped cells. Bar, 100l um.

Horizontal neurons were seen in the lower region oflamina VIb. The soma of these cells were flattened and one or more horizontally oriented, sparsely spined dendrites emerged at each pole. Horizontal cell size varied from 250 to 450 jum in length. The axon frequently originated at the lower edge of the soma and immediately changed its direction to a horizontal course which, in some of the large neurons, could be traced for 600-700 #sm. At times the axon appeared to enter the white matter, but if traced further along its trajectory it followed a horizontal course alongside the superficial white matter (Fig. 3A). Horizontal pyramids. Although oblique pyramidal neurons were occasionally found in the superficial region of the sixth layer, horizontal pyramidal cells were concentrated in lamina VIb. They exhibited a triangular soma from which a large horizontal dendrite approximately 400,um long originated. The basilar dendrites sprouted at angles of 900 and arborised symmetrically; the dendrites which 222 I. FERRER, I. FABREGUES AND E. CONDOM

Fig. 4 (A-C). Martinotti cells with characteristic ascending axons (A); multipolar neurons with variable axonal spreads (B) and multipolar neurons with locally arborizing axons resembling basket cells (C) in layer VI in a 21 days old rat. Two different types of multipolar neurons may be distinguished according to the trajectory of their axons. One multipolar type is a projection neuron with a long descending axon penetrating the white matter (1); the other multipolar type is a local circuit neuron with a short axon arborising within the sixth layer (2). Bar, 100 sm. projected upwards were longer than those which projected towards the white matter (Fig. 3 B). From the large horizontal dendrite, thin ascending collateral branches emerged, but they did not extend beyond the upper boundary of layer VI. Other rare horizontal neurons. Some horizontal cells resembled horizontal pyramidal neurons, but large numbers of vertically oriented collateral branches (200 ,um long) emerged from the main horizontal dendrite. Other neurons had radially ramifying dendrites stretching across the full width of layer VI and the lower region of layer V. These fan shaped neurons had a broad dendritic arbor which covered an area measuring 350 x 300 ,um (Fig. 3 C). The axons of these cells penetrated into the white matter but the majority also showed an additional horizontal collateral. Neurons ofcerebral cortex. I 223

Fig. 5. Main neuronal types in layer VI ofthe cerebral cortex of the adult rat (Rattus norvegicus). P, pyramidal neuron; IP, inverted pyramidal neuron; *, multiapical pyramidal neuron; Fs, fan shaped neuron; SH, small horizontal neuron; M, Martinotti cell; HP, horizontal pyramidal neuron; LH, large horizontal cell; BT, bi-tufted neuron; S, multipolar neuron with short axonal spread; FP, flattened pyramidal neuron; ax, axons. Arrows indicate trajectories ofthe axons and collaterals. Bar, 100 um.

Martinotti cells were present in lamina VIa; they exhibited a globular soma and frequently had radially ramifying dendrites. In some cells, the dendrites branched upwards while in others arborisation was downwards. Some cells mimicked inverted pyramids. However, a characteristic feature ofthese cells was the very long, ascending axon stretching across the upper layer of the cortex (Fig. 4A); on occasion, the trajectory of the axon was traced over a distance of 900-1000 .um. Multipolar spiny neurons with axons deeply penetrating into the white matter were seen in the deeper region of the sixth layer. They had polygonal soma with radially arborising spiny dendrites approximately 150-200,m long. The axon descended towards the white matter, which it penetrated to a considerable depth (Fig. 4B); before entering the white matter a few local collaterals emerged. 224 I. FERRER, I. FABREGUES AND E. CONDOM

Fig. 6. Main neuronal types in layer VI of the cerebral cortex of the mouse (Mus musculus). LP, large pyramidal neuron; SP, small pyramidal neuron; IP, inverted pyramidal neuron; FP, flattened pyramidal neuron; HP, horizontal pyramidal cell; H, horizontal neuron; S, multipolar neuron with locally arborising axon, M, Martinotti cell; ax, axons; asterisks, multiapical pyramidal cells. Bar, 100 em. Multipolar sparsely spined or spine-free neurons with locally ramifying axons and bi-tufted neurons. Multipolar neurons were seen throughout layer VI while bi-tufted neurons were confined to the upper region (VIal. Multipolar neurons had polygonal soma with 150-200 ,tm long, thin, radiate dendrites showing few secondary branches. The bi-tufted neurons were spindle shaped and vertically aligned with sparsely ramified, vertically oriented thin dendrites emerging at both poles. The boundaries between multipolar and bi-tufted neurons were not always clearly defined, and intermediate types may well have existed. Both were either sparsely spined or spine- free. The axon originated from the soma or from the proximal segment of a dendrite and followed an irregular course with extensive arborisation within layer VI. Some multipolar neurons exhibited axonal arbors similar to those seen in small (basket- like) cells in other layers of the cortex (Fig. 4B, C). Neurons of cerebral cortex. I 225

ax ------

h Fig. 7. Some neuronal types in layer VI of the cerebral cortex of the hamster (Cricetus cricetus). P, pyramidal neuron; *, multiapical pyramidal neuron; H, horizontal neuron; S, multipolar neuron with short axons; M, Martinotti cell, ax, axons; arrows, course ofthe axons. Bar, 100 /m.

All these cell types also were observed in the adult rat (Fig. 5), although the axons in myelinated fibres failed to stain.

Layer VI in other rodents: the mouse, hamster and vole Most neuronal types encountered in the rat were also seen in other rodents (Figs. 6, 7). In young mice (15-20 days), impregnated axons exhibited characteristics similar to those seen in the rat. In the adult mouse, hamster and vole (only adult animals were studied in the last two species), the axons often were not impregnated, but the different cell types were easily distinguished with the rapid Golgi method.

Layer VI in the rabbit The cerebral cortex in the rabbit was approximately 1800 ,cm thick, with layer VI accounting for 400 ,um; the supragranular layers (laminae II and III) were slightly better developed in the rabbit than in the rat and mouse. 226 I. FERRER, I. FABREGUES AND E. CONDOM

ax HP Fig. 8. Main neuronal types in layer VI ofthe cerebral cortex ofthe rabbit (Oryctolagus cuniculus). P, pyramidal neuron; *, multiapical pyramidal neuron; M, Martinotti cell; HP, horizontal pyramidal neuron; S, multipolar neuron with short axon; H, horizontal neuron; ax, axons; SP, small pyramidal cell. Arrows show the course of the axons and collaterals in some cells. Bar, 1OOtzm.

With the Golgi method, neuronal types similar to those described in the rat were seen: pyramidal neurons (large, medium, atypical and flattened), multiapical pyramidal neurons, inverted pyramids, fusiform neurons, horizontal pyramidal neurons, Martinotti cells, large and small spinous and sparsely spined horizontal neurons and sparsely spined multipolar cells with locally arborising axons (Fig. 8). Fan shaped neurons were stained only twice in a 20 days old rabbit, while multipolar neurons with long descending axons were never impregnated. The horizontal cells in layer VI in the rabbit usually were larger than those in the rat, and dendrites were more spinous than in the latter species; however, small horizontal neurons with sparsely spined dendrites were also observed. The axons of the large horizontal cells were traced over distances of 700 ,um in 20-22 days old Neurons ofcerebral cortex. I 227

Fig. 9. Neuronal types in layer VI of the cerebral cortex of the hedgehog (Erinaceus europaeus). P, pyramidal neuron; *, multiapical pyramidal neuron; HP, horizontal pyramidal neuron; sP, small pyramidal neuron; S, multipolar neuron with locally arborising axon; M, Martinotti cell; IP, inverted pyramidal neuron; H, horizontal neuron; ax, axons. The arrows indicate the trajectories of the axons and collateral branches. Bar, 100 /,m. rabbits. Martinotti cells stained in large numbers in the rabbit; they often had an inverted pyramid-like appearance, but the axon had the typical ascending course and reached the supragranular layers. Layer VI in the hedgehog The cerebral cortex in the hedgehog has characteristics not found in other mammals. The upper regions or 'accentuated layers II' (Sanides & Sanides, 1974) are occupied by large, spiny stellate cells resembling those found in palaeocortical formations (Valverde & Lopez-Mascaraque, 1981). The medium pyramidal and polymorphous neurons below this layer were interpreted by Valverde & Lopez- Mascaraque (1981) as forming laminae III-IV. Layers V and VI appear to be similar to those in other animals. The cortex of the hedgehog was 800 ,um thick and layer VI measured approximately 150,zm in thickness. The cells in the deeper region were separated by myelinated fibres. In Golgi sections, medium and large pyramidal cells, small pyramidal neurons, multiapical pyramidal cells and obliquely oriented pyramidal 228 I. FERRER, I. FABREGUES AND E. CONDOM

1 2

8 ax

Fig. 10. Pyramidal neurons (1, 2, 3, 4), multiapical neurons (5, 6), multipolar neurons (7) and horizontal neurons (8) in layer VI of the cerebral cortex of the bat (Pipistrellus pipistrellus). ax, axons. Bar, 100 /m. cells were seen. These cells had large descending axons entering the white matter; some of the neurons had horizontal or oblique collaterals and thin, long, ascending collaterals stretching as far as the upper boundary of layer V. Fan shaped cells, fusiform neurons and multipolar neurons with descending axons did not stain, but inverted pyramidal neurons, Martinotti cells, horizontal neurons with horizontal axons and sparsely spined and spine-free multipolar neurons with locally arborising axons were impregnated in great detail (Fig. 9). The morphological characteristics of these neurons were similar to those of the rat.

Layer VI in the dwarfbat The detailed study of the cerebral cortex of the bat is the subject of a future report but a brief summary of findings related to layer VI is included here. The sixth layer of the parietal lobe was composed ofcell types similar to those seen in other species (Fig. 10). Pyramidal neurons, multiapical pyramidal cells, sparsely spined multipolar neurons with locally arborising axons, Martinotti cells and horizontal neurons were the morphological types most frequently seen. Neurons ofcerebral cortex. I 229

DISCUSSION General comments A basic uniformity in the structure of layer VI of the cerebral cortex has been observed in species with lissencephalic brains. The main neuronal types include: (1) pyramidal neurons (large, medium, small or flattened and atypical or triangular); (2) multiapical pyramidal cells; (3) inverted pyramids; (4) fusiform neurons; (5) large and small horizontal neurons with horizontal axons; (6) horizontal pyramidal cells; (7) fan shaped neurons; (8) Martinotti cells; (9) multipolar spinous neurons with long descending axons penetrating into the white matter; (10) sparsely spined and spine-free multipolar neurons with short axons and (11) bi-tufted neurons. The distribution of these cells is not homogeneous,but varies between the two laminae ofthe sixth layer. Large pyramidal neurons, triangular or atypical pyramidal cells, multiapical pyramidal neurons, inverted pyramids, fusiform neurons, Marti- notti cells and bi-tufted cells predominate in lamina VIa; medium sized, flattened pyramids, large and small horizontal neurons, horjzontal pyramidal cells, fan shaped neurons and multipolar spinous neurons with long descending axons predominate in lamina VIb; sparsely spined and spine-free multipolar neurons with short axons are present in both laminae, while sparsely spined neurons with axons similar to those found in basket cells in other layers of the cortex are seen mainly in lamina VIa. In the mouse cerebral cortex, Lorente de No (1922, 1933) described several neuronal types in the sixth layer: polygonal or globular neurons with long apical dendrites and long descending axons penetrating the white matter, globular cells with axons ascending towards the upper layers of the cortex and small cells with short axons and horizontal neurons. Horizontal neurons were also described by Meller et al. (1969) in electron micro- scopic studies of the brain of the mouse. Parnavelas et al. (1977) demonstrated the presence of properly orientated pyramidal cells and inverted or obliquely orientated pyramidal neurons in the sixth layer of the rat cortex. Feldman & Peters (1978) and Peters & Proskauer (1980) described sparsely spined and spine-free multipolar neurons, horizontal multipolar neurons and bi-tufted cells in the sixth layer of the rat. Oblique pyramidal neurons in the cerebral cortex of the rabbit have been reported by McMullen & Glaser (1982). Multipolar neurons, large and small horizontal neurons and cells with long ascending axons directed towards the upper layers were illustrated by Tombol (1978a) in the sixth layer of the rabbit. Valverde & Lopez- Mascaraque (1981) described pyramidal cells and polymorphic neurons in layer VI of the cortex of the hedgehog. So far, little is known about the deep layer of the cerebral cortex of the bat (Brown, 1982). Morphological characteristics of most of the cellular types in the present study are similar to those described by other authors with minor differences in nomenclature, the result of personal choice; in the present study, the preference is for the term horizontal neurons of the sixth layer (Tomb6l, 1976, 1978 a) rather than horizontal multipolar neurons (Feldman & Peters, 1978), because the structural shape and axonal course of these neurons is entirely different from that of other multipolar neurons. The terms multiapical pyramidal cells and fan shaped cells are also intro- duced to identify two types of pyramidal neurons observed only in the sixth layer 230 I. FERRER, I. FABREGUES AND E. CONDOM because of their unique, broad apical dendritic arbor; likewise, triangular neurons or atypical pyramidal cells (Ramon y Cajal, 1911) with their long asymmetrical basilar dendrite may have a particular receptive dendritic field. Tombol (1984) has recently reviewed comprehensively and in detail the structure of the sixth layer, but because it is mainly devoted to gyrencephalic species a comparative analysis of results is discussed by Ferrer et al. (1986).

Morphological bases for projective and associative functions in layer VI Projective cells Over the last decade, several studies using horseradish peroxidase as tracer in the cerebral cortex of the rat have demonstrated that the source of the majority of cortico-thalamic projections and of a large number of the contralateral corticocortical (callosal) connections emerge from pyramidal neurons in layers V and VI (Jacobson & Trojanowski, 1974, 1975; Yorke & Caviness, 1975; Wise & Jones, 1976, 1977; Cipolloni & Peters, 1979; Sefton, MacKay-Sim, Baur & Cottee, 1981; Swadlow & Weyand, 1981; Zabousky & Wolff, 1982; Olavarria & Sluyters, 1983). Nevertheless, other neurons in layer VI with axons penetrating deeply into the white matter may also possess long projection fibres. Such putative projection neurons, revealed with the Golgi method, are multiapical pyramidal cells, inverted pyramidal neurons, fan shaped neurons, fusiform cells and spinous multipolar neurons with long descending axons. Although it is possible that these different cell types represent different functional classes projecting towards specific target sites, the foregoing morphological findings are not sufficient to confirm this possibility. Ipsilateral, long, corticocortical connections were also seen in the cerebral cortex of the rat with the Fink-Heimer method or using horseradish peroxidase as tracer. Layers II-VI participate in the reciprocal connections between striate and extrastriate areas of the cortex (Montero, Bravo & Fernandez, 1973; Olavarria & Sluyters, 1983) but the supragranular laminae appear to be the main source for cortical connections between striate and extrastriate areas and between visual and other (i.e. motor, association, temporal and perirhinal) regions (Miller & Vogt, 1984). However, the large horizontal neurons with their long axons extending across the lower boundary of layer VI may be the source of long corticocortical connections projecting within the same layer. Local circuit neurons With the Golgi method three intracortical subsystems may be distinguished in the sixth layer. The ascending interlaminar fibrillar system is formed by axons of Martinotti cells reaching towards the supragranular layers of the cortex and by ascending axonal collaterals of pyramidal neurons and multiapical pyramidal neurons which reach different levels of the contiguous upper levels and even the upper region of layer V. The horizontal interlaminar fibrillar system is formed by axons of horizontal neurons and of horizontal axonal collaterals of pyramidal neurons and multiapical cells. The arrangement ofthese horizontal neurons is noteworthy because in common with the Cajal-Retzius neurons in the molecular layer and with basket-like neurons in different layers of the cortex they appear to be the only cells not directly contri- buting to the basic columnar organisation of the neocortex (Szentigothai, 1975, 1978; Eccles, 1981). Neurons ofcerebral cortex. I 231 The local non-horizontalfibrillar system originates in the sparsely spined multipolar neurons and bi-tufted neurons. Horizontal neurons, sparsely spined, spine-free multipolar neurons and bi-tufted neurons may have an important function in the modulation of layer VI activity since the inhibitory neurotransmitter y-aminobutyric acid as well as somatostatin, cholecystokinin and avian pancreatic polypeptide-like activity has been demonstrated in these cells (Ribak, 1978; Chronwall & Wolff, 1980; McDonald et al. 1982a, b, c; Peters, Miller & Kimerer, 1983).

Receptor fields in layer VI neurons Various afferent fibres terminate in the deep region of the cortex and may establish connections with neurons of the sixth layer. In horseradish peroxidase tracer studies in the rat, Peters & Saldanha (1976) demonstrated projections ofthe lateral geniculate nucleus to layer VI of area 17; Herkenham (1980) has reported projections of the medial and geniculate nuclei to layers IV and VI of the auditory and visual regions respectively and projections of the intralaminar nuclei in layers V and VI of the cortex. Using similar techniques, Ribak (1977) found projections of layers V and VI of area 18a into the infragranular layers of the contralateral hemisphere. Further- more, nonadrenergic fibres from the locus coeruleus and serotoninergic fibres from the raphe nuclei, although distributed throughout all layers of the cortex, also terminated in layer VI (Parnavelas & McDonald, 1983). The pyramidal neurons of the polymorphic cell layer, however, may have receptor fields which are not limited to these terminals within layer VI. The classification of pyramidal neurons and their different subtypes (i.e. large, medium and flattened) are not only descriptive, but express the degree of specialisation of their respective receptor fields. The small pyramids have receptor fields limited to localised afferents within layer VI only, while medium and large pyramids may have afferent receptor fields in the fifth and upper layers of the cortex. A particular type of pyramidal cell, the multiapical pyramidal neuron (and also the fan shaped neuron) may, according to the degree of arborisation of its apical dendrites, occupy a field 250-300 ,sm wide extending across the lower and middle regions of layer V. Besides exogenous afferent fibres, the neurons of layer VI may have receptor sites for intracortical fibres from the same layer and from the upper layers of the cortex, mediated by axonal collaterals originating in the pyramidal and non-pyramidal neurons of layers II, III and V as seen in Golgi preparations. Although the present results based on Golgi techniques illustrate certain general features of the organisation of layer VI, further studies with a combination of different methods (Fairen, Peters & Saldanha, 1977; Peters & Fairen, 1978; Fairen, De Felipe & Martinez-Ruiz, 1981; Cipolloni & Peters, 1983; White, Bensaholm & Hersch, 1984) are needed to obtain more accurate information on interneuronal connections. SUMMARY A study of the morphological characteristics of the neurons in layer VI of the cerebral cortex was carried out using the rapid Golgi method in several lissencephalic species including Rodentia (rat, mouse, vole (Microtus agrestis) and hamster), Lagomorpha (rabbit), Insectivora (hedgehog) and in the Chiroptera the dwarf bat (Pipistrellus pipistrellus). There was a basic uniformity in the structure of the sixth layer. Main neuronal types in lamina VIa were large pyramidal neurons, triangular or atypical pyramidal 232 I. FERRER, I. FABREGUES AND E. CONDOM cells, multiapical pyramidal neurons, inverted pyramids, fusiform neurons, Marti- notti cells and bi-tufted cells. Main neuronal types in lamina VIb were medium sized, flattened pyramids, large and small horizontal neurons, horizontal pyramidal cells, fan shaped neurons and multipolar spinous neurons with long descending axons. Sparsely spinous and spine-free multipolar neurons with short axons were present in the two laminae of layer VI, but sparsely spinous neurons with axons similar to those found in basket cells of other layers of the cortex were observed mainly in lamina VIa. Neuronal subsystems were tentatively classified on the basis of the course of the axons. Pyramidal neurons, fusiform neurons, multiapical pyramidal cells, inverted pyramidal cells, fan shaped neurons and multipolar neurons with large descending axons were interpreted as being the main source of long projection and association connections. Large horizontal neurons were interpreted as possible ipsilateral association neurons because the horizontal course of the axons over long distances followed the boundary of the deeper region of the sixth layer. Three intracortical (association) subsystems were included. Axons of Martinotti cells and collateral ascending axons of pyramidal neurons (including multiapical pyramidal neurons) formed the ascending interlaminar fibrillary subsystem. Axons of small horizontal cells and horizontal collaterals of pyramidal neurons formed the horizontal intracortical subsystem. Sparsely spinous and spine-free multipolar neurons and bi-tufted cells were the main source of the local, non-horizontal fibrillary subsystem.

REFERENCES BROWN, J. W. (1982). Telencephalon of Chiroptera. In Comparative Correlative Neuroanatomy of the Vertebrate Telencephalon (ed. E. C. Crosby & H. N. Schnitlein), pp. 435-462. New York: Macmillan. CHRONWALL, B. & WOLFF, J .R. (1980). Prenatal and postnatal development of GABA-accumulating cells in the occipital neocortex of rat. Journal ofComparative Neurology 190, 187-208. CIPOLLONI, P. B. & PETRs, A. (1979). The bilaminar and banded distribution of the callosal terminals in the posterior neocortex of the rat. Brain Research 176, 33-47. CIPOLLONI, P. B. & PErERs, A. (1983). The termination of callosal fibres in the auditory cortex of the rat. A combined Golgi-electron microscope and degeneration study. JournalofNeurocytology 12, 713-726. ECCLES, J. C. (1981). The modular operation of the cerebral neocortex considered as the material basis of mental events. Neuroscience 6, 1839-1856. FAIREN, A., DE FELIPE, J. & MARTINEz-RuIz, R. (1981). The Golgi-EM procedure: a tool to study neocortical interneurons. In Glial and Neuronal Cell Biology (ed. E. Acosta Vidrio & S. Fedoroff), pp. 291-301. New York: Liss. FAIREN, A., PETERS, A. & SALDANHA, J. (1977). A new procedure for examining Golgi impregnated neurons by light and electron microscopy. Journal ofNeurocytology 6, 311-337. FELDMAN, M. L. & PETERS, A. (1978). The forms of non-pyramidal neurons in the visual cortex of the rat. Journal of Comparative Neurology 179, 761-794. FERRER, I., FABREGUES, I. & CONDOM, E. (1986). A Golgi study of the sixth layer of the cerebral cortex. II. The gyrencephalic brain of Carnivora, Artiodactyla and Primates. Journal ofAnatomy (in Press). FERRER, I. & MARTINEZ MATOS, J. A. (1981). Development ofnon-pyramidal neurons in the rat sensory motor cortex during the fetal and early post-natal periods. Journalffir Hirnforschung 22, 555-562. HERKENHAM, M. (1980). Laminar organization of the thalamic projections in the rat neocortex. Science 207, 532-535. JACOBSON, S. & TROJANOWSKI, J. Q. (1974). The cells of origin of the corpus callosum in rat, cat and Rhesus monkey. Brain Research 74, 149-155. JACOBSON, S. & TROJANOWSKI, J. Q. (1975). Corticothalamic neurons and thalamo-cortical fields: an investigation in rat using HRP and autoradiography. Brain Research 85, 385-401. JONES, E. G. (1975). Varieties and distribution of non-pyramidal cells in the somatic sensory cortex of the squirrel monkey. Journal of Comparative Neurology 160, 205-268. KOWALSKI, K. (1976). Mammals (an outline of theriology), pp. 229-239. Warszawa: Panstwowe Wydawnictwo Naukowe. (Spanish translation, ed. Blume, Madrid, 1981). LORENTE DE N6, R. (1922). Le corteza cerebral del raton. Trabajos del Laboratorio de investigaciones biologicas de la Universidad de Madrid 20, 41-78. Neurons of cerebral cortex. I 233 LORENTE DE N6, R. (1933). Studies on the structure of the cerebral cortex. Journalfur Psychologie und Neurologie 45, 381-438. LORENTE DE N6, R. (1949). Cerebral cortex: architecture, intracortical connections, motor projections. In Physiology ofthe Nervous System (ed. J. F. Fulton), pp. 288-330. London: Oxford University Press. McDONALD, J. K., PARNAVELAS, J. G., KARAMANLIDIS, A. N., BRECHA, N. & KOENIG, J. A. (1982a). The morphology and distribution of peptide-containing neurons in the adult and developing visual cortex of the rat. I. Somatostatin. Journal ofNeurocytology 11, 809-824. McDONALD, J. K., PARNAVELAS, J. G., KARAMANLIDIS, A. N., ROSENQUIST, G. & BRACHA, N. (1982b). The morphology and distribution ofpeptide-containing neurons in the adult and developing visual cortex of the rat. III. Cholecystokinin. Journal ofNeurocytology 11, 881-895. MCDONALD, J. K., PARNAVELAS, J. G., KARAMANLIDIS, A. N. & BREcHA, N. (1982c). The morphology and distribution of peptide-containing neurons in the adult and developing visual cortex of the rat. IV. Avian pancreatic polypeptide. Journal ofNeurocytology 11, 985-995. MCKENNA, M. C. (1975). Toward a phylogenetic classification of the mammals. In Phylogeny of the Primates (ed. W. P. Luckett & F. S. Szalay), pp. 31-46. New York: Plenum Press. MCMULLEN, N. T. & GLASER, E. M.,(1982). Morphology and laminar distribution of non-pyramidal neurons in the auditory cortex of the rabbit. Journal of Comparative Neurology 208, 85-106. MELLER, K., BREIPOHL, W. & GLEES, P. (1969). Ontogeny of the mouse motor cortex. The polymorph cell layer or layer VI. A Golgi and electron microscopical study. Zeitschrift fur Zellforschung und mikro- skopische Anatomie 99, 443-458. MILLER, M. W. & VOCT, B. A. (1984). Direct connections of rat visual cortex with sensory, motor and association cortices. Journal of Comparative Neurology 226, 184-202. MIODONSKI, A. (1974). The angioarchitectonics and cytoarchitectonics structure of the fissural frontal neocortex in dog. Folia biologica 22, 237-279. MONTERO, V. M., BRAvo, H. & FERNANDEZ, V. (1973). Striate-peristriate cortico-cortical connections in the albino and grey rat. Brain Research 53, 202-207. OLAVARIUA, J. & VAN SLUYTERS, R. C. (1983). Widespread callosal connections in infragranular visual cortex of the rat. Brain Research 279, 233-237. O'LEARY, J. L. & BISHOP, G. H. (1938). The optically excitable cortex of the rabbit. Journal of Com- parative Neurology 68, 423-477. O'LEARY, J. L. (1941). Structure of the area striata of the cat. Journal of Comparative Neurology 75, 131-164. PARNAVELAS, J. G., LIEBERMAN, A. R. & WEBSTER, K. E. (1977). Organization of neurons in the visual cortex, area 17, of the rat. Journal ofAnatomy 124, 305-322. PARNAVELAS, J. G. & MCDONALD, J. K. (1983). The cerebral cortex. In Chemical Neuroanatomy (ed. P. C. Emson), pp. 505-549. New York: Raven Press. PETERS, A. & FAIREN, A. (1978). Smooth and sparsely spined stellate cells in the visual cortex of the rat: a study using a combined Golgi-electron microscope technique. Journal ofComparative Neurology 181, 129-172. PETERS, A., MILLER, M. & KIMERER, L. M. (1983). Cholecystokinin-like immunoreactive neurons in rat cerebral cortex. Neuroscience 8, 431-448. PETERS, A. & PROSKAUER, D. C. (1980). Smooth and sparsely spined cells with myelinated axons in rat visual cortex. Neuroscience 5, 2079-2092. PETERS, A. & REGIDOR, J. (1981). A reassessment of the forms of non-pyramidal neurons in Area 17 of cat visual cortex. Journal of Com;parative Neurology 203, 685-716. PETERS, A. & SALDANHA, J. (1976). The projection of the lateral geniculated nucleus to Area 17 of the rat cerebral cortex. III. Layer VI. Brain Research 105, 533-537. RAMON Y CAJAL, S. (1911). Histologie du Systeme Nerveux de l'Homme et des Vertibrds, vol. ii, pp. 571- 575. (Reprint 1972). Madrid: Consejo Superior de Investigaciones Cientificas. RIBAK, C. E. (1977). A note on the laminar organization ofthe rat visual cortex projections. Experimental Brain Research 27, 413-418. RIBAK, C. E. (1978). Aspinous and sparsely-spinous stellate neurons in the visual cortex of rats contain glutamic acid decarboxylase. Journal ofNeurocytology 7, 461-478. SANIDES, D. & SANIDES, F. (1974). A comparative Golgi study of the neocortex in insectivores and rodents. Zeitschrift fur mikroskopisch-anatomische Forschung 88, 5 S, 957-977. SEFTON, A. J., MACKAY-SIM, A., BAUR, L. A. & COTrEE, L. J. (1981). Cortical projections to visual centers in the rat: an HRP study. Brain Research 215, 1-13. SWADLOW, H. A. & WEYAND, T. G. (1981). Efferent systems of the rabbit visual cortex: laminar distribution of the cells of origin, axonal conduction velocities and identification of axonal branches. Journal of Comparative Neurology 203, 799-822. SZENTAGOTHAI, J. (1975). The "module concept" in cerebral cortex architecture. Brain Research 95, 475-496. SZENTAGOTHAI, J. (1978). The local neuronal apparatus of the cerebral cortex. In Cerebral Correlates of Conscious Experience (ed. P. Buse & A. Rougeul-Buser), INSERM symposium no. 6, pp. 131-138. Amsterdam: Elsevier/North Holland. 234 I. FERRER, I. FABREGUES AND E. CONDOM TOMBOL, T. (1976). Golgi analysis of the internal layers (V and VI) of the cat visual cortex. Experimental Brain Research, Suppl. I, 292-295. TOMBOL, T. (1978a). Comparative data on the Golgi architecture of interneurons of different cortical areas in cat and rabbit. In Architectonics ofthe Cerebral Cortex (ed. M. A. B. Brazier & H. Petsche), pp. 59-76. New York: Raven Press. TOMBOL, T. (1978b). Some Golgi data on visual cortex of the Rhesus monkey. Acta morphologica Academiae scientiarum hungaricae 26, 115-138. T6MBOL, T. (1984). Layer VI cells. In Cerebral Cortex, vol I, (ed. A. Peters & E. G. Jones) pp. 479-519. New York: Plenum Press. TuTruRI, A. R. (1971). Classification of neurons in the ectosylvian auditory cortex of the dog. Journal of Comparative Neurology 203, 685-716. VALVERDE, F. (1978). The organization of Area 18 in the monkey. A Golgi study. Anatomy and Embryology 154, 305-334. VALVERDE, F. & LOPEZ-MASCARAQUE, L. (1981). Neocortical endeavor: basic neuronal organization in the cortex of hedgehog. In Glial and Neuronal Cell Biology (ed. E. Acosta Vidrio & S. Fedoroff), pp. 281-289. New York: Alan R. Liss. VAN DER Loos, H. (1965). The improperly oriented pyramidal cell in the cerebral cortex and its possible bearing on problems of neuronal growth and cell orientation. Bulletin ofthe Johns Hopkins Hospital 117, 228-250. WHITE, E. L., BENSHALOM, G. & HERSCH, S. M. (1984). Thalamocortical and other synapses involving nonspiny multipolar cells of mouse SmI cortex. Journal of Comparative Neurology 229, 311-320. WISE, S. P. & JoNEs, E. G. (1976). The organization and postnatal development of the commissural projections of the rat somatic sensory cortex. Journal of Comparative Neurology 168, 313-344. WISE, S. P. & JONES, E. G. (1977). Cells of origin and terminal distribution of descending projections of the rat somatosensory cortex. Journal of Comparative Neurology 175, 129-158. YORKE, C. H. & CAvINmSS, V. S. (1975). Interhemispheric neocortical connections of the corpus callosum in the normal mouse: a study based on anterograde and retrograde methods. Journal of Comparative Neurology 164, 233-246. ZABOUSKY, L. & WOLFF, J. R. (1982). Distribution pattern and individual variations of callosal connections in the albino rat. Anatomy and Embryology 169, 45-59.