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Brain Research, 244 (1982) 9-16 Elsevier Biomedical Press 9 A Type of Basket Cell in Superficial Layers of the Cat Visual Cortex. A Golgi-Electron Microscope Study JAVIER DeFELIPE and ALFONSO FAIRI~N* Unidad de Neuroanatomia, Instituto Cajal, CSIC, Veldzquez 144, Madrid 6 (Spain) (Accepted December 24th, 1981) Key words: cerebral cortex - - interneurons - - basket cells - - Golgi-EM technique The axonal arborizations of the basket cells in the cerebral neocortex have long been considered as the source of the presynaptic terminals contacting the cell bodies of pyramidal cells. Given that the concept of the cortical basket cell is based upon indirect evidence only, it was deemed worthwhile to re-investigate this problem using the Golgi-EM technique. This approach permits one to trace the presynaptic terminals back to their parent cell body, so that it allows for a positive identification of basket cells, i.e. cells which produce axosomatic synapses by preference. A type of interneuron in layer l I - l l l of the cat visual cortex is described. Its axon terminals form multiple synaptic contacts, of the symmetrical type, on cell bodies and proximal dendrites of pyramidal and non-pyramidal cells. On the basis of this efferent synaptic pattern, this interneuron is considered to be a basket cell. The possible correspondence of this interneuronal type with other putative basket cells described in previous Golgi studies is discussed. In addition, a simple re-sectioning method for semithin sections is described, which has been used to identify individual Golgi-impregnated axonal boutons in electron microscopy. INTRODUCTION Cell bodies o f p y r a m i d a l cells in the cerebral n e o c o r t e x receive synapses which are o f the symmetrical type2,6A2,17,18,19. All evidence indicates that these a x o s o m a t i c synapses are GABAergicg,22, 24 a n d thus, inhibitoryg,ls, 23. M o r e o v e r , symmetrical G A B A e r g i c 23 synapses have been r e p o r t e d on cell bodies o f n o n - p y r a m i d a l cells12,17,12, 2°. The pericellular plexuses f o r m e d by the axons o f b a s k e t cells have been considered as the source o f these a x o s o m a t i c synapses on p y r a m i d a l cells~, 10, 13,14,22,27. These plexusesS, 15 are s u p p o s e d l y form e d b y the convergence o f an i n d e t e r m i n a t e n u m b e r o f b a s k e t cell axonsl,8,12,15, b u t it is implied that each i n d i v i d u a l i n t e r n e u r o n m a k e s a substantial c o n t r i b u t i o n to the pericellular plexus. To date, however, there is no direct evidence to substantiate such assumptions. G o l g i - E M studies4,7,12A7,18,25,26 have shown that s y m m e t r i c a l synapses derive from the a x o n terminals o f aspinous n o n - p y r a m i d a l cells. One class o f these i n t e r n e u r o n s are m u l t i p o l a r cells * To whom correspondence should be addressed. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press whose axons f o r m descending arcades is. Their a x o n terminals c o n t r i b u t e to the a x o s o m a t i c synapses, but, in addition, an a m p l e variety o f p o s t s y n a p t i c elements are contacted. M o r e o v e r , no multiple axosomatic contacts on a given p y r a m i d are f o r m e d t h a t derive f r o m a single interneuron. This r e p o r t presents G o l g i - E M evidence for a type o f b a s k e t cell, located in layers I I - I I I o f the cat area 17. The axons o f this i n t e r n e u r o n a l type f o r m multiple synaptic contacts on cell bodies and proxim a l dendrites o f p y r a m i d a l a n d n o n - p y r a m i d a l cells. MATERIALS AND METHODS Seven cats, 3 m o n t h s old, were used in this study. U n d e r N e m b u t a l anaesthesia all cats were perfused with a solution o f l ~ g l u t a r a l d e h y d e - l ~o p a r a f o r m a l d e h y d e in a p h o s p h a t e buffer 16. Pieces o f cerebral cortex were rinsed in buffer and then G o l g i i m p r e g n a t e d 6. Slices, 150/zm thick, were o b t a i n e d with a sliding m i c r o t o m e a n d stored in a n h y d r o u s 10 I, 2 3 t lO.um'] Fig. 1. C a m e r a lucida d r a w i n g o f an i n t e r n e u r o n in layer I I - I I I of the cat area 17. The arrow points to the origin of the axon. See text for details. Fig. 2. Enlarged view of the boxed area in Fig. I. Note the axonal swellings I. 2 a n d 3 a n d a connecting axom~l braid {arrowhead L A n ascending dendrite is labelled as d. 11 glycerol. Those slices containing well impregnated interneurons were illuminated while in glycerol at 24 °C for 90 rain, using a 150 W photoflood lamp ,~. Then they were gold-toned, de-impregnated, and embedded in Epon-Araldite 6. Besides illumination of the slices prior to gold toning, some technical modifications were used 5,7 which significantly increase the yield of axonal arborizations that can be followed in their entirety after de-impregnation. After plastic embedding, drawings were made using a camera lucida, and then the slices were serially sectioned at 3 # m in an ultramicrotome. Semithin sections were picked up individually from the trough and mounted on slides made from the same EponAraldite used for embedding. For that purpose, the sections were placed on the slides with a drop of distilled water and allowed to dry at 60 °C for at least 1 h; no special adhesive was used. Later on, they were stained with l ~ toluidine blue in 1 ~o borax. The sections were inspected in the light microscope to ascertain which parts of the axonal arborization would appear in each section. Using this method, absolute certainty as to which Golgistained boutons belong to the axonal plexus under study was obtained (Figs. 1-6). With the semithin sections facing out, the plastic slide pieces were attached to plastic blanks using cyanoacrylic glue and trimmed with glass knives. Serial ultrathin sections were then obtained. When compared to previous techniques for re-sectioning semithin sections 2,s,11, the present one offers the advantage of its obvious simplicity. RES U LTS The interneurons that were considered for the present study were multipolar neurons with local axonal arborizations, located in layer l l - l l l of the cat visual cortex. These cells were systematically inspected in semithin sections to ascertain whether their axonal boutons were preferentially located at a perisomatic site. When this was the case, the semithin sections were re-sectioned in the ultrathin range and examined under the electron microscope in order to prove the existence of axosomatic synapses effected by these axons, and to define the form of the synaptic contacts. One example has been chosen to illustrate the features of these cells that give origin to perisomatic axonal boutons. Fig. 1 depicts a layer l I - I I l cell that has a multipolar appearance with a rather wide dendritic field. The axon (arrow) emerges from the base of a major dendritic trunk. It is primarily descending and originates as a dense plexus in the lower region of the dendritic field. Some collaterals turn upwards and spread in a fan-like fashion to distribute in the upper part of the dendritic domain. Boutons, mainly 'en passant', are seen along the axonal branches. It is noteworthy that the axonal arborization shows no signs of target selectivity (see ref. 7), i.e. no basket-like formations are visible when the Golgi sections are examined at the light microscope level. Under the electron microscope, however, labelled neurons belonging to the axonal plexus of this interneuron are consistently seen against the cell bodies of pyramidal cells (Figs. 4, 7, 9 and 10). Such distribution is also evident in semithin sections (Fig. 3). One distinguishing characteristic of this interneuronal type is that its axonal arborizations form multiple axosomatic contacts on a given postsynaptic cell. The number of pericellular boutons each single pyramidal cell body receives from the cell shown in Fig. 1 can be rather large; for example, 7 in the case shown in Figs. 9 and 10. Pyramidal cell bodies are not the only targets for this axonal arborization. The perikaryon of a cell, which, according to established criteriaas,2 0, is classified as an aspinous non-pyramidal cell, is shown in Figs. 13 and 14. This cell, located in the bottom of Fig. 3. A selected area ofa semithin section, is photographed here at the same scale as Fig. 2. Fragments of dendrite d, boutons 2 and 3, and the axonal braid (arrowhead) can be recognized. Axonal boutons 2 and 3 are against the perikaryon of a pyramidal cell (P) of layer 11. Fig. 4. After re-sectioning the semithin section shown in Fig. 3., the same structures can be recognized in an ultrathin section. Fig. 5. In an adjacent section, boutons 2 and 3 are seen surrounding the perikaryon, close to the axon hillock, is - axon initial segment. Fig. 6. In another section of the series, bouton 2 is seen at a higher magnification. The symmetrical synapse (arrow) formed by this bouton on the soma has been sectioned obliquely. 12 Fig. 7. L o w power electron m i c r o g r a p h of a n ultrathin section obtained f r o m a semithin section adjacent to the one s h o w n in Fig, 3, B o u t o n s 1 a n d 2 (see Fig. 2) are in contact with the perikaryon of the s a m e p y r a m i d a l n e u r o n (P) as in Fig. 4, Fig. 8. Symmetrical synapse (arrow) formed by bouton t on the pyramidal cell perikaryon. Figs. 9-12. A x o n a l b o u t o n s fi'om the interneuron of Fig. I, s u r r o u n d i n g the perikaryon of a layer 11 pyramidal cell, T h e low powe~ m i c r o g r a p h s of Figs. 9 a n d 10 belong to ultrathin sections taken f r o m two adjacent semithin sections. A ~ot~fl of 7 boutoTls (I 7) ~re visible. I n Figs. 1 1 a n d 12 the symmetrical synapses (arrows) formed by bouton~ I and 4 are show~. 13 . Figs. 13-18. Synapses on the perikaryon of a smooth stellate cell. Fig. 13. Low power micrograph of the postsynaptic neuron. The nucleus is eccentrically located, and the cytoplasm is rich in organelles. (continued on page 14) 14 Fig. 19. Low power micrograph of a fusiform cell (A). The perikaryal cytoplasm is dark and contrasts with the appearance of that of a contiguous neuron (B), which is the same shown in Fig. 13. A labelled bouton contacts the perikaryon of the fusiform neuron (arrow). Fig. 20. In a serial section, a narrow band of the perikaryat cytoplasm of the fusiform neuron is seen (A). Note the high number of clusters of ribosomes. A band of cytoplasm of cell B is seen. Two additional labelled boutons synapse on the fusiform cell perikaryom layer II1, receives 5 stained b o u t o n s on its cell body which e m a n a t e from the same source. In addition, 3 labelled b o u t o n s contact the p e r i k a r y o n of a cell with a different m o r p h o l o g y (Figs. 19 and 20). It is a fusiform cell with a dark cytoplasm due to its high c o n t e n t of free ribosomes grouped into clusters. The p e r i k a r y o n receives only a few unlabelled synapses. The nucleus is elongated a n d shows, in other sec- tions t¥om the series, a n i n d e n t e d envelope. These features make this cell c o m p a r a b l e to the bitufted cell described by Peters et al. "~'t in the rat visual cortex. Moreover, proximal dendrites of pyramidal cells have also been identified as postsynaptic. W i t h o u t exceptions, the b o u t o n s form symmetrical synapses on the cell bodies and the proximal dendrites they contact (Figs. 6, 8, 11, 12, 15b, 17, 18 Fig. 14. A large number of synapses (arrowheads) contact the perikaryon. Four boutons (14), labelled by their content of gold particles, are seen in this ultrathin section. Fig. 15. In two consecutive sections from the series, an additional bouton (5) contacts the same perikaryon, The symmetrical synapse (s2) formed by this bouton is seen in b; in a, an unlabelled asymmetrical synapse (sl) is visible On the perikaryon. Fig. 16. The perikaryon receives both asymmetrical (s~) and symmetrical (s2) synapses. Bouton 4 (see Fig. 14) is against the membrane; synaptic contact is not visible in this ultrathin section. Fig. 17. In a consecutive section, bouton 4 forms a symmetrical synapse (arrow) on the perikaryot~ Fig. 18. Axosomatic synapse (arrow) of the symmetrical type, formed by bouton I (see Fig. 14) 15 and 20). These synapses show a very thin postsynaptic density and are similar in m o r p h o l o g y to those formed by the axon o f other types of smooth stellate cells4,7,12,17,18,25,26. DISCUSSION This paper presents, for the first time, evidence for a type of interneuron whose axon terminals form multiple synaptic contacts o f the symmetrical type on cell bodies of both pyramidal and non-pyramidal cells in superficial layers of the cat visual cortex. On the basis of this efferent synaptic pattern, this interneuron is considered to be a basket cell. Although it is clear that convergence must play a role in the building up of a pericellular basket1,8,13, is, the existence of multiple synaptic contacts of a c o m m o n origin indicates that each basket cell axon makes a substantial contribution to the pericellular plexus. Thus, a high degree of selectivity with regard to the postsynaptic partners is apparent which could be compared to that shown by chandelier cells7, 25. If, as postulated, basket and chandelier cells exert an inhibitory action7,9,18,zz,24, 25, the selective distribution of their axon terminals would make these two neuronal types very efficient functionally. A n interesting finding, moreover, is that the axons of the present type of basket cell form multiple synaptic contacts on cell bodies of aspinous non-pyramidal cells, which are considered to be inhibitorylS,20, 23. Such a synaptic arrangement is consistent with physiological data revealing inhibition of first-order REFERENCES 1 Cajal, S. R., Histologie du SystOme Nerveux de l'Homme et des Vertdbrds, Vol. lI, A. Maloine, Paris, 1911, 993 pp. 2 Christensen, B. N., Morphological correlates of synaptic transmission in lamprey spinal cord, J. NeurophysioL, 39 (1976) 197 212. 3 Colonnier, M., Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study, Brain Research, 9 (1968) 268-287. 4 DeFelipe, J. and Fair6n, A., Interneurones with axonal arcades in the cat visual cortex. A Golgi-EM study, Neurosci. Lett., Suppl. 7 (1981) S 399. 5 Fair6n, A., DeFelipe, J. and Martinez-Ruiz, R., The Golgi-EM procedure: a tool to study neocortical interneurons. In E. Acosta Vidrio and S. Fedoroff (Eds.), Glial and Neuronal Cell Biology, Progress in Clinical and Biological Research, Vol 59A, Alan R. 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