THE ANATOMICAL RECORD 228:327-338 (1990)

Morphometry and Frequency of Afferent Synaptic Terminals in the Rabbit Dorsal-Lateral Geniculate Nucleus RAMON CARMONA, RUTH CALVENTE, FRANCISCO ABADfA-MOLINA, AND FRANCISCO ABADf A-FENOLL Departamento de Biologia Celular, Facultad de Ciencias, Campus Fuentenueva, Universidad de Granada, E-18071 Granada, Spain

ABSTRACT

Morphological and morphometric features of the retinal synaptic terminals (RLP) and cortical synaptic terminals (RSD) were analyzed in the aE sector of the rabbit dorsal-lateral geniculate nucleus (dLGN). A methodological approach was selected which allowed us to determine volume of the neuropil and elsewhere record variations in the size and distribution of the two types of terminals found in the three zones (superior, middle, and inferior) from up to down into which the aE sector of the dLGN was divided. After obtaining a n isotropic, uniform, and pseudorandom (IUR) sample, the terminals were examined on the basis of a set of morphometric parameters. An analysis of these data showed the retinal terminals (RLP) to be more numerous and to occupy a greater total area of the neuropil in the dorsal (superior) zone of the nucleus, whereas the number and total area occupied by cortical terminals (RSD) did not vary in the superior, middle, and inferior zones. Upon comparing the two types of terminals, the RLP were larger and more widely distributed, the greatest differences between the two appearing in the dorsal (superior) zone of the dLGN.

As previously reported in the literature, the dorsal lateral geniculate nucleus (dLGN) in the rabbit is made up of two sectors: the a and the p (Rose and Malis, 1965). They differ in terms of cytoarchitecture and distribution of the retinogeniculate projection, which reaches the a but not the p sector (Rose and Malis, 1965; Giolli and Guthrie, 1969; Holcombe and Guillery, 1984). The OL sector can be subdivided into two portions (Holcombe and Guillery, 1984): one external (aE)and one internal (aI) Two types of neurons are found in the a sector of the rabbit dLGN: thalamocortical relay neurons and interneurons. Morphologically, the two cellular types can be clearly distinguished by their size and the features of their processes (Guillery, 1966; Szentagothai et al., 1966; Wong-Riley, 1972; Famiglietti and Peters, 1972; Rafols and Valverde, 1973; Lieberman and Webster, 1974; Kriebel, 1973, 1975; Caballero et al., 1986). Despite the many publications on the dLGN which have appeared, a number of aspects of the fine structure of the synaptic elements and their distribution in the nucleus have yet to be analyzed in detail. In addition to the studies cited above, other authors who have further examined and catalogued the synapses include Guillery (1969), Ralston and Chow (1973), Hajdu et al. (1982), Wilson and Hendrickson (19811, and Hamos et al. (1985). The descriptive information provided by these authors would be of greater use if complemented by studies of the distribution of synaptic elements in the different nuclei. The present article contributes to the current knowledge of the structure of the rabbit dLGN by analyzing the distribution of RLP and RSD terminals in the aEsector. 0 1990 WILEY-LISS, INC

MATERIALS AND METHODS

Six adult New Zealand rabbits weighing approximately 3 kg each were anesthetized with urethane and perfused via the carotid artery with saline solution followed by a mixture of 2% glutaraldehyde and 2% paraformaldehyde in 0.2 M sodium cacodylate buffer (pH 7.4, 340 mosmol). After perfusion the brains were removed, and the diencephala were dissected and divided sagittally into two parts, which were immersed in the same fixative solution as that used in the perfusion step, at 4°C for 2 h. The next step was to extract “cylinders” of dLGN tissue from each piece of diencephalon with a flat-tipped hypodermic needle (i.d. 0.3 mm). The needle inserted into the dLGN gave us the “cylinders” in which we studied the three parts of equal length from up to down in the aEsector. This division allowed us to analyze the contents, at different levels of this sector in the nucleus. These parts were designated superior (S),middle (M), and inferior (I) (Fig. la). The procedure is described in detail in a n earlier publication (Carmona et al., 1990). To ensure that the samples were IUR (isotropic, uniform, and pseudorandom), the method illustrated schematically in Figure 1was used. For each zone of study (S, M, and I), the height h was determined. Two per-

Received July 20, 1989; Accepted March 19, 1990. Address reprint requests to Ramon Carmona, Departamento de Biologia Celular, Facultad de Ciencias, Campus Fuentenueva, Universidad de Granada, E-18071 Granada, Spain.

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hl

a

b

C

e

f

Fig. 1 , Schematic drawings of the sampling system used to obtain IUR (isotropic, uniform, and pseudorandom) sections. (a)Three zones of equal height along the dorsoventral axis of the cxE sector of the nucleus are determined (S = superior, M = middle, I = inferior). (b) Two levels in each zone are chosen at random to determine the thickness h of the slab. (c)Slab thickness limits the type of section obtainable: Thinner slabs (below) provide a narrower range of possible an-

gles of section than thicker slabs (above). (d)To obtain an IUR plane of section, orientators are placed on the upper and lower surfaces of the slab; one division in the lower plate and two in the upper one are chosen at random to determine (e),a plane of section containing all three points of intersection with the slab. (0Material for electron microscopic examination is obtained from a cylinder drawn diagonally across the first section cut through the plane defined in d and e.

pendicular levels along this parameter were chosen randomly (Fig. l b ) for sectioning in order to obtain slabs of random thickness. The variable thickness of these slabs limited the type of section obtainable in future cuts (Fig. lc). Two orientators (Mattfeldt, 1988) were placed on the upper and lower surfaces of the slabs. The zpper one was divided into equidistant sectors, while the lower one contained nonequidistant sine-weighted divisions. One sector of the lower and two in the upper plate were randomly chosen (Fig. Id), and a plane (or section) was drawn to contain the three points chosen (Fig. le). This plane represented a n IUR

section, being randomly determined on the basis of the h parameter as shown in Figure l b . If the values of h obtained a t random were equal or nearly so, the slab would have the same thickness as the section. The second randomizing step, illustrated in Figure Id, results from the use of the two orientating circles. In the plane of section, the height h l will represent the vertical direction of the section, and its perpendicular would be horizontal (normal with respect to the base of the slab). From this plane the parallel cuts required for light microscopic observations can be obtained. For electron microscopic observation, a cylinder was drawn diago-

A F F E R E N T SYNAPTIC TERMINALS IN THE RABBIT dLGN

nally through t h e plane defined by the orientator (Fig. If). In practice, the needle is inserted along the diagonal indicated by the plane of section under consideration. This tissue can be either taken directly as IUR material or subjected to the procedure of Gundersen et al. (1988). These steps can be repeated for as many animals a s necessary, sampling from 100 to 200 events measured per zone. The cylinders thus obtained were kept in fixative for 2 h, then washed several times over a period of 2 h in 0.2 M (pH 7.4, 340 mosmol) sodium cacodylate buffer. After postfixation in 1.5% osmium tetroxide in the same buffer as t h a t used with the fixative, the cylinders were embedded in Spurr’s resin and sectioned for electron microscopic examination. Once the cylinders had been obtained from the dLGN, the rest of the diencephalon pieces were postfixed in Carnoy’s solution for 48 h, embedded in paraffin, and serially sectioned (15 km) according to the method described above, and stained with Kliiver-Barrera’s technique (1953) for light microscopic observation. From these sections, the volume of each zone and of the corresponding neuropils were determined according to Gundersen et al. (1988), based on Cavalieri’s principle. To estimate the neuropil volume we considered a sufficient number of microscopic fields (50) whose distribution was weighted in relation to the area between the sections for each zone and each animal. These sections were observed through a 4 0 objective ~ with a 100 point grid, and, using a point counting method (Glagolev, 19331, two relations were estimated for each zone and each dLGN: the fraction of the zone occupied by cellular soma (VJV,) and the fraction of the zone occupied by the myelin fiber bundles (VdV,). From these two values the neuropil fraction was derived. The series of sections of diencephalon containing the dLGN were used to determine the exact zone traversed by the cylinders in each level (superior, middle, and inferior). The eventual goal of our study was to obtain information on the detailed structural composition of the aE section of the rabbit dLGN in terms of the RLP and RSD afferent synaptic terminals. After completing the calculations described above, the synaptic terminals were analyzed zone by zone, by estimating the following parameters: 1. number of RLP and RSD profiles per unit area 2. mean size of RLP and RSD profiles 3. surface density of RLP and RSD profiles per unit area. The unit of area considered was lo4 km2. All measurements were taken in electron photomicrographs (30,000 x ) of lead citrate-contrasted thin (approximately 700 A) sections from cylinders. Photomicrographs or each section were selected by the systematic process of Weibel (1979)) by aligning the image along the left corners of the support grid. Finally, the EM sections from each cylinder were chosen by weighting throughout the length of the aE sector, allowing sufficient distance between the sections to obtain a greater number of random measurements and to avoid the same structures being measured twice.

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RESULTS Qualitative Observations

In the present work we concentrated mainly on two aspects of the dLGN: first and most importantly, the characteristics of the neuropil elements constituting the afferent synaptic structures, and second the morphometrical and quantitative distribution of these elements throughout the aEsector of the dLGN from up to down. We examined retinal (RST) and cortical (CST) synaptic terminals. This terminology, introduced by Guillery (19691, was preferred because it describes the features specific to each terminal. The other abbreviations used in the text below are as follows: RSD, round vesicles, small profiles, and dark mitochondria corresponding to cortical synaptic terminals (CST); RLP, round vesicles, large profiles, and pale mitochondria, corresponding to retinal terminals (RST); F1, flattened or pleomorphic vesicles, dark cytoplasm, and numerous vesicles corresponding to interneuron axonal terminals; F2, flattened or pleomorphic vesicles (less numerous than in F l ) , of moderate electron density, corresponding to interneuron specialized dendritic appendages. Before analyzing the morphometric features of the RLP and RSD synaptic terminals, their structural characteristics will be briefly reviewed. The RLP terminals are relatively large (2-4 ym in diameter) and contain numerous round synaptic vesicles, among which are scattered a few larger vesicles (up to 100 nm in diameter) with a n electron-dense center. Inside the RLP terminals, several mitochondria (3 to 10 per profile) of low electron density are seen. The RLP terminals (Fig. 2) establish asymmetric synaptic contact with two types of process: (1)conventional dendrites (D), identifiable by the presence of ribosomes, and (2) processes containing pleomorphic synaptic vesicles lacking ribosomes, considered presynaptic dendrites of interneurons (F2) (Mize e t al., 1986). In addition to the synaptic contacts, interrupted desmosome-like contacts (Guillery et al., 19881, also called filamentous contacts (Colonnier and Guillery, 1964), are frequently seen between RLP terminals and dendrites (Fig. 3). These desmosome-like contacts, apparently without synaptic significance, were observed in our material as zones of up to 1 km in length in which the pre- and postsynaptic membranes were discontinuously thickened with a dense material. In general, these contacts were seen near dendritic bulbs (B), i.e., the site of synaptic contact. The filamentous contacts were also noteworthy in that one or more mitochondria in the dendritic profiles were aligned along the surface of contact. An amorphous dense material as well as a few cisternae of endoplasmic reticulum were also seen between the mitochondria and the filamentous contact per se. Another aspect of interest observed in our material was the relationship between the RLP terminals and the postsynaptic profiles (dendrites and F2). In our material, RLP terminals made contacts with single dendrites or with other complex elements in the glomerulae first described by Szentagothai (1963) in the cat. The morphology of the glomerulae in the rabbit is varied, ranging from highly complex zones (Fig. 4) involving many profiles, in which the predominant element was

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Fig. 2. Inferior zone of the rabbit dLGN (a,sector). Retinal terminal (RLP) with abundant rounded synaptic vesicles, including several dense-cored vesicles (unlabelled black arrowhead). Several mitochondria of low electron density (m) in the RLP terminal are clearly distinguishable from the dark mitochondria of the adjacent processes.

The RLP terminal establishes synaptic contact with a n F2 profile (unlabelled open arrowhead) and a dendrite (D) at the level of a dendritic bulb (B). There is also interrupted filamentous contact (fc) with the dendrite. Glial lamellae are seen in contact with the entire complex (asterisks). X 30,800.

usually the RLP terminal, to glomerulae or synaptic In some glomerular formations, normally surrounded zones involving a single RLP terminal, one dendrite by numerous glial lamellae, the dendritic elements (usually as the main element), and a single F2 profile. were not clearly distinguishable; the glomerular forFigures 2 and 3 show a n incomplete relationship be- mations appeared to consist mainly of RLP terminals tween the three elements of the glomerulus. Although, which were presynaptic with respect to several F2 teras pointed out by Famiglietti and Peters (1972) and minals (Fig. 6). This situation may arise in a n occaGuillery et al. (1988), all three element make contact in sional section taken from the top level of a glomerular these triadic units, images of such multiple contact were formation, from which the dendrite has already exited. rarely found i n a given section in our material. To detect Our material also showed a set of small profiles conthis event, it would be necessary to follow a n extensive taining spherical, densely packed synaptic vesicles series of EM sections throughout the specimen, or to which established intimate asymmetrical contacts almake three-dimensional reconstructions. Figure 2 most exclusively with dendrites (Figs. 3 and 7). Their shows the synaptic contacts between RLP and F2, and appearance matches that described by Guillery (1969) the contact between F2 and dentrite D; the contact be- in the cat, in which species the cortical origin of these tween F2 and D does not appear in this picture. This terminals has been demonstrated (Robson, 1983). In type of contact appears in Figure 3, where the relation- general, these terminals contained no mitochondria; in ship between F2 and D is established through special the few cases where they were seen, they showed a n dendritic bulbs or postsynaptic thorns (Famiglietti and electron-dense matrix (dark mitochondria). We therefore designate these profiles as RSD terminals, in Peters, 1972). The F1 terminals (axonal terminals of interneurons) agreement with Guillery (1969). occasionally established synaptic contacts in the vicinQuantitative Study ity of the triadic units, at the level of the dendrite. This As explained in Materials and Methods, our quantioften occurred when the triadic unit was close to the neuronal soma and in the absence of glial lamella in tative study consisted of calculating the fraction reprethe immediate vicinity of the F1 profile (Fig. 5). sented by the neuropil in the aE sector of the rabbit

AFFERENT SYNAPTIC TERMINALS IN THE RABBIT dLGN

Fig. 3. (a)Middle zone of the rabbit dLGN (a, sector) showing a synaptic zone composed of two RLP profiles, a dendritic profile (D) and a n F2 terminal in contact with glial lamellae and processes (stars). Note the central position of the dendrite and the presence of dendritic bulbs (B). Some profiles correspond to corticogeniculate terminals (RSD),which establish synaptic contact with a dendrite (D). x 18,200. (b) Detail of a illustrating some of the features of the triadic relation between the retinal terminal, the F2 terminal and the dendrite. The

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retinal terminal (RLP) makes synaptic contact with the dendrite (D) (unlabelled black arrow), as does the F2 terminal (unlabelled open arrow). Note the synaptic contacts upon dendritic bulbs, among which a filamentous contact can be seen (fc). The arrangement of the mitochondria (m) along the filamentous contact and the dense material between these two elements stand out. Thin glial lamellae (asterisks) are seen in intimate contact with the entire complex. x 39,500.

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Fig. 4. Middle zone of the rabbit dLGN (a, sector) showing a complex synaptic zone with a large retinal terminal (RLP) and other smaller terminals, together with several profiles of dendrites (D) and F2 terminals. A dendritic bulb (B) is intercalated in a characteristic filamentous contact (unlabelled black arrowhead). The thin glial

lamellae (asterisks) are seen, together with a glial process (As) containing bundles of glial filaments (f). A profile of a characteristic corticogeniculate terminal (RSD) is seen in the lower right corner. X 18,200.

dLGN, and determining the number and mean and total area in pm2 of the profiles corresponding to RLP and RSD terminals. Table 1 gives the results for V, (total volume of the a,-dLGN zones studied), V, (volume occupied by the neuronal perikarya), and Vf (volume of the fiber fascicles). These values, the neuropil value derived from them, and the areas of the synaptic terminal profiles are used to determine and compare the features of each of the three zones. We calculated and used only mean profiles areas of the presynaptic bulbs, not the volume per unit of element, because the former satisfied our aim of comparing the situation in the three zones. The neuropil volume is easily obtained from V,, V,, and Vf with the formula: neuropil volume = V, - (V, + Vf). The percentage volumes occupied by the neuropil in each zone, together with the P values, are also shown in Table 1. The percentages found in each of the three zones are very similar, hence the differences between them were not significant, especially with respect to the middle and inferior zones. Columns 1, 2, and 3 in Table 1 present mean values of the volumes of the zones analyzed (V,), the volumes occupied by the neu-

ronal soma (V,), and the volumes of the myelin fiber fascicles (Vf). Table 2 contains mean values of the areas (a)of RLP and RSD profiles in each zone, together with total areas (A) occupied by each type of terminal per lo4 pm2. Mean area (a)of the RLP profiles was significantly greater in the middle zone than in the rest of the sector, while mean areas of the RSD profiles differed significantly in all three zones. With respect to the total area (A) occupied by the profiles in each zone per unit area (lo4 pm2) those belonging to the RSD type did not significantly (Table 2), whereas RLP terminals occupied a larger proportion of the nucleus in superior zones than in inferior zones. In other words, the area of neuropil occupied by RSD terminals remained more or less constant throughout the aEsector while the area of RLP profiles decreased gradually as more inferior levels were approached. Finally, Table 3 shows the number and percentage of RLP and RSD profiles in each zone. Statistically significant differences were found between the superior zone and the other two zones for the number of both types of terminal. The percentage values reflect a degree of similarity between the middle and inferior zones, where the proportion of RLP to RSD profiles was

AFFERENT SYNAPTIC TERMINALS IN THE RABBIT dLGN

Fig. 5. (a) Overall view of the superior zone of the rabbit dLGN (a, sector). A glomerular formation is seen in the neuropil near neuronal somata and a capillary. x 8600. (b) Enlarged detail of a illustrating the glomerular formation and a central dendrite (D), a retinal terminal (RLP) which makes synaptic contact a t two different points (unlabelled open arrow) with the dendrite, and several F profiles which

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also establish synaptic contact with the dendrite (unlabelled black arrowhead). Note the axonal terminal of the interneuron (Fl), characterized by dense mitochondria and pleomorphic synaptic vesicles, which are more densely packed than in F2 profiles. Thin glial lamellae (asterisks) partially surround the glomerulus, but not the F1 terminal. x 32,400.

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Fig. 6. Inferior zone of the rabbit dLGN (a, sector) showing a glomerular formation surrounded by many glial lamellae (asterisks). No central dendrite, a characteristic of other glomerulae, is seen. One process contains numerous rounded vesicles possibly of retinal na-

ture, lacking in membrane condensations (unlabelled black arrow). RLP, retinal terminal; F2, dendritic terminal of a n interneuron; As, glial process; f, glial filaments. x 38,400.

approximately 1:l. In the superior zone, the resulting proportion was nearly 2:l for both types of terminals.

fascicles in each zone. These values are necessary to obtain quantitative information about the neuropil. The volume occupied by the large myelin fiber fascicles was not taken into account in order to avoid introducing bias in the possible value of the grey cell coefficient or in the relationship between the griseum and neuropil, which was calculated as V, - (V, Vf). The relevance of obtaining and using these values lies in that this provides us with information about the relationships between the griseum and the neuropil, and the possible consequences in elucidating the function and distribution of the elements constituting the structure under study. Such values have been used in the stereological literature as parameters, but, a s Haug (1986) has pointed out, “it is too early to give a historical survey.” We can, however, use those dimensions which “estimate the volume density of the neuropil by light-microscope . . . (and) details of the finer structures can only be investigated with electron-microscopical morphometry.” No attempt was made to distinguish between the volume occupied by the capillary network and that occupied by the neuropil, because the regular distribution of the capillary network throughout the tissue was assumed to obviate any significant inf luence on the values obtained in each of the three zones.

COMMENT AND DISCUSSION On the Parameters Analyzed

In a n attempt to broaden our understanding of the composition and function of the dorsal-lateral geniculate nucleus in the rabbit, we aimed to analyze the spatial relationships of the retinal (RLP) and cortical (RSD)terminals a s afferent contacts impinging on the nucleus. In the main, we directed our attention to the features of the presynaptic boutons rather than the contacts themselves (pre- and postsynaptic thickenings). Mean areas ( a and A) were used as relative estimators, since we basically wanted to compare the zonal distribution of the afferent projections and the space occupied by RLP and RSD terminals. We did not analyze the number or size of the synaptic thickenings, since the available methodology is insufficiently accurate for this purpose, and the problems associated with these measurements make such a n analysis impracticable (see De Groot and Bierman, 1986). We calculated V, as the reference volume (i.e., the zone analyzed), V, a s the volume occupied by neurons in a given zone, and Vf as the volume occupied by fiber

+

AFFERENT SYNAPTIC TERMINALS IN THE RABBIT dLGN

Fig. 7. (a) Superior zone of the rabbit dLGN (a, sector) showing several profiles of retinal terminals (RLP) of different size, and profiles of corticogeniculate terminals (asterisks). x 13,650. (b)Enlarged detail of a. Two corticogeniculate terminals (RSD) establish asym-

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metric synaptic contact (unlabelled open arrowheads) with a dendrite (D). A retinal terminal (RLP) is related with a dendrite which presents two small dendritic bulbs (B). The contacts are of a filamentous type. x 33,000.

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TABLE 1. Mean volumes (expressed in mm3) of the zones studied in the aE sector of the rabbit dLGN (V ), and mean volumes expressed as percentage V, for the space occupied by celldar soma (V,) and the s p y e occupied by fiber fascicles (Vf) (n = 12 dLGN) cip Zone

Superior Middle

V, t SE 1.20 t0.03 2.15

Vo t SE 0.106 t0.005 0.192 20.007 0.055 20.004

k0.05

Inferior

0.84 20.02

V, t SE 0.085 k0.005

0.169 k0.006 0.089 k0.005

Neuropil Volume 1.02 '0.02 1.79 20.03 0.72 20.02

%

84.1' t0.5 83.2' t0.3 83.0' k0.4

'The fourth column shows mean volumes occupied by the neuropil in absolute values and percentages (with respect to VJ, including that occupied by the capillary network but excluding the volume occupied by the myelinized fascicles. 'Student's t test failed to reveal significant differences between neuropil volume in any two zones (P