The Lateral Reticular Nucleus of the Opossum (Didelphis virginiana) I. CONFORMATION, CYTOLOGY AND SYNAPTOLOGY JOSEPH A. ANDREZIK AND JAMES S. KING Department of Anatomy, The Ohio State Uniuersity, College of Medicine, 1645 Neil Avenue, Columbus, Ohio 43210
When viewed in Nissl preparations, the lateral reticular nucleus (LRN) of the opossum can be divided into three subgroups: a medial internal portion, a lateral external portion and a rostral trigeminal division. Neurons within the internal division measure 13-45p in their greatest dimension whereas those within the external and trigeminal portions measure 11-32 p and 14-27 p respectively. Golgi impregnations reveal that many neurons in all three subdivisions display a radial dendritic pattern although some of the nerve cells within the external division have dendrites which orient mainly in a ventromedial to dorsolateral direction. The cell bodies of LRN neurons are relatively spine-free. However, a small percentage of neurons exhibit clusters of sessile spines on proximal and more distal dendritic segments. No locally ramifying axons or axon collaterals were found within the LRN. Synaptic terminals within the LRN were divided into four categories: (1) small terminals measuring 2.5 p or less containing agranular spherical vesicles; (2) small terminals (2.5 p or less) with agranular pleomorphic synaptic vesicles, i.e., a mixture of spherical and elliptical synaptic vesicles; (3)small terminals (2.5 p or less) containing agranular spherical or pleomorphic vesicles with a variable number (4-27) of dense core vesicles; and (4) large terminals (greater than 2.5 p ) which contain agranular spherical synaptic vesicles and a variable number of dense core vesicles (1-17). Dendritic diameters were measured from Golgi impregnations and correlated with crosssectioned profiles in electron micrographs to help determine the post-synaptic distribution of synaptic endings. Small terminals containing agranular spherical or pleomorphic synaptic vesicles contact the soma and entire dendritic tree in each portion of the nucleus, whereas the small terminals containing dense core vesicles are usually located on distal dendrites or spines. Some large terminals make multiple synaptic contacts with a cluster of spines, others contact groups of small (distal) dendrites. In order to identify two of the major afferent systems to the LRN, 15 adult opossums were subjected to either a cervical spinal cord hemisection or a stereotaxic lesion of the red nucleus. Two days subsequent to spinal hemisection, large terminals in the caudal part of the ipsilateral LRN exhibit either a n electron dense or filamentous reaction. Their postsynaptic loci are spines and shafts of proximal dendrites or a number of distal dendrites and spines. In addition, small terminals containing spherical agranular synaptic vesicles undergo a n electron dense reaction in the same areas. Their postsynaptic loci are proximal or distal dendrites. Two days subsequent to rubral lesions, small terminals containing agranular spherical synaptic vesicles undergo a dark reaction in rostral portions of the contralateral nucleus. They contact intermediate or distal dendrites and occasionally spines.
' This work was submitted by Joseph A. Andrezik in partial fulfillment of requirementsfor the degreeDoctorof Philosophy a t TheOhiostate U n i ~ versity. A portion of this study was presented a t the eighty-ninth session of t h e American Association of Anatomists in Louisville, April. 1976. Present address: Department of Anatomy, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts, 02115 (sendreprint requests to this address) "his investigation was supported in part by United States Public Health Service Grant NS-08798 to Dr. James S. King. J . COMP. NEUR.. 174: 119-150.
JOSEPH A. ANDREZIK AND JAMES S. KING
The lateral reticular nucleus, (LRN), situated in the ventrolateral portion of the caudal medulla, was first described by Clarke (1858) and subsequently by Dean (1864) who called it the anterolateral nucleus. Later Ramon y Cajal ('09) using Nissl preparations of adult rabbit, subdivided the LRN into three portions, a medial nuyau interne, a lateral noyau externe, and a dorsally directed column of neurons termed the foyer lineaire. For further details related to the early history and nomenclature of the LRN the reader is referred to Walberg's ('52) study. The cytological features of LRN neurons as revealed by Golgi impregnations have been described by a number of authors (Mannen, '60, '66; Valverde, '61; Ramon-Moliner, '62; Leontovich and Zhukova, '63; Ramon-Moliner and Nauta, '66). Recently, accounts of LRN ultrastructure in the cat have been reported by Mizuno and Nakamura ('73) and Mizuno et al. ('73, '75). The intent of the present study is to correlate the ultrastructure of the opossum LRN with details seen in Golgi and Nissl preparations. This report and previous studies (Bowman and King, '73; Mihailoff and King, '75) are part of a continuing effort to detail the cytology and synaptic organization of certain precerebellar nuclei. These results also provide, in part the structural substrate for the accumulating electrophysical data of afferents to the LRN (Bruckmoser et al., '70a,b; Burton et al., '71; Clendenin et al., '74b,c, '75; Ekerot and Oscarsson, '75, '76; Kitai et al., '74b; Rosen and Scheid, '72, '73a,b,c; Zangger and Wiesendanger, '73) which point out its importance as a precerebellar nucleus (Azzena and Ohno, '73; Bloedel, '73; Clendenin et al., '74a; Kitai et al., '74a). MATERIALS AND METHODS
Animals utilized for Nissl preparations were anesthetized with sodium pentobarbital (36 mg/kg) and subsequently sacrificed via intracardiac perfusion of 10% buffered formalin. The brains were immediately removed from the skull and remained in fixative for a t least five days. The brains were then cut into blocks 8-10 mm in length and placed into a n aqueous solution of 30% sucrose overnight. The following day, sections 40-p thick were cut on a freezing microtome in the transverse, horizontal or sagittal plane. The sections were serially mounted on slides and stained for Nissl substance using the method of either
Windle ('43) or Fernstrom ('58). The conformation of the LRN and the cytology of individual neurons were studied in each plane. Paraffin sections cut in the transverse plane were also used to study and photograph individual neurons. Neuronal dimensions were measured in both Nissl stained frozen sections and 1-p plastic sections stained with toluidine blue. Additional brains were impregnated using either the Kopsch (Colonnier, '64) or del Rio Hortega modification (Stensaas and Stensaas, '68) of the Golgi technique. Sections were cut between 100 and 300 p in either the transverse or horizontal plane and impregnated neurons were drawn using a Leitz projection microscope. For electron microscopic study, the brains of 23 adult opossums were fixed according to the procedure described by Mihailoff and King ('75). Subsequent to embedding, 1p semi-thin sections were cut in the transverse plane from each block and stained with toluidine blue. The location of the nucleus was determined in each block and, as the tissue block was trimmed prior to thin sectioning, orientation was maintained. Thin sections were cut on either a Sorvall Porter-Blum MT-2 or a Reichert Om-U2 ultramicrotome, picked up on copper grids, stained with lead citrate Wenable and Coggeshall, '65) and observed with a Philips EM 300 electron microscope. Using criteria of size, synaptic vesicle shape and number of dense core vesicles, synaptic terminals were classified as: (1) small spherical, (2) small pleomorphic (mixture of spherical and flattened vesicles), (3) small dense core or (4) large spherical. Vesicle size was determined by using the electronic measuring system described by King ('76). The synaptic vesicles measured were randomly selected and 30 vesicles in each terminal were traced. In terminals containing six or more dense core vesicles (dcv) each dcv was measured in addition to 30 agranular synaptic vesicles. Electron microscopic study of degenerating axon terminals involved 15 adult opossums which were subjected to either a spinal hemisection a t the second cervical level or a stereotaxic lesion of the red nucleus. Spinal hemisections were accomplished with a scalpel, whereas stereotaxic lesions of the red nucleus were performed by thermocoagulation using a Grass LM-3 lesion maker. The coordinates used in positioning the electrode
LRN: CONFORMATION, CYTOLOGY AND SYNAPTOLOGY
were our own, complemented by those of Oswaldo-Cruz and Rocha-Miranda (‘68). The LRN targets of spinal and rubral fibers were first determined by light microscopic methods (Martin et al., ’77) so that tissue samples for electron microscopy could be taken from areas showing maximal distribution of the fiber systems in question. Survival times for spinal lesions ranged from 28 hours to 4 days (11 cases), whereas animals with rubral lesions survived from 48 to 72 hours (4 cases). OBSERVATIONS
Conformation of the lateral reticular nucleus The LRN is situated in the ventrolateral quadrant of the opossum medulla and can be separated into a large internal division, a smaller external division (fig. 1) and a rostral trigeminal division (fig. 2). The rostral to caudal extent of the nucleus is 4.1 mm. Its caudal pole is situated approximately 0.5 mm caudal to the inferior olivary nucleus (fig. 3) and it extends to about the level of the rostral tip of the same nucleus (fig. 8). The caudal portion of the LRN is populated by neurons which are compactly arranged (fig. 3: small arrow) and variable in both size (15-27 p in their largest dimension) and staining quality. Slightly rostral to its caudal pole, the LRN contains a group of larger and more darkly staining neurons which appear medial and ventral to the compact cluster (fig. 3: solid block arrow) and are included as a part of the internal division. In progressively more rostral sections the internal division enlarges and the external division first appears a t the ventrolateral margin of the brainstem (fig. 4: open block arrow). At the level shown in figure 5, the internal division is expanded dorsomedially (fig. 5: solid block arrow), but the compact area is smaller (small arrows). Neurons continuous with the medial part of the internal division are now interposed between the external division and the remainder of the compact group forming longitudinal strands oriented in a ventromedial to dorsolateral direction (fig. 5: open triangles). At the same level the external division has increased in size forming a prominent bulge on the ventrolateral surface of the brainstem (fig. 1: EXT.; fig. 5: open block arrow). This division consists of closely packed neurons which are lighter staining than most of those comprising the internal division. In serial sections through the region be-
tween figures 5 and 6, cell bridges two or three neurons wide extend laterally from the dorsal part of the internal division and form a prominent cell group a t the rostral dorsolateral pole of the external division (figs. 1, 6, 7: large arrowhead). Because of its position, we consider the latter cell group as part of the external division. At more rostral levels (fig. 6) the compact portion of the internal division is absent and the remaining part is segregated into three columns of neurons (fig. 6 : solid block arrows). The dorsolateral part of the external division is diminished a t this level (fig. 6: large arrowhead) and from it, strands of neurons extend in a dorsomedial direction (fig. 6: open arrows). These dorsomedially directed strands are the beginning of the trigeminal division which is evident only in the rostral one-fourth of the nucleus (figs. 7 and 8: open and curved arrows). The neurons composing the trigeminal division are intimately associated with the descending trigeminal tract and nucleus. Some of these neurons form small islets ventral to the descending trigeminal tract (fig. 9: small solid block arrow). The majority of them, however, are dispersed within the descending tract and they usually traverse its entire expanse (fig. 9: large solid block arrow). The most dorsal and rostrally situated neurons of the trigeminal division occupy a position on the ventromedial margin of the descending trigeminal nucleus (figs. 7, 8, 9: curved arrow). Some neurons of this latter group are actually incorporated within the ventromedial portion of the descending trigeminal nucleus. Examination of the nuclear subdivisions in 1 p toluidine blue stained sections (figs. 10, 11) reveals that the internal division is composed of strands of neuropil, punctuated by neuronal and glial somata, which course between fascicles of myelinated fibers (fig. 10: INT.). As stated by Brodal (‘43) this gives the nucleus a reticular appearance and hence, its name. The external division, however, is somewhat different and it appears as a compact mass of somata and neuropil with relatively few intervening myelinated fibers (fig. 10: EXT.). At more rostral levels (fig. 11) the trigeminal division consists of isolated clusters of neurons with associated neuropil. Neurons found within these three divisions are shown a t a higher magnification in figures 12 to 14.
JOSEPH A. ANDREZIK AND JAMES S. KING
Cytology A typical neuron found within the internal division is indicated by the solid white arrow in figure 10 and is enlarged in figure 12. Neurons of this division range in size from 13-45p in their greatest dimension and their soma may be oval, multipolar or pear shaped. In Nissl preparations internal division neurons are usually darkly stained and possess prominent Nissl granules which may extend for some distance into the proximal dendritic tree (fig. 15). Nerve cells which comprise the compact portion of the internal division are usually smaller and lighter staining than those making up the remainder of the internal division. Neurons of the external division are generally smaller than those of the internal division and measure 11-32 p in their greatest dimension. Two examples of nerve cells from this division are indicated by the solid and open block arrows in figure 10 and are enlarged in figure 13. In Nissl preparations neurons populating the ventral and caudal portions of this division are generally lighter and more evenly stained than those found throughout the remainder of the nucleus (compare fig. 16 with figs. 15 and 17). Nerve cell bodies within the caudal and ventral portions of the external division are usually elongate, fusiform or spindle shaped with the long axis oriented in a ventromedial to dorsolateral direction following the orientation of the external arcuate fibers (Martinet al., '77: fig. 1). Neurons of the trigeminal division are more uniform in both size and shape. Most of them measure 14-27 p in their greatest dimension, but larger ones (up to 34 p ) have been observed. The shape of the soma is usually round or triangular. Two such neurons are indicated by the solid block arrows in figure 11 and are enlarged in figure 14. Nissl preparations reveal distinct aggregates of Nissl substance within the perikaryon giving neurons of this division a prominent and darkly stained appearance (figs. 9, 17).
Golgi preparations Many neurons within the trigeminal division exhibit a radial dendritic pattern (fig. 18). At their origin, dendrites usually measure 3.0-5.0 p in diameter, and generally branch two or three times. Daughter branches typically are longer than the parent stems.
The dendrites rapidly taper and except for occasional bulges appear uniform throughout the intermediate range measuring 1.5-2.5 p. The distal one-third of the dendritic tree gradually tapers to dimensions of 0.5 p or less. The somata and dendrites of these neurons are generally spine free (fig. 181, although some nerve cells show a rare spine on their soma and clusters of spines on proximal dendrites. Dendrites of neurons forming the bulk of the external division exhibit a more restricted geometry (fig. 19) and are usually oriented in a ventromedial to dorsolateral direction. Occasional branches are directed perpendicular to the main dendritic polarity (fig. 19) and some extend into the internal division. Spines are seen only rarely on the dendritic surface and have not yet been observed on cell bodies. Dendrites measure 2.0-5.0p in the most proximal portions and gradually taper to 0.5 p or less. Neurons composing the more rostral and dorsolateral part of the external division display a more radiate and extensive arborization. The proximal dendrites of some of these neurons are laden with clusters of spines. The dendrites of many internal division neurons branch in a radial fashion as they course between myelinated fascicles (fig. 20). In well impregnated Golgi sections, numerous dendrites are seen to converge and follow a similar course around myelinated bundles (Martin et al., '77: fig. 1). Some neurons display dendritic excrescences which form clawlike structures on the proximal and intermediate protions of the dendritic arbor, but spines are only occasionally observed. Another neuron characteristic of the internal division is shown in figure 23. The neuron differs from the former in that its dendrites have a more restricted arborization and its proximal dendritic tree displays clusters of sessile spines (Westrum, '70) measuring 1.0 p in length (fig. 24: open block arrow). Somatic spines are occasionally seen and measure up to 2.0 p in length. The remainder of the dendritic tree also exhibits spines, but they are not clustered as seen on more proximal portions. The majority of these spines measure 1.0 p in length, but some as long as 2.5 p have been observed. This type of neuron is similar to some of those residing in the rostral dorsolateral part of the external division and to some neurons of the trigeminal division. The dendritic spread of neurons in the three subdivisions is directed mainly in the trans-
LRN: CONFORMATION, CYTOLOGY AND SYNAPTOLOGY
verse plane with dendrites extending as much a s 660 p from t h e soma. The greatest rostrocaudal extent observed in the horizontal plane is 320 p from the neuronal soma. Axons were generally not impregnated beyond the initial segment, but in a few cases (7) the myelinated portions were followed through the nucleus for distances up to 310 p. In no instance were collaterals observed. All impregnated axons coursed in a ventrolateral direction through the nucleus to collect as external arcuate fascicles. Only portions of afferent axons have been impregnated in our Golgi material and two classes are apparent. The “beads” of one axon type measure between 1.5 and 2.0 p in their greatest dimension and are separated by distances of 1-5p . Enlargements measuring 3.03.5 p are found on a second type of axon. These varicosities are separated by distances of 2.5 -7.0 p along the axon.
the surface. Somatic spines or protrusions are sometimes seen (fig. 26: asterisk) and when present, are approximated to a synaptic terminal.
Using criteria of terminal size, synaptic vesicle shape and number of dense core vesicles, four categories of synaptic terminals can be distinguished. Synaptic endings designated as small, measure 2.5 p or less in their greatest dimension and contain either: (1)agranular spherical synaptic vesicles (figs. 26, 27); (2) agranular pleomorphic synaptic vesicles, i.e., a mixture of spherical and elliptical synaptic vesicles (figs. 30-32); or (3) agranular spherical or pleomorphic synaptic vesicles plus a distinct population of dense core vesicles (fig. 28). Large terminals measure 2.5-5.0 p in their greatest dimension and always contain spherical agranular synaptic vesicles (figs. 25,291. A variable number of dense core Fine structure vesicles also may be present in these endings Neuronal somata (fig. 29: arrows). A few terminals measuring With the exception of size, the neuronal up to 9 p in length are found contacting the perikarya from all portions of the nucleus soma and proximal dendrites when cut in lonappear similar in electron micrographs. The gitudinal section. These synaptic profiles were nucleus conforms to the shape of the cell, infrequently observed, measured less than 2.5 being either round or elongate and i t is most p in width and lacked obvious cytological feafrequently found in a slightly eccentric posi- tures distinguishing them from the majority tion. The nuclear outline is smooth in appear- of smaller terminals. Thus, some of the small ance with only occasional indentations (fig. synaptic terminals may simply represent 21: open block arrows). A prominent nucleo- cross sections of these more elongate endings. lus is present (n), as are isolated clusters of Endings containing spherical synaptic vesiheterochromatin (h) (fig. 21). The Golgi appa- cles usually terminate in a Gray’s type 1 ratus with its associated vesicles (g) is dis- (Gray, ’59) or asymmetric (Colonnier, ’68) tributed throughout the cytoplasm in a cir- junction (figs. 26, 27). Terminals containing cumnuclear pattern. Distinct Nissl bodies pleomorphic vesicles end with a junctional (nb) are evident in most neurons although morphology intermediate (Akert et al., ’72) smaller cells (18 p or less) display isolated between Gray’s type 1and type 2 (figs. 30-32). strands of rough endoplasmic reticulum and In order to determine the size of synaptic only occasional groupings of Nissl substance. vesicles, the area of 30 synaptic vesicles was The remaining cytoplasm of all neurons is measured in 59 small terminals containing populated by numerous rosettes of free poly- spherical vesicles and in 56 terminals conribosomes (p) and mitochondria. Lipofuscin taining pleomorphic vesicles. A mean vesicle granules (1) are usually present and are quite area was calculated for each terminal. The prominent in neurons whose dimensions fall sample was selected from all portions of the within the upper range of the size spectrum. nucleus and was distributed among terminals Few synapses occur on the soma of LRN contacting either the soma or isolated parts of neurons (fig. 21) and when found, most often the dendritic tree. The mean vesicle areas for occur on the larger neurons of the internal terminals containing spherical vesicles range and trigeminal divisions. The somatic surface from 616 nm to 1,757 nm with the mean of is typically encapsulated by thin glial lamel- the 59 terminals being 1,046 nm2. These lae. Occasionally, myelinated axons (fig. 2 1) measurements when converted to diameters or the soma of a perineuronal glial cell contact and expressed in Angstroms range from 280 A
JOSEPH A. ANDREZIK AND JAMES S. KING
to 473 A and the mean size is 365 A. The range of mean vesicle areas for synaptic endings containing pleomorphic vesicles is 665 nm to 1,787 nm and the mean of the 56 terminals is 1,014 nm 2. If this area were considered to be that of a circle, the diameters ex ressed in Angstroms would range from 290 to 476 A with a mean of 359 A. In terminals of large size the mean of 30 synaptic vesicles ranges from 711 n m 2 to 1,084 nm2. When converted to Angstroms their ran e is 301 A to 420 A with a mean size of 358 In synaptic endings containing dense core vesicles, variability in the quality of dense cores is readily apparent (figs. 28,291. One type of vesicle contains a very dark core and is rather irregular in shape (fig. 28) whereas the other type is usually round and contains a much lighter core (fig. 29: arrows). Small or large terminals may contain both types of dense core vesicles but individual endings clearly contain a majority of one or the other type. The size of the dark irregular dense core vesicles ranges from 479A to 1,308 A with a mean of 982 8.The lighter more regular dense core vesicles range in size from 455 A to 1,086 A with a mean of 723 A. Moreover, dense core vesicles in terminals found in the internal division are smaller than in the other two divisions. The mean of the darker vesicles is 833 A, whereas the lighter cored vesicles measure 640 A.
Distribution of synaptic profiles Throughout the nucleus, small terminals containing spherical synaptic vesicles (figs. 26, 27) contact the soma and all parts of the dendritic tree. A t present, correlations between the postsynaptic localization of axodendritic terminals and vesicle size is not possible. However, terminals with a mean vesicle area greater than 1,250 nm (diameter of 399 A) do not contact the soma. Endings containing pleomorphic vesicles also are observed in all divisions of the nucleus. They contact the soma and all parts of the dendritic tree (figs. 30-321,but in terms of mean vesicle size, there appears to be no segregation relative to postsynaptic locus. Figure 22 is a drawing of the ventrolateral quadrant of a Golgi impregnated section from the caudal brainstem of the opossum and indicates the position of a neuron shown a t a higher magnification in figures 23 and 24. This type of neuron is characterized by clusters of sessile spines on the proximal dendritic tree,
particularly near points where the dendrities bifurcate into secondary branches. Large terminals make multiple contacts with clusters of spines (fig. 25: asterisks) and sometimes with spines and shafts of dendrites measuring 1 p or less (fig. 29). Since the three profiles adjacent to the terminal shown in figure 25 are comparable in size to spines seen in Golgi impregnations, and since clusters of these spines are apparent only on the proximal dendrites, i t is concluded that a t least some of the large terminals synapse a t these sites. Small terminals containing dark irregular dense core vesicles only occasionally display synaptic contacts (fig. 28). When synaptic contacts are present, they usually occur on dendritic spines, although contacts with the soma and all parts of the dendritic tree have been observed. Small terminals containing dense core vesicles of the smaller and less dense variety display active synaptic sites more fequently than the former. Likewise, their postsynaptic loci are usually distal dendrites or spines.
Experimental results Results from experimental material reveal that different populations of synaptic endings degenerate following spinal cord hemisection or stereotaxic lesion of the red nucleus. Terminals of spinal origin end in the caudal portion of the ipsilateral LRN (Martin et al., '77). Small, electron dense terminals (1.3-2.3 p ) containing spherical synaptic vesicles are observed as early as 28 hours subsequent to a spinal cord hemisection. Degenerating terminals also are apparent a t a survival time of 46 hours, although by that time some of them have become crenated and engulfed in glial cytoplasm. Small degenerating profiles were observed contacting the shafts of proximal or distal dendrites (fig. 34). In addition, larger terminals (2.5-4.0p ) exhibit either an electron dense or filamentous reaction to axotomy (figs. 33,35) and some of them contain a number of dense core vesicles. These profiles contact spines andlor the shafts of proximal dendrites (fig. 331, or clusters of spines. The large filamentous terminal shown in figure 35 measures 3.0 /*, contains no dense core vesicles and contacts small dendrites via Gray's type 1 junctions. At a survival times of four days all of the degenerating axon terminals observed were dark, crenated and surrounded by glial cytoplasm. Also, numerous electron dense, small
LRN: CONFORMATION, CYTOLOGY AND SYNAPTOLOGY
diameter myelinated axons peppered the neuropil. Two and three days subsequent to stereotaxic lesions of the red nucleus small terminals (1.0-1.6 p ) which contain spherical synaptic vesicles exhibit an electron dense reaction (fig. 36). These terminals contact spines andfor the shafts of intermediate and distal dendrites of neurons in the rostral portion of the contralateral LRN. DISCUSSION
Conformation of the lateral reticular nucleus The opossum LRN was first divided into two nuclei which contribute to the external arcuate bundles and project to the cerebellum (Voris and Hoerr, '32). The divisions we describe in the present account as internal, external and trigeminal correspond to the noyau interne, noyau externe and the foyer lineaire of Cajal ('09) and to Berman's ('68) internal, external and infratrigeminal nuclei. Brodal ('43) described the three portions of the LRN in newborn rabbits and kittens and named them magnocellular, parvicellular and subtrigeminal. In the opossum, however, the range of neuronal size is great within each subdivision and overlaps between the three nuclear divisions. Thus, nomenclature based on location within the brainstem rather than cell body size was utilized. Traditionally, the most rostral portion of the nucleus has been described as lying beneath the descending trigeminal tract (Verhaart, '57; '70; Taber, '61; Petrovicky, '66 and Berman, '68). However, as Brodal points out, in the rabbit scattered cells often connect this subgroup with the nucleus of the descending trigeminal tract (Brodal, '43: p. 178). Gerhard and Olszewski ('69) also found this to be the case in the primates Perodicticus potto and Macaca mulatta. It seems that of all the mammals studied thus far, this tendency of neurons to course through the trigeminal complex reaches its zenith in the opossum (fig. 9). Since, in the opossum, some neurons are located ventral and medial to the descending trigeminal tract, and since some neurons are actually within the descending trigeminal tract and nucleus, this entire complex is referred to simply as the trigeminal division. Verhaart ('57, '70) described the parcellation of the LRN with reference to the passing fiber systems of the lateral funiculus. In the present study, the neurons comprising the dorsolateral portion of the external division
(figs. 1, 6, 7: large arrowhead) may be comparable to those described by Verhaart as lying within the dorsal spinocerebellar tract, whereas those of the trigeminal portion are probably analogous to cells within the rubrospinal tract. In the present account, each division of the LRN has been further divided into subgroups. This parcellation of the major divisions of the LRN is based on distinguishing cytoarchitectonic characteristics and the analysis of the terminal zones of a number of afferent fiber systems (Martin et al., '77).
Golgi impregnations The dendrites of LRN neurons are relatively short and unbranched. The dendritic arborization is directed mainly in the transverse plane similar to that described for neurons of the reticular core (Scheibel and Scheibel, '58). For the most part, dendrites follow a course which is dictated by the arrangement of fibers within the immediate area. This is especially true of neurons in the caudal three-fourths of the nucleus. The dendrites of many neurons within the internal division are sequestered into bundles which course between fascicles of myelinated axons (Martin et al., '77: fig. 1).This arrangement is quite striking and its ultrastructural correlate can be seen in figure 29 of the present account. Such bundling of dendrites is similar to t h a t described by Valverde ('61), but a n arrangement of neurons into discrete packages or glomeruli is not evident in our material. Most neurons found in the external division and those along the interface between this portion and the internal division display dendrites that are primarily oriented in a ventromedial to dorsolateral direction, an arrangement paralleling the external arcuate fibers. However, some dendrites course a t right angles to the preferred polarity and may traverse the entire expanse of the external division (fig. 19, inset). Moreover, the dendritic arborization of the individual neurons does not honor the boundaries of degeneration which result from lesions of the spinal cord a t different levels (Brodal, '49; Kiinzle, '73; Martin et al., '77). Since the dendrites of a single neuron can occupy more than one terminal zone, axons arising from various cord segments may converge on the dendritic tree of one neuron. This organization correlates well with the electrophysiological data which also suggests convergence of impulses within
JOSEPH A. ANDREZIK AND JAMES S. KING
the LRN originating a t different cord levels (Rosen and Scheid, ’73c; Clendenin et al., ’74b). Although similar, the dendritic expanse of neurons within the opossum LRN is not as elaborate as that reported in carnivora (Leontovich and Zhukova, ’63).Because of the relatively simple arborization and directional arrangement of dendrites, neurons of the opossum LRN may be categorized as allodendritic according to the classification of the RamonMoliner and Nauta (‘66). The dendrites of neurons in the internal and external divisions rarely extend beyond the nuclear border. However, due to the variability in conformation and the ill-defined borders of the trigeminal division, dendrites of neurons in this portion are not confined within the specific boundries. Hence, the results of this study reaffirm the observations of Mannen (‘60, ’66) that the caudal portion of the LRN is “closed” (his nqyuu ferrne),but a t more rostra1 levels, it is “open” &is noyuu ouuert). In our material we have not found convincing morphological evidence for an interneuron. Axons which have been impregnated beyond the initial segment have not shown collaterals. This is in keeping with the original observations of Cajal (‘09)who concluded that all neurons in the LRN project out of the nucleus. In all species studied thus far, neither axon collaterals from projection neurons nor interneurons have been identified in the LRN. In addition, there is evidence that a t least some neurons throughout the entire size range possess an axon which projects to the cerebellum. After cerebellar cortical lesions in the opossum, LRN neurons throughout the entire size range undergo chromatolysis (unpublished observations). In cases where horseradish peroxidase (HRP) was injected into the anterior lobe, paramedian lobule, pyramis, lobulus simplex or ansiform lobules of the cerebellar cortex, HRP labelled neurons ranging in size from 13-35p have been observed in all three divisions of the nucleus (Martin et al., ’77). These results can not be taken as compelling proof for the absence of intranuclear connections since both the Golgi material and the HRP technique provide only negative evidence. In other areas of the nervous system, a t least some small neurons are known to possess axons that project beyond their nuclear borders. Upon injection of HRP into the inferior olivary nucleus, small cells measuring 11-15
p are labelled in the lateral cerebellar nucleus
(Martin et al., ’76).With injections of HRP into the cerebellar cortex, labelled cells as small as 13 p are found in the basilar pontine gray (G. A. Mihailoff, personal communication). This, of course, does not exclude the possibility that the axons of small neurons collateralize within the nucleus as well as project to more distant areas. In fact, such a case exists within the lateral cerebellar nucleus, where virtually all neurons have axons which collateralize within the nucleus (Chan-Palay, ’73). Hence, it has become increasingly more difficult to distinguish between interneurons and projection neurons based upon the criterion of cell size.
Fine structure One striking feature of the LRN is the paucity of synaptic terminals which contact the cell body of resident neurons. This same observation has been reported in the cat (Mizuno et al., ’75), and as pointed out by those authors, i t is a common feature of all the brainstem precerebellar nuclei studied to date (see Bowman and King, ’73 and Mihailoff and King, ’75). In the opossum, the maximum number of axosomatic terminals seen in random electron micrographs is fewer than reported in the cat (6 vs. 20) (Mizuno et al., ’75). Somatic contacts are most numerous in the internal and trigeminal divisions of the opossum. Mizuno and Nakamura (‘73) reported that axosomatic terminals were least frequent on neurons of their parvicellular division, an area which is analogous to our external portion. Thus, it appears that the relative occurrence and distribution of axosomatic terminals is similar within the nuclear subdivisions of these two species. As seen in figures 25, 33 and 35, the postsynaptic loci of large terminals are usually clusters of spines, shafts of proximal dendrites or shafts of small diameter dendrites. Golgi impregnations reveal that the clusters of spines are frequently located on proximal dendrites. It has been suggested that these small spines may function as current injection devices (Llinas and Hillman, ’69).Since spines seen in both Golgi impregnations and electron micrographs are sessile, the flow of electrotonus generated by synaptic contacts on these structures will be minimally reduced, relative to that flowing through pedunculated spines or stalked appendages. Thus, the stra-
LRN: CONFORMATION, CYTOLOGY AND SYNAPTOLOGY
tegic proximal placement of these terminals would not only effectively modulate the electrotonus reaching the soma from the entire dendritic branch, but could supply the cell body with a "functionally powerful" synaptic potential as well (see Shepherd, '74 for discussion of dendritic electrotonus). The present results establish that some of these large synaptic endings are the terminals of ascending spinal fibers. No attempt was made to quantify the frequency of each category of synaptic terminal, but i t is clear that all categories of terminals are found in each subdivision of the nucleus. Moreover, endings containing dense core vesicles were seen with greatest frequency in the trigeminal division. This area also is found to be rich in fluorescent axon terminals which contain catecholamines (Martin et al., '77). Recently, studies using radioactively labelled neurotransmitters have identified termimals that contain a biogenic amine (Descarries and LaPierre, '73; Descarries et al., '75; and ChanPalay, '75, '76). Although terminals of our dense core category are similar to some of those containing norepinephrine or serotonin, the unequivocal identification of neurotransmitters and their correlation with the fine structure of synaptic endings within the LRN must await radioactive labelling. Small terminals containing spherical synaptic vesicles seem to be the most frequently occurring type in all areas of the nucleus. Profiles containing pleomorphic synaptic vesicles, large terminals and small terminals of the dense core category appear to occur in a decreasing order of frequency. These features are similar to those observed by Mizuno et al. ('75) who found that axodendritic terminals containing round (spherical) synaptic vesicles outnumbered those with pleomorphic vesicles by about 2:l; however, they appeared with almost equal frequency on the soma. The size of synaptic vesicles was measured in a number of small terminals containing either spherical or pleomorphic synaptic vesicles and a wide range continuum of mean vesicle size was revealed within each category. These results may reflect the variety of axon systems which terminate within the LRN (Kitai e t al., '74b; Clendenin e t al., '74b,c, '75; Ekerot and Oscarsson, '75; and Martin et al., '77). Our findings demonstrate that small terminals containing spherical synaptic vesicles whose mean vesicle area is greater than 1,250 nm (399 do not termi-
nate on the soma of LRN neurons. Although areal measurements do not afford information relative to shape, distribution, density or other aspects of synaptic morphology, if mean vesicle size is one of the fine structural parameters that can be correlated with a specific axon system, then there may be a t least one afferent system that does not synapse on the cell bodies of LRN neurons. Experimental fine structure The distribution of both spinal and rubral fibers within the LRN of the opossum has been described by Martin et al. ('77). The present results establish that some terminals of spinal origin are large and undergo a filamentous andlor dark reaction two days following a cervical hemisection (figs. 33,351. In addition, small terminals with spherical synaptic vesicles degenerate. It is apparent that some of the large terminals end proximally on the dendritic tree, whereas most of the small ones terminate more distally. At least two separate spinal-LRN axon systems have been indentified by physiological techniques and both ascend in the ipsilateral ventrolateral funiculus (Clendenin et al., '74b,c). Although denied by Ekerot and Oscarsson ('76), LRN neurons have been reported to receive collaterals from the dorsal spinocerebellar tract (Burton et al., '71). The differences in both synaptic terminal morphology and in their postsynaptic distribution may reflect the various axon systems which compose the totality of the spinal projection. At a survival time of four days, all degenerating terminals of spinal origin observed were electron dense. Therefore the large filamentous terminal in figure 35 probably represents an early form of degeneration. A similar progression from early filamentous to later dark reaction has been reported for fibers of cerebellar origin which terminate in the opossum red nucleus (King et al., '73). Monosynaptic EPSPs and IPSPs have been recorded from LRN neurons after spinal stimulation (Rosen and Scheid, '73a; Kitai et al., '74b; Ekerot and Oscarsson, '75). Ekerot and Oscarsson ('75) have localized the source of a t least some of these IPSP's to the ipsilateral forelimb tract (iF), one of the pathways which course through the ipsilateral ventrolateral funiculus. Correlations between shape of synaptic vesicles and postsynaptic activity were first made by Uchizono in the cerebellar cortex of the cat (Uchizono, '65). He stated that
JOSEPH A. ANDREZIK AND JAMES S. KING
terminals which are excitatory in nature contained round (spherical) synaptic vesicles, whereas inhibitory terminals contained flattened (pleomorphic) vesicles. If Uchizono's proposal can be extended throughout the mammlian central nervous system, one would expect both terminals containing spherical synaptic vesicles and terminals containing pleomorphic (flattened) synaptic vesicles to degenerate in the LRN after spinal lesions. Such is not the case in our material. We have not observed degenerating terminals which contained pleomorphic synaptic vesicles subsequent to spinal hemisection, a finding also reported by Mizuno and Nakamura ('73) and Mizuno et al. ('75). Negative findings are tenuous a t best, and these observations could simply reflect a sampling bias. In some areas of the nervous system there is a correlation between synaptic vesicle shape and postsynaptic action (Uchizono, '65; Peters et al., '76). However, since correlative morphology and physiology is lacking in most parts of the nervous system, this correlation is only a hypothesis which must be substantiated for each area studied. McLaughlin et al. ('75) suggest that in the spinal cord, synaptic vesicle shape and the morphology of synaptic contacts are not necessarily correlated with proposed postsynaptic action. The fact that terminals containing pleomorphic vesicles were not observed degenerating in the LRN after spinal hemisections may mean that the correlation between synaptic vesicle shape and postsynaptic action does not hold true for the LRN as well. Terminals which are inhibitory in function may contain spherical synaptic vesicles or conversely, terminals which contain spherical synaptic vesicles may be excitatory, inhibitory or both, depending on the postsynaptic site (see Peters et al., '76 for review). All degenerating rubral terminals observed in the present account were small (less than 2.5 p ) and contained spherical synaptic vesicles. The postsynaptic loci were usually intermediate or distal dendrites and occasionally a solitary spinous process. These observations confirm those of Mizuno et al. ('75) in the rabbit. This synaptic terminal morphology seems to be common to projection neurons of the red nucleus as evidenced by degenerating terminals observed in the spinal cord of the rat, cat and opossum (Brown, '74; Kostyuk and Skibo, '75; Goode, '75) and in the facial motor nucleus of the rabbit (Mizuno et al., '73). How-
ever, Kostyuk and Skibo ('75) have reported that terminals containing flattened or ovoid vesicles (F-type) and terminals containing dense core vesicles (DC-type) also degenerate within the spinal cord after lesions of the red nucleus. We have seen degenerating synaptic profiles within the LRN which contain either pleomorphic (flattened) vesicles or dense core vesicles (1-8)after rubral lesions which extend well into the tegmentum dorsal to the red nucleus and when the brainstem was hemisected a t a rostra1 pontine level. Many synaptic terminals remain normal after spinal or rubral lesions. As stated earlier, quantitative data as to the relative occurrence of degenerating synaptic terminals is not available. However, the number of degenerating terminals observed after either lesion is conservatively estimated to be well under 5% of the total population. Since our lesions interrupted the majority of afferent fibers from spinal and rubral areas, this estimate is not thought to be biased by the relative sparing of fibers. Moreover, Mizuno et al. ('75) found that the number of degenerating synaptic terminals of spinal origin never exceeded 3%of the total, even in areas where maximum amounts of degeneration were found. Information concerning the terminal distribution of other afferent systems to the opossum LRN is now available (Martin et al., '77) and additional relevant details of its synaptic organization awaits further experimental analysis. ACKNOWLEDGMENTS
The authors thank Ms. Barbara Diener for her excellent technical assistance, Ms. Malinda Amspaugh for typing the manuscript and Mr. Gabriel Palkuti for his photographic assistance. The counsel and time given by Doctor George F. Martin throughout the entirety of this study is gratefully acknowledged. LITERATURE CITED Akert, K., K. Pfenninger, C. Sandri and H. Moor 1972 Freeze-etching and cytochemistry of vesicles and membrane complexes in synapses of the central nervous system. In: Structure and Function of Synapses. G. D. Pappas and D. P. Purpura, eds. Raven Press, New York. Azzena, G. B., and T. Ohno 1973 Influence of spino-reticulo-cerebellar pathway on PurkynE cells of paramedian lobule. Exp. Brain Res., 17;63-74. Berman, A. L. 1968 The brain stem of the cat. A cytoarchitectonic atlas with stereotaxic coordinates. The University of Wisconsin Press, Madison. Bowman, M. H., and J. S. King 1973 The conformation, cy-
LRN: CONFORMATION, CYTOLOGY AND SYNAPTOLOGY tology and synaptology of the opossum inferior olivary nucleus. J. Comp. Neur., 148: 491-524. Bloedel, J. 1973 Cerebellar afferent systems: A review. Prog. Neurobiol., 2: 1-68. Brodal, A. 1943 The cerebellar connections of the nucleus reticularis lateralis (nucleus funiculi lateralis) in rabbit and cat. Experimental investigations. Acta Psychiat. Neurol. (Kbh), 18: 171-233. 1949 Spinal afferents to the lateral reticular nucleus of the medulla oblongata in the cat. J. Comp. Neur., 91: 259-295. Brown, L. T. 1974 Rubrospinal projections in the rat. J. Comp. Neur., 154: 164-188. Bruckmoser, P., M.-C. Hepp-Reymond and M. Wiesendanger 1970a Cortical influence on single neurons of the lateral reticular nucleus of the cat. Exp. Neurol., 26: 239252. 1970b Effects of peripheral, rubral and fastigial stimulation on neurons of the lateral reticular nucleus of the cat. Exp. Neurol., 27: 388-398. Burton, J. E., J. R. Bloedel and R. S. Gregory 1971 Electrophysiological evidence for an input to lateral reticular nucleus from collaterals of dorsal spino-cerebellar and cuneocerebellar fibers. J. Neurophysiol., 34: 885-897. Chan-Palay, V. 1973 A light microscopic study of cytology and organization of neurons in the simple mammalian nucleus lateralis: Columns and swirls. Z. Anat. Entwickl-Gesch., 141: 125-150. 1976 Serotonin axons in the supra- and subependymal plexuses and in the leptomeninges; their role in local alterations of cerebrospinal fluid and vasomotor activity. Brain Res., 102: 103-130. Clarke, J. L. 1858 Researches on the intimate structure of the brain, human and comparative. First series. On the structure of the medullaoblongata. Phil. Trans. Roy. Soc. London., 148: 231-259. Clendenin, M., C.-F. Ekerot and 0. Oscarsson 1 9 7 4 ~The lateral reticular nucleus in the cat. 111. Organization of component activated from ipsilateral forelimb tract. Exp. Brain Res., 21: 501-513. 1975 The lateral reticular nucleus in the cat. IV. Activation from dorsal funiculus and trigeminal afferents. Exp. Brain Res., 24: 131-144. Clendenin, M., C.-F. Ekerot, 0. Oscarsson and I. Rosen 1974a The lateral reticular nucleus in the cat. I. Mossy fibre distribution in cerebellar cortex. Exp. Brain Res., 21: 473-486. 1974b The lateral reticular nucleus in the cat. 11. Organization of component activated from bilateral ventral flexor reflex tract (bVFRT). Exp. Brain Res., 21: 487-500. Colonnier, M. 1964 The tangential organization of the visual cortex. J. Anat. (London), 98: 327-344. 1968 Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscopic study. Brain Res., 9: 268-287. Dean, J. 1864 The gray substance of the medulla oblongata and trapezium. Smithsonian Contributions to Knowledge, 16: 1-75. Descarries, L., A. Beaudet and K. C. Watkins 1975 Seretonin nerve terminals in adult r a t neocortex. Brain Res., 100: 563-588. Descarries, L., and Y. LaPierre 1973 Noradrenergic axon terminals in the cerebral cortex of rat. I. Radioautographic visualization after topical application of DL-13HI norepinephrine. Brain Res., 51: 141-160. Ekerot, C.-F., and 0. Oscarsson 1975 Inhibitory spinal paths to the lateral reticular nucleus. Brain Res., 99: 157-161. 1976 The lateral reticular nucleus in the cat.
V. Does collateral activation from the dorsal spinocerebellar tract occur? Exp. Brain Res., 25: 327-337. Fernstrom, R. C. 1958 A durable Nissl stain for frozen and paraffin sections. Stain Tech., 33: 175-177. Gerhard, L., and J. Olszewski 1969 Medulla oblongata and pons. Primatologia. Vol. 11. H. Hofer, A. H. Schultz and D. Stark, eds., S. Karger, Basel. Goode, G. E. 1975 An electron microscopic study of rubrospinal projections to the lumbar spinal cord of the opossum. Neuroscience Abstracts, 1: 269. Gray, E. G. 1959 Axosomatic and axodendritic synapses of the cerebral cortex: An electron microscopic study. J. Anat. (London)., 93: 420-433. King, J. S. 1976 The synaptic cluster (glomerulus) in the inferior olivary nucleus. J. Comp. Neur., 165: 387400. King, J. S., R. M. Dom, J. B. Conner and G. F. Martin 1973 An experimental light and electron microscopic study of cerebellorubral projections in the opossum, Didelphis marsupialis uirgininm. Brain Res., 52: 61-78. Kitai, S. T., J. F. Defrance, K. Hatada and D. T. Kennedy 1974b Electrophysiological properties of lateral reticular nucleus cell. 11. Synaptic activation. Exp. Brain Res., 21: 419-432. Kitai, S. T., D. T. Kennedy, J. F. DeFrance and K. Hatada 1974a Electrophysiological properties of lateral reticular nucleus cells. I. Antidromic activation. Exp. Brain Res., 21: 403-418. Kostyuk, P. G., and G. G. Skibo 1975 An electron microscopic analysis of rubrospinal tract termination in the spinal cord of the cat. Brain Res., 85: 511-516. Kiinzle, H. 1973 The topographic organization of spinal afferents to the lateral reticular nucleus of the cat. J. Comp. Neur., 149: 103-116. Leontovich, T. A,, and G. P. Zhukova 1963 The specificity of the neuronal structure and topography of the reticular formation in the brain and spinal cord of carnivoa. J. Comp. Neur., 121: 347-381. Llinh, R., and D. E. Hillman 1969 Physiological and morphological organization of the cerebellar circuits in various vertebrates. In: Neurobiology of Cerebellar Evolution and Development. R. Llinas, ed. AMAERF, Chicago, pp. 43-73. Mannen, H. 1960 Noyau ferme et noyau ouvert. Arch. ital. biol., 98: 333-350. 1966 Contribution of the morphological study of dendritic arborization in the brain stem. In: Correlative Neurosciences Part A: Fundamental Mechanisms. Prog. Brain Res., Vol. 21A. T. Tokizane and J. P. Schade, eds. pp. 131-162. Martin, G. F., J. A. Andrezik, K. Crutcher, M. Linauts and M. Panneton 1977 The lateral reticular nucleus of the opossum (Didelphis uirginiana). 11. Connections. J. Comp. Neur., 174: 151-186. Martin, G. F., C. K. Henkel and J. S. King 1976 Cerebelloolivary fibers: Their origin, course and distribution in the North American opossum. Exp. Brain Res., 24: 219-236. McLaughlin, B. J., R. Barber, K Saito, E. Roberts and J. Y. Wu 1975 Immunocytochemical localization of glutamate decarboxylase in rat spinal cord. J. Cornp. Neur., 164: 305-322. Mihailoff, G. A,, and J. S. King 1975 The basilar pontine gray of the opossum: A correlated light and electron microscopic analysis. J. Comp. Neur., 159: 521-552. Mizuno, N., A. Konishi and Y. Nakamura 1975 An electron microscope study of synaptic organization in the lateral reticular nucleus of the medulla oblongata in the cat. Brain Res., 94: 369-381. Mizuno, N., K. Mochizuki, C. Akimoto, R. Matsushima and Y. Nakamura 1973 Rubrobulbar projections in the rab-
JOSEPH A. ANDREZIK AND JAMES S. KING
bit. A light and electron microscopic study. J. Comp. Neur., 147: 267-279. Mizuno, N. and Y. Nakamura 1973 An electron microscope study of the spinal afferents to the lateral relicular nucleus of the medulla oblongata in the cat. Brain Res., 53: 187-191. Oswaldo-Cruz, E., and C. E. Rocha-Miranda 1968 The brain of the opossum (Didelphis marsupialis) Instituto de Biofisica, Universidade Federal do Rio de Janeiro, Rio de Janeiro. Palay, S. L., and V. Chan-Palay 1974 Cerebellar Cortex, Cytology and Organization. Springer-Verlag, Berlin. Peters, A,, S. L. Palay and H. deF. Webster 1976 The Fine Structure of the Nervous System: The Neurons and Supporting Cells. Second ed. W. B. Saunders Co., Philadelphia. Petrovicky, P. 1966 A comparative study of the reticular formation of the guinea pig. J. Comp. Neur., 128: 85108. Ramon-Moliner, E. 1962 An attempt a t classifying nerve cells on the basis of their dendritic patterns. J. Comp. Neur., 119: 211-227. Ramon-Moliner, E., and W. J. H. Nauta 1966 The isodendritic core of the brain stem. J. Comp. Neur., 126: 311336. Ramon y Cajal, S. 1909 Histologie du systeme nerveu de l’homme et des vertebres. Vol. I. A. Maloine, Paris, pp. 934-959. Rosen, I., and P. Scheid 1972 Cutaneous afferent responses in neurones of t h e lateral reticular nucleus. Brain Res., 43: 259-263. 1973a Patterns of afferent input to the lateral reticular nucleus of the cat. Exp. Brain Res., 18: 242-255. 197313 Responses of nerve stimulation in the bilateral ventral flexor reflex tract (bVFRT) of the cat. Exp. Brain Res., 18: 256-267. 1973c Responses in the spino-reticulo-cerebellar pathway to stimulation of cutaneous mechanoreceptors. Exp. Brain Res., 18: 268-278. Scheibel, M. E., and A. B. Scheibel 1958 Structural substrates for integrative patterns in the brain stem reticu-
lar core. In: Reticular Formation of the Brain. H. H. Jasper et al., eds. Little Brown andCo., Boston, pp. 31-55. Shepherd, G. M. 1974 The Synaptic Organization of the Brain. An Introduction. Oxford University Press, New York. Stensaas, L. J., and S. S. Stensaas 1968 Astrocytic neuroglial cells, oligodendrocytes and microgliocytes in the spinal cord of the toad. I. Light microscopy. Z. Zellforsch., 84: 473-489. Taber, E. 1961 The cytoarchitecture of the brain stem of the cat. J. Comp. Neur., 116: 27-69. Uchizono, K. 1965 Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat. Nature (London), 207: 642-643. Valverde, F. 1961 Reticular formation of the pons and medulla oblongata. A Golgi study. J. Comp. Neur., 116: 71-99. Venable, J. H., and R. Coggeshall 1965 A simplified lead citrate strain for use in electron microscopy. J. Cell Biol., 25: 407-408. Verhaart, W. J. C. 1957 The lateral reticular nucleus of the medulla oblongata and the passing fiber systems of the lateral funiculus. Acta Psychiat. Scand., 32: 211-229. 1970 Comparative Anatomical Aspects of the Mammalian Brain Stem and Spinal Cord. Vols. I and 11, Van Gorcum, Assen. Voris, H. C., and N. L. Hoerr 1932 The hindbrain of the opossum, Didelphis uirginiana. J. Comp. Neur., 54: 277355. Walberg, F. 1952 The lateral reticular nucleus of the medulla oblongata in mammals. J. Comp. Neur., 96: 283337. Westrum, L. E. 1970 Observations on initial segments of axons in the prepyriform cortex of the rat. J. Comp. Neur., 139: 337-356. Windle, H. 1943 A Nissl method using buffered solutions of thionin. Stain Tech., 18: 77-86. Zangger, P., and M. Wiesendanger 1973 Excitation of lateral reticular nucleus neurones by collaterals of the pyramidal tract. Exp. Brain Res., 17: 144-151.
Abbreviations d, small diameter dendrite Dd, dendrite D Dd., distal dendrite EXT., external division of the lateral reticular nucleus g, Golgi apparatus
h, heterochromatin I Dd., intermediate dendrite INT., internal division of lateral reticular nucleus 10, inferior olivary nucleus 1, lipofuscin
n, nucleolus nb, Nissl body p, polyribosomes P Dd., proximal dendrite TR., trigeminal division of the lateral reticular nucleus
PLATE 1 EXPLANATION OF FIGURES
1 Photomicrograph of a horizontal section through the mid dorsoventral extent of the LRN. The internal (INT.) and external (EXT.) divisions are labelled. The arrowhead indicates a group of neurons at the rostral dorsolateral pole of the external division. Broken lines labelled 3-8 indicate corresponding levels of the transverse sections seen in figures 3 to 8. (Frozen section, Nissl preparation). 2 Photomicrograph of a horizontal section through the dorsal portion of the LRN. The trigeminal division (TR.) is labelled. Broken lines labelled 3-8 indicate corresponding levels of the transverse sections seen in figures 3 to 8. (Frozen section, Nissl preparation). 3 Photomicrograph of a transverse section near the caudal pole of the LRN. Only the internal division is present. The small arrow indicates a compact group of neurons. The solid block arrow points to larger neurons located ventral and medial to the compact group. (Frozen section, Nissl preparation). 4
Photomicrograph of a transverse section near the level of the caudal pole of the inferior olivary nucleus (10). The small arrow points to the continuation of the compact group of neurons labelled figure 3. The solid block arrow indicates the group of larger neurons which form the medial boundry of the LRN. The open block arrow marks the caudal tip of the external division. (Frozen section, Nissl preparation).
5 Photomicrograph of a transverse section near the midportion of the external division of the LRN. The external division (open block arrow) forms a prominent bulge on the ventrolateral surface of the medulla. The two small arrows point to all that remains of the compact group of neurons of the internal division. The remainder of the nucleus consists of the enlarged medial group of neurons (solid block arrow) and small cords of neurons which form the border between the internal and external divisions (open triangles). The inferior olivary nucleus (10)is labelled for reference. (Frozen section, Nissl preparation). 6 Photomicrograph of a transverse section near the rostral tip of the external division (open block arrow) of the LRN. The internal division consists of three columns of neurons (solid block arrows) which are connected with one another a t their ventral margins. The group of neurons forming the rostral dorsolateral pole of the external division is marked by the arrowhead. Open arrows indicate strands of neurons which course in a ventrolateral to dorsomedial direction and are part of the trigeminal division. The inferior olivary nucleus is labelled for reference. (Frozen section, Nissl preparation).
7 Photomicrograph of a transverse section at the caudal tip of the trigeminal division. The arrowhead marks a rostral extension of the large neurons populating the rostral dorsolateral pole of the external division. A strand of neurons (open arrow) courses dorsomedially from this group and constitutes part of the trigeminal division. The curved arrow marks a cluster of neurons lying on the ventromedial margin of the descending trigeminal nucleus. The inferior olivary nucleus (10) is labelled for reference. (Frozen section, Nissl preparation).
8 Photomicrograph of a transverse section near the rostral pole of the LRN. Only the trigeminal division is present. It consists of a compact group of neurons situated a t the ventromedial border of the descending trigeminal nucleus (curved arrow) and neurons scattered among the fibers of the descending trigeminal tract (open arrow). (Frozen section, Nissl preparation).
LRN: CONFORMATION, CYTOLOGY AND SYNAPTOLOGY Joseph A. Andrezik and James S. King
PLATE 2 EXPLANATION OF FIGURES
9 Photomicrograph of a transverse section through the rostral portion of the LRN which illustrates the various components of the trigeminal division. The arrowhead marks the rostral extreme of the dorsolateral portion of the external division. Some neurons of the trigeminal division collect along the ventral margin of the descending trigeminal tract (small solid block arrow). The majority of neurons in this division are dispersed among fibers of the descending trigeminal tract (large solid block arrow). One neuron which lies on the ventromedial border of the descending trigeminal nucleus is marked by the curved arrow. (Frozen section, Nissl preparation, 4 X objective). 10 Photomicrograph of a transverse section near the midpoint of the rostro-caudal extent of the LRN. Dotted lines indicate the boundries of the internal (INT.). and external (EXT.) divisions. The solid white arrow indicates a neuron of the internal division which is enlarged in figure 12. The solid block and open block arrows point to two neurons of the external division which are enlarged in figure 13. (Semi-thin section, toluidine blue preparation, 4 X objective). 11 Photomicrograph of a transverse section through the rostral portion of the LRN. Dotted lines encircle the trigeminal division (TR.) and two neurons enlarged in figure 14 are marked by the solid block arrows. (Semi-thin section, toluidine blue preparation, 4 X objective).
LRN: CONFORMATION, CYTOLOGY AND SYNAPTOLOGY Joseph A. Andrezik and James S.King
PLATE 3 EXPLANATION OF FIGURES
12 Photomicrograph of a neuron from the internal division of the LRN. Solid white arrow corresponds to the position of the similar arrow in figure 10. (Semi-thin section, toluidine blue preparation, 54 X oil immersion objective).
13 Photomicrograph of two neurons from the external division of t h e LRN. The variation in perikaryal size is evident. Solid and open block arrows correspond to the position of similar arrows seen in figure 10. (Semithin section, toluidine blue preparation, 54 X oil immersion objective). 14 Photomicrograph of three neurons from the trigeminal division of t h e LRN. The solid block arrows point to the two neurons similarly marked in figure 11. (Semi-thin section, toluidine blue preparation, 54 X oil immersion objective). 15 Photomicrograph of a neuron from t h e internal division. This neuron is comparable in size to t h a t shown in figure 12 and distinct Nissl granules are evident in the perikaryon. In addition, the basophilia extends well into the proximal dendrites. (Paraffin section, Nissl preparation, 54 X oil immersion objective). 16 Photomicrograph of a neuron from the external division of t h e LRN which is comparable to t h e neurons shown in figure 13.The Nissl pattern within neurons of this division is less distinct t h a n i n neurons of the internal and trigeminal divisions and t h e basophilia is less marked (compare with figures 15 and 17). (Paraffin section, Nissl preparation, 54 X oil immersion objective).
17 Photomicrograph of neurons from the trigeminal division which are comparable to those illustrated in figure 14.Neurons of this division display prominent Nissl granules. (Paraffin section, Nissl preparation, 54 X oil immersion objective). 18 Camera lucida drawing of a neuron from the trigeminal division. The line drawing at t h e left indicates the position of this neuron within the LRN. The dendrites are distributed in a radial pattern and few spines are apparent. (Golgi-Kopsch preparation, 54 X oil immersion objective).
LRN: CONFQRMATION, CYFOLOGY AND SYNAPTOLOGY Joseph A. Andrezik and James S. King
PLATE 4 EXPLANATION OF FIGURES
19 Camera lucida drawing of a neuron from the external division of the LRN. The line drawing a t the lower left indicates the location of this neuron within the brainstem. The dendrites of this neuron emanate from opposite poles of the perikaryon and are primarily oriented in a ventromedial to dorsolateral direction. However, one dendrite courses perpendicular to the majority of the arborization and extends to the interface between the external and internal divisions. The neuronal surface is relatively free of spines. (Golgi-Hortega preparation, 54 X oil immersion objective). 20 Camera lucida drawing of a neuron from the internal division of the LRN. The line drawing in the lower portion of the figure indicates the location of this neuron within the brainstem. The dendrites emanate in a radial pattern from the cell body. (Golgi-Kopsch preparation, 54 X oil immersion objective).
LRN: CONFORMATION, CYTOLOGY AND SYNAproLOGY Joseph A. Andrezik and James S.King
PLATE 5 EXPLANATION OF FIGURES 2 1 A neuron from the rostra1 dorsolateral part of the external division. No synaptic ter-
minals contact the soma in this plane of section and t h e neuronal surface is bordered by astrocytic lamellae or myelinated axons. The open block arrows point to small invaginations of the nuclear membrane. A prominent nucleolus (n) is present as well as isolated clusters of heterochromatin (h). The usual complement of cytoplasmic organelles is present. Nissl bodies (nb) formdistinct aggregates and the Golgi apparatus (g) is arranged in a circumnuclear pattern. Free polyribosomes (p) and lipofuscin (1) are labelled. (Electron micrograph, X 6,284).
LRN: CONFORMATION. CYTOLOGY AND SYNAPTOLOGY Joseph A. Andrezik and James S.King
25 Large synaptic terminal adjacent to three spinous processes (asterisks) in t h e internal division of the LRN. A longitudinally sectioned dendrite (Dd.) is seen a t the left and probably represents the source of the three spines. (Electron micrograph, X 30,000).
24 Montage photomicrograph of a portion of t h e dendritic tree of the neuron drawn in figures 22 and 23. The open block arrow indicates the portion of the dendritic tree which is correspondingly labelled i n figure 23. Clusters of sessile spines occur near the branching points of the proximal dendrites.
23 Camera lucida drawing of the neuron illustrated in figures 22 and 24. In addition to the clusters of spines on the proximal dendrites (open block arrow) the more distal segments exhibit a greater number of spines than seen on other LRN neurons. (Golgi-Hortega preparation, 54 X oil immersion objective).
22 Camera lucida drawing of the ventrolateral quadrant of the caudal brainstem. The location of t h e neuron shown a t a higher magnification in figures 23 and 24 is indicated. The majority of t h e dendritic a r h r i z a t i o n is oriented in a ventromedial to dorsolateral direction. (Golgi-Hortega preparation, 4 X objective).
EXPLANATION OF FIGURES
LRN: CONFORMATION, CYTOLOGY AND SYNAPTOLCGY
Joseph A. Andrezik and Jamea S.King
PLATE 7 EXPLANATION OF FIGURES
26 A small synaptic terminal which contains agranular spherical vesicles. This terminal is synapsing with the soma and a small profile (probably a somatic spine) of a neuron within the internal division of the LRN. The open block arrow indicates a Gray’s type 1 synaptic junction. A small somatic appendage (asterisk) lies adjacent to the terminal. (Electron micrograph, X 30,000). 27 A small synaptic terminal which contains agranular spherical vesicles synapsing with a distal dendrite in the internal division of t h e LRN. The open block arrow indicates a Gray’s type 1 synaptic junction. (Electron micrograph, X 30,000). 28 A small synaptic terminal which contains several large irregular dense core vesicles and agranular pleomorphic vesicles. (Electron micrograph, X 30,000).
29 A large synaptic terminal which contains agranular spherical vesicles and several larger dense core vesicles (small arrows). This ending contacts a small dendritic profile in the internal division of the LRN. A bundle of small diameter dendrites (d) lies adjacent to the terminal. (Electron micrograph, X 30,000)
LRN: CONFORMATION, CYTOLOGY AND SYNAPTOLOGY Joseph A. Andrezik and James S.King
PLATE 8 EXPLANATION OF FIGURES 30 A synaptic ending containing pleomorphic synaptic vesicles which contacts the soma of a neuron in the internal division of the LRN. Open block arrows indicate synaptic junctions which are intermediate between Gray’s type 1 and type 2. (Electron micrograph, X 30,000). 31 A synaptic ending containing pleomorphic vesicles which contacts an intermediate dendrite (I Dd.) within the external division of the LRN. The open block arrow indicates a synaptic junction intermediate between Gray’s type 1 and type 2. (Electron micrograph, X 30,000). 32 A synaptic ending containing pleomorphic vesicles which contacts a distal dendrite (D Dd.) within the internal division of the LRN. The open block arrow indicates a synaptic junction intermediate in morphology between Gray’s type 1 and type 2. (Electron micrograph, X 30,000).
LRN: CONFORMATION, CYTOLOGY AND SYNAPTQLOGY Joseph A. Andrezik and James S. King
PLATE 9 EXPLANATION OF FIGURES
33 A large synaptic terminal undergoing degeneration after ipsilateral spinal cord hemisection a t C, (survival time 46 hours). This ending is in the internal division, displays a dark reaction and contains several dense core vesicles. The postsynaptic loci of this terminal are a spine (asterisk) and the shaft of a proximal dendrite (P Dd). (Electron micrograph, X 30,000) 34 A small synaptic terminal undergoing degeneration after ipsilateral spinal cord hemisection a t C, (survival time 46 hours). This terminal is from the internal division and is in the beginning stages of degeneration. Its postsynaptic site is the shaft of a distal dendrite (D Dd.). (Electron micrograph, X 30,000). 35 A large synaptic terminal undergoing degeneration after an ipsilateral spinal cord hemisection (survival time 46 hours). This terminal is from the internal division of the LRN and is undergoing a filamentous reaction. The postsynaptic loci are two small diameter dendrites (d) and a neuronal process of undetermined nature (unlabelled). (Electron micrograph, X 30,000). 36 A small synaptic terminal undergoing degeneration after a stereotaxic lesion of the contralateral red nucleus (survival time 48 hours). This ending is from the trigeminal division and contacts a dendritic shaft (Dd.). (Electron micrograph, X 30,000).
LRN: CONFORMATION, CYTOLOGY AND SYNAPTOLOGY Joseph A. Andrezik and James S.King