Golgi Material Research Articles

Organization of the GABAergic system in the rat hippocampal formation: a quantitative immunocytochemical study.

Specific antibodies against gamma aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) were used to study the organization of the GABAergic system in the rat hippocampal formation. Both the number of GABA-like-immunoreactive (Li) somata and neuropil density were assessed in semithin sections. Cell counts revealed that approximately 11% of the hippocampal neuronal population showed GABA-Li within the various planes of section. Each layer in the hippocampal formation had a characteristic organization of GABA-Li elements. In Ammon's horn, 80-95% of the neuronal somata within the apical and basal dendritic regions were GABA-Li positive. Within the pyramidal cell layer 5-8% of the cells were GABA-Li in the CA1 to CA3 subfields of Ammon's horn and only 3% were GABA-Li within that portion of the pyramidal cell layer that inserts into the hilus. Only slight differences in the density of the GABA-Li neuropil were observed within the CA1-CA3 dendritic regions. Restricted to the stratum lucidum was a dense band of GABA-Li label. Counts of immunoreactive grains localized on the perimeter of pyramidal (CA1-CA3) and granule somata revealed more terminal boutons on the CA3 cells than on CA1 and granule neuronal somata. A topographical distribution of GABA-Li somata and neuropil was found in the fascia dentata: There the label particularly concerned its suprapyramidal and rostrolateral portions. Approximately 40% of neurons in the molecular layer, 60% in the polymorph layer, and 18% within the hilar region were GABA-Li. Within the granule cell layer only 2% of the neurons were GABA-Li positive. Distinct differences in the density of the GABA-Li neuropil were present in the molecular, pericellular granular, and hilar regions of the fascia dentata. While the morphology of GABA-Li neuronal somata varied according to their hippocampal layer, the most heterogeneous cell types were found in the regio inferior of the hippocampus. There we have identified neurons that are reminiscent of the inferior region interneuron described in Golgi material by Amaral and Woodward (Brain Res. 124:225-236, '77). Moreover, particularly in the sagittal plane, we have identified oval, triangular, and round cells and that have processes oriented in a parallel arrangement, appearing to be aligned along the granule cell mossy fibers.

Early postnatal development of the monkey globus pallidus: A Golgi and electron microscopic study

The globus pallidus of 20 monkeys ranging in age from newborn to 4 months was examined in Golgi-impregnated material and ultrastructurally. There was no discernible difference between the lateral and medial segments of the structure. At the light microscope level, all neuronal types described in the adult are found at birth. The most common, the large fusifom cell, shows initial signs of immaturity such as blunt protrusions and dendritic dilations at bifurcation points, as well as growth cones, filopodia, and filiform processes. These features become more rare with age, and by 4 months, the neurons appear fully mature save for the terminal dendritic arborizations which are still underdeveloped. From the earliest ages examined, the large globular cells and the interneurons are more mature than the previous type. The afferent radial fibers of striatal origin are observed from birth, but they are grouped in bundles only after 8 weeks. The density of their climbing branches increases over time, reaching a mature appearance by 16 weeks. Afferents entering from the ventral surface do not yet show clusters of varicosities at 2 weeks. At the latter age, plexuses of fine beaded fibers are already seen covering large extensions of the nucleus. The fine structure correlates well with the Golgi material. The basic features of the neuropil are present at birth, albeit with immature characteristics such as the incomplete covering of the dendrites with axonal boutons and the low level of myelination of the radial fibers. Growth cones and profiles with signs of degeneration are observed during the first month. In the early ages examined, most dendrites show large varicosities and protrusions, some of which are spinelike and can be postsynaptic to multiple terminals. The other dendritic type, with only an occasional axodendritic synapse, is also seen from birth and increases in size as a function of time. The type I axonal boutons, of probable striatal origin, are quite immature at birth, and their characteristic interdigitations are seen only after the first week. The type II, III, IV, and V boutons appear mature at all ages examined but crest synapses formed by the type III terminals are observed in the later stages of the study. Finally, postsynaptic vesicle-containing profiles are present at 4 weeks, but triadic synaptic arrangements are formed only by 16 weeks.(ABSTRACT TRUNCATED AT 400 WORDS)

Populations of GABAergic neurons and axons in layer I of rat auditory cortex

Neurons and axon terminals (puncta) immunostained by an antibody against glutamic acid decarboxylase were studied in layer I of adult rats in architectonically identified area 41 of auditory cortex. The borders of area 41 and the laminar subdivisions of cortex were established in normal material and in other studies of cortical connections. Vibratomed or frozen sections were immunostained. The objectives of the study were to classify the types of (i) glutamic acid decarboxylase-positive neurons and (ii) puncta, and (iii) to examine their spatial distribution within layer I. Control sections were devoid of specific immunostaining. More than 90% of layer I cells are glutamic acid decarboxylase-positive. Four types of neuron were identified in Golgi material, including small neurons with stellate dendritic fields, horizontal cells with laterally projecting arbors, medium-sized neurons with stellate, widely ramifying dendritic fields, and large neurons with broad dendritic fields spanning the depth of layer I or branching laterally. In the glutamic acid decarboxylase material, examples with a somatodendritic shape matching each of these types were found. The average somatic diameter of glutamic acid decarboxylase-positive neurons ( X¯= 59μm 2, S.D. = 21μm 2) suggests that the small and medium-sized neurons predominate. Glutamic acid decarboxylase-positive neurons occur throughout the depth of layer I, but are far more numerous in the deeper half (68% in layer Ib) than in the superficial part (32% in layer Ia). Glutamic acid decarboxylase-positive neurons form small clusters of three to five cells across the cortical surface, with a range of 0–9/100 μm across the cortex. Most glutamic acid decarboxylase-positive neuronal perikarya were intensely immunostained, and the dendrites of the medium-sized and large neurons were traced as far as 50–75 μm beyond their initial branching point. Glutamic acid decarboxylase-positive puncta also had variable shapes. Both small, fine puncta (< 0.5 μm in diameter) and larger, coarser ones (>1.5 μm in diameter) were present, though the former were much more common. In traverses from the pia to the layer II border, the puncta average about 40/100 μm 2 (range: 20–80), and the shape of individual pia-layer II traverses is multipeaked, often with a slight trough at −80μm depth, then rising slowly in number toward layer II. These anatomical arrangements may have physiological consequences for cortical functional organization: (i) the GABAergic influences of layer I cells are directed laterally, toward other layer I neurons, or at the apical dendritic arbors of deep-lying commissural or corticofugal neurons, whose activity might be modulated by GABAergic layer I influences; (ii) the axons of layer II glutamic acid decarboxylase-positive neurons or of corticocortical interneurons may affect layer I. While many different transmitter molecules have been identified in layer I, it is probable that their activity influences a largely GABAergic neuronal population.

Mamillary body in the rat: a cytoarchitectonic, Golgi, and ultrastructural study.

The present study provides a comprehensive light and electron microscopic analysis of the anatomical organization of the rat mamillary body. The cytoarchitecture and morphology of mamillary neurons were investigated with the aid of Nissl-stained and Golgi-impregnated sections cut in transverse, horizontal, and sagittal planes. The ultrastructural features of the mamillary nuclei were correlated with observations made on Golgi material. The mamillary body is comprised of a lateral and a medial nucleus, the latter being subdivided into five major subnuclei: pars lateralis, pars basalis, pars medialis, pars medianus, and pars posterior. The perikarya are medium-sized or small with the proportions of each differing among subnuclei. The largest perikarya are found in the lateral mamillary nucleus (cell area 257.0 microns2) and have 2-5 radially oriented aspiny dendrites that are often beaded. Small cells predominate in the pars lateralis (cell area 116.3 microns2) and pars basalis (cell area 118.3 microns2), whereas the pars medialis (cell area 196.7 microns2), pars medianus (cell area 136.5 microns2), and pars posterior (cell area 154.6 microns2) contain mainly medium-sized cells. The dendrites of most cells in the medial nucleus are radially oriented and exhibit a variety of spines including numerous short stubby spines, spines with thin necks that end in spherical swellings, and long thin spines. Neuronal somata are often closely apposed with no intervening glial processes and contain eccentrically located nuclei with one or more invaginations of the nuclear envelope. Two main classes of axon terminals were identified in the mamillary body. One type contains round vesicles and forms asymmetric synaptic junctions (RA) with dendrites and dendritic spines. RA terminals rarely contact neuronal somata and proximal dendrites in the MB. The second type contains pleomorphic vesicles and forms mainly symmetric synaptic junctions (PS) with neuronal somata as well as dendrites and spinous processes. Dense-cored vesicles were frequently seen in both types of terminals. Both types of terminals often synapse with two adjacent dendrites and are also found near or adjacent to each other on the same dendrite. A quantitative analysis indicated that the numbers of RA terminals in the medial nucleus almost equals the numbers of PS terminals, whereas the lateral mamillary nucleus contains considerably more PS (64%) than RA terminals (36%).

Alterations and recovery of dendritic spine density in rat hippocampus following long-term ethanol ingestion

Neuronal loss and dendritic pathology are often observed in humans and animals after long-term ethanol ingestion. It is not known, however, if surviving but damaged neurons can recover normal structure during ethanol anstinence. We quantified dendritic spine density in two neuronal populations in rat hippocampus to investigate whether reversibility from the cellular neurotoxic sequelae of chronic ethanol exposure was possible. Male Long-Evans rats were maintained for 20 weeks on an ethanol-containing liquid diet. Controls were pair-fed a liquid diet with sucrose substituted isocalorically for ethanol. One-half of each group was sacrificed at the end of the 20-week treatment and one-half was given a 20-week ethanol-free recovery period prior to sacrifice. Analysis of rapid Golgi material revealed a decreased spine density in CA1 pyramidal cells that increased to control level during abstinence, and an increased spine density in dentate gyrus granule cells that was reduced toward control level during abstinence. Thus, despite the fact that chronic ethanol exposure produced differential initial effects, the return toward normal spine density in each region is consistent with the concept of neuronal recovery and reorganization during abstinence from ethanol.

Morphology, number, and distribution of putative GABA-ergic neurons in the basilar pontine gray of the monkey.

We used an antibody raised against the inhibitory transmitter gamma-aminobutyric acid (GABA) in the basilar pontine gray (bpg) of the monkey. Somata, dendrites, and a plexus of probably axonal fibers exhibited GABA-like immunoreactivity. Labeled neurons were small with oval, triangular, or circular soma shape. They gave rise to 2 to 4 dendrites with little branching. No axons were seen issuing from the soma. Occasionally, appendages consisting of spheroidal bodies positioned at the distal end of tenuous stalks and (in 1 cell) axonlike processes were observed to originate from dendrites. According to their morphology, we suggest that these putative GABA-ergic neurons may correspond to the type II neurons observed in Golgi material. The average number of putative GABA-ergic cells in 40-micron sections was about 30/mm2. When compared with Nissl-stained sections, these amounted to about 5% of all cells. There was no substantial variation in average density in different parts of the bpg. However, labeled cells tended to lie in clusters, perhaps related to the well-established input-output compartments of the bpg. The demonstration of a significant population of putative inhibitory neurons challenges the traditional view of the bpg, which suggests that this brainstem cell group functions as a simple relay exchanging signals between cortex and cerebellum.

Synaptic organization of the nucleus of the optic tract in the rabbit: a combined Golgi-electron microscopic study.

The organization of the nucleus of the optic tract was investigated with light and electron microscopy in combination with Golgi impregnation. In Golgi material, neurons ranged in size from 10 to 25 microns with three to seven principal dendrites extending predominantly parallel to the fibres of the optic tract, irrespective of their location within the nucleus. In some areas dendrites extended into the neuropil of the adjacent dorsal terminal nucleus of the accessory optic system and the posterior pretectal nucleus. Occasionally spines and appendages were observed. The fine structure of the nuclei, perikarya and the dendritic arborization did not allow a well-defined distinction between interneurons and projection neurons. The synaptic organization of the nucleus of the optic tract showed great resemblance to the neuropil of the lateral geniculate nucleus and the superior colliculus. Similar types of presynaptic terminals were noticed: (i) R-terminals were either large and scalloped or small and regular in outline with spherical vesicles and electron-lucent mitochondria, and showed asymmetric contact zones; (ii) F-terminals with flattened vesicles, opaque mitochondria and symmetric contact zones; (iii) RLD-terminals with spherical vesicles and electron-dense mitochondria and asymmetric contact zones; (iv) P-terminals with pleomorphic vesicles and electron-lucent or opaque mitochondria and asymmetric synaptic thickenings. These different types of terminal were found isolated in the neuropil or in clusters of synapses. The most striking differences between the nucleus of the optic tract and the lateral geniculate nucleus were the relative scarcity of F-terminals in the clusters, the paucity of triadic arrangements and the relatively small size of the R-terminals. The differences in ultrastructure may be related to retinal W-type ganglion cells, which form the main retinal input to the nucleus of the optic tract and could also be related to the physiologically identified direction-selective units within the nucleus of the optic tract.

Autoradiographic and Golgi study on the early development of n. isthmi principalis and adjacent grisea in the chick embryo: a tridimensional viewpoint.

Neurogenesis, cell migration and early histogenesis of the isthmic nuclear complex in chick embryos were investigated in autoradiographic and Golgi material. The aim of the experimental observations was to detect whether the apparent origin of different grisea of this complex at separate matrix territories (neuromeres) was accompanied by peculiar generation patterns, consistent with predictions of neuromeric theory. Differential birthday patterns were indeed obtained for a) n. semilunaris--born in the rh1 a rhombomere, b) n. isthmi principalis pars parvocellularis, nn. lemnisci lateralis dorsalis and ventralis, and n. isthmi ventralis--born in the isthmic rhombomere, and c) n. isthmi principalis pars magnocellularis--born at the m1 mesomere. Only the nuclear group at (b) shows a clear-cut gradient of generation. The morphological analysis aimed to describe isthmic neuroblast cell form before, during and immediately after migration into the mesencephalic optic lobe. Golgi data indicate that isthmic neuroblasts emerge as free cells from the matrix and aggregate into a dense superficial mantle layer. Between stages HH26 and 30, the whole mass of cells translocates tangentially in a rostrolateroventral direction, invading the m2 mesomere. The individual migrating neuroblasts have a leading axonal process which rapidly grows into the tectum in advance of the cell body, which follows at a slower pace. As the migration runs to an end the neuroblasts start to differentiate, sprouting dendritic processes. A joint origin in the isthmic mantle primordium is proposed for the nuclear group at (b) (above), whereas n. isthmi principalis pars magnocellularis is formed separatedly from the rest, and shows no tangential migratory behaviour of its neuroblasts. The complex histogenetic and morphogenetic processes at the isthmo-mesencephalic boundary may be explained on the basis of these new data, but this requires a tridimensional viewpoint that is exposed in the Discussion.

The timing of granule cell differentiation and mossy fiber morphogenesis in the opossum.

The timing of developmental events may be important for the orderly formation of neuronal interconnections. In the present study, the timing of granule cell migration is compared with the arrival and maturation of mossy fiber projections. The opossum was chosen as the experimental animal because its protracted postnatal development enables the examination of developmental sequences not as easily recognized in other more commonly used mammalian species. It is shown that all areas that project to the cerebellum as mossy fibers in the adult opossum do so by postnatal day (PD) 30. Their major target, the granule cells begin inward migration from the external germinal layer (EGL) prior to PD 30, but do not form a distinct internal granular layer (IGL) until PD 35. Migrating granule cells penetrate into the IGL deep to granule cells that have begun dendritic differentiation. By PD 50, Golgi impregnations reveal that many granule cells have numerous immature processes, somal spines and dendritic growth cones. After this age these structures are rare and the vast majority of granule cells exhibit short dendrites with digiform endings. Dendritic differentiation subsequent to PD 54 involves an increase in the length of the shaft and the further maturation of terminal digits. Also from Golgi material, immature mossy fiber endings can be identified in the IGL by PD 35 and exhibit mature characteristics at PD 73. Thus, the formation and maturation of granule cell dendrites and their afferents (mossy fibers) occur over an extended period of time (PD 35-73). Moreover, granule cells exhibit a sequence of development similar to that of Purkinje cells: early arrival of their primary afferent projections in the cerebellar anlage; a period of exuberant dendritic growth; and a protracted and overlapping period for dendritic and synaptic maturation.

Stabilization of Silver Chromate Golgi Impregnation in Rat Central Nervous System Neurons Using Photographic Developers. II. Electron Microscopy

The silver chromate precipitate present in neurons impregnated according to the Golgi-rapid and Golgi-Kopsch procedures can be stabilized by treatment with a photographic developer. In a complementary light microscopic study the stabilizing properties of various photographic developers were tested. Kodalith, Elon-ascorbic acid, HC-110, D-19 and Neutol proved to be the most successful. In the present electron microscopic study, we studied the distribution, shape and size of the particles found in Golgi-rapid and Golgi-Kopsch-impregnated neurons by treatment with each of these developers and, simultaneously, the effect of the developer on the preservation of the ultrastructural details. The reaction product after developer-treatment of Golgi-rapid material is sufficiently stable to withstand embedding and thin sectioning, whereas in Golgi-Kopsch material additional gold chloride "toning" is necessary. In Golgi-impregnated, Kodalith-, Elon-ascorbic acid-, or HC-110-treated material the formed particles are small and located in the cytoplasm, limited by the plasma membranes of the impregnated profiles. In Golgi-impregnated, D-19 treated neurons, the formed particles are relatively coarse. The majority of these particles are within cytoplasm, but particles may also lie either across or entirely outside the plasma membranes of the impregnated profiles. A large number of the small particles in Golgi impregnated, Neutol-stabilized neurons can be seen partly or entirely outside the plasma membranes of the impregnated profiles. Good original ultrastructural preservation seems to be unaffected by developer treatment. Treatment of Golgi material with sodium bromide before stabilization (bromide substitution) results in the formation of small silver particles both inside and outside the impregnated profiles. The sodium bromide step of this procedure has an adverse effect on the preservation of ultrastructural detail.

Cytoplasmic organization during pattern formation in the alga Micrasterias.

Using an improved fixation for electron microscopy, we have observed an ordered cytoplasmic organization during pattern formation in the alga Micrasterias. At the earliest stages of morphogenesis (the bulge to 3-lobe stage), as observed previously, electron-dense vesicles and endoplasmic reticulum were found towards the cell periphery, while mitochondria and Golgi material were located internally. In contrast to previous studies, which found no organization in the cytoplasm of growing lobes, we observed a single cluster of electron-dense vesicles at the tip of each lobe at or beyond the 5-lobe stage. In lobes about to branch, two separate clusters of electron-dense vesicles were present in each lobe, apparently foreshadowing the sites of new lobe outgrowth. Individual lobes also possessed an ordered distribution of organelles. Immediately basal to the lobe tip, the concentration of electron-dense vesicles decreased and rough endoplasmic reticulum was more prevalent, followed more deeply in the cytoplasm by increased concentrations of mitochondria and then Golgi material. This cytoplasmic organization in individual lobes is characteristic of tip growth, thus suggesting that individual lobes elongate by this process. We have also investigated the effect of the ionophore A23187 on this cytoplasmic organization. The polarization of rough endoplasmic reticulum, mitochondria and Golgi material was destroyed by ionophore treatment in the presence of 2 mM-MgCl2 or 2 mM-CaCl2. The tip-associated clusters of electron-dense vesicles were dispersed by A23187 in 2 mM-CaCl2 but not significantly affected by A23187 in 2 mM-MgCl2. The latter observation indicates that the vesicle pattern can be disrupted by high calcium, but not high magnesium concentrations.

Ultrastructural analysis of synaptic relationships of intracellularly stained pyramidal cell axons in piriform cortex

Axons of pyramidal cells in piriform cortex stained by intracellular injection of horseradish peroxidase (HRP) have been analyzed by light and electron microscopy. Myelinated primary axons give rise to extensive, very fine caliber (0.2 micron) unmyelinated collaterals with stereotyped radiating branching patterns. Serial section electron microscopic analysis of the stained portions of the collateral systems (initial 1-2 mm) revealed that they give rise to synaptic contacts on dendritic spines and shafts. These synapses typically contain compact clusters of large, predominantly spherical synaptic vesicles subjacent to asymmetrical contacts with heavy postsynaptic densities. On the basis of comparisons with Golgi material and intracellularly stained dendrites, it was concluded that dendritic spines receiving synapses from the proximal portions of pyramidal cell axon collaterals originate primarily from pyramidal cell basal dendrites. Postsynaptic dendritic shafts contacted closely resemble dendrites of probable GABAergic neurons identified in antibody and [3H]-GABA uptake studies. Electron microscopic examination of pyramidal cell axon initial segments revealed a high density of symmetrical synaptic contacts on their surfaces. Synaptic vesicles in the presynaptic boutons were small and flattened. It is concluded that pyramidal cells synaptically interact over short distances with other pyramidal cells via basal dendrites and with deep nonpyramidal cells that probably include GABAergic cells mediating a feedback inhibition. This contrasts with long associational projections of pyramidal cells that terminate predominantly on apical dendrites of other pyramidal cells.

Maturation of rat visual cortex. III. Postnatal morphogenesis and synaptogenesis of local circuit neurons

The postnatal development of 3 types of local circuit neurons in rat visual cortex was examined in Golgi and electron microscopic preparations. During the first postnatal week, smooth and sparsely spinous stellate, bitufted and bipolar neurons were identified in Golgi material by their characteristic dendritic arborizations. Morphological differentiation begins during this week, as each neuron sprouts dendrites which extend, branch and produce spines, and ends by day 21. This differentiation was traced by quantifying the somatic area and number of primary dendrites on stellate, bitufted and bipolar neurons in layer II/III or layer V. Neurons in deep cortex differentiate earlier than those in superficial laminae. On day 3, axons are evident as short, straight processes, however, by day 6, many axons have branches and varicosities. The increase in the complexity of the axonal trees continues during the second and third postnatal weeks. Since the axons of stellate and bitufted neurons form synapses with the somata of pyramidal neurons, an index of the synaptogenesis of these neurons was traced by counting the numbers of synapses on the somata of pyramidal neurons. The mean number of axosomatic synapses increases steadily from day 3 to day 30. Layer V pyramidal neurons form axosomatic synapses before pyramidal neurons in layer II/III. In conclusion, the morphology of local circuit neurons develops during the period after they migrate into cortex. The principle that cortical local circuit neurons develop after projection neurons only applies for the synaptogenesis of the axon, but not for the maturation of the cell body and dendrites.

Maturation of rat visual cortex. III. Postnatal morphogenesis and synaptogenesis of local circuit neurons

The postnatal development of 3 types of local circuit neurons in rat visual cortex was examined in Golgi and electron microscopic preparations. During the first postnatal week, smooth and sparsely spinous stellate, bitufted and bipolar neurons were identified in Golgi material by their characteristic dendritic arborizations. Morphological differentiation begins during this week, as each neuron sprouts dendrites which extend, branch and produce spines, and ends by day 21. This differentiation was traced by quantifying the somatic area and number of primary dendrites on stellate, bitufted and bipolar neurons in layer II/III or layer V. Neurons in deep cortex differentiate earlier than those in superficial laminae. On day 3, axons are evident as short, straight processes, however, by day 6, many axons have branches and varicosities. The increase in the complexity of the axonal trees continues during the second and third postnatal weeks. Since the axons of stellate and bitufted neurons form synapses with the somata of pyramidal neurons, an index of the synaptogenesis of these neurons was traced by counting the numbers of synapses on the somata of pyramidal neurons. The mean number of axosomatic synapses increases steadily from day 3 to day 30. Layer V pyramidal neurons form axosomatic synapses before pyramidal neurons in layer II/III. In conclusion, the morphology of local circuit neurons develops during the period after they migrate into cortex. The principle that cortical local circuit neurons develop after projection neurons only applies for the synaptogenesis of the axon, but not for the maturation of the cell body and dendrites.

Intrinsic connections of macaque striate cortex: afferent and efferent connections of lamina 4C

We have studied the intrinsic organization of macaque striate cortex by tracing the pattern of horseradish peroxidase (HRP)-labeled axons and cell bodies produced by microinjections of HRP into single cortical laminae. Both anterograde and retrograde transport results were used to examine: (1) the pattern of projections from lamina 4C to the superficial layers; (2) the projection from lamina 4C to deeper cortical layers; and (3) the projections to lamina 4C from other cortical laminae. Laminae 4C alpha and 4C beta differ in their pattern of projections to the superficial layers of striate cortex. Axons from neurons in lamina 4C beta ascend through lamina 4B without giving off collaterals and terminate in lamina 4A and in the base of lamina 3. By contrast, axons from neurons in lamina 4C alpha terminate in lamina 4B and less densely in the 4A/3B region. The projection from lamina 4C beta to lamina 4A is particularly dense and is distributed in a patchy fashion immediately above each injection site. The projection from lamina 4C beta to lamina 3B appears less dense and more widespread; we estimate that individual 4C beta axons may spread laterally for more than 400 micron. Furthermore, the pattern of HRP-labeled cell bodies in lamina 4C beta following injections into laminae 4A and 3B provides evidence for a subdivision within 4C beta. These injections always produce a large number of labeled neurons in the upper part of lamina 4C beta, whereas the lower portion contains few labeled neurons that are located immediately under the center of the injection site. Both lamina 4C alpha and lamina 4C beta also contribute less dense projections to the deeper layers of cortex. Lamina 4C beta projects mainly to lamina 6, whereas lamina 4C alpha contributes axon terminals to both lamina 5A and lamina 6. Neurons in lamina 6 provide the bulk of the intracortical projections to lamina 4C. The axons of these neurons are fine in caliber and have a delicate side-spine morphology that is quite distinct from lateral geniculate axon arbors. Neurons in lamina 5A also project onto lamina 4C, but the projections of these neurons appear concentrated in lamina 4C alpha. These results confirm or refine many conclusions about intrinsic connections of striate cortex drawn from Golgi material and suggest new patterns of connections not suspected from previous work.

A Golgi study of the periaqueductal gray matter in the cat. Neuronal types and their distribution.

In Golgi material, the neurons of the periaqueductal gray matter (PAG) of the cat have been classified into five types, according to the following criteria: number of dendrites per cell, characteristics of secondary arborization, frequency of spines and axon caliber. Type 1 cells, which are multipolar and rich in spines are the most frequent, and are probably intranuclear neurons. Type 4 cells have a short axon which ends in the PAG, but they differ from Type 1 in that their dendritic ramification is of a different type and there are few spines. Type 2 and 3 neurons have a thick axon which runs outside the PAG, and dendrites rich in spines. Type 2 cells have more primary dendrites, while Type 3 neurons have dendrites which may spread outside the PAG. Type 5 cells have dendrites with few spines and no secondary ramification. Their thick and long axon projects outside the PAG. Type 2, 3 and 5 cells have been considered projective neurons. The various neuron types are present in every area of the PAG, although in the ventral region there is a predominance of Type 2 and 5 neurons, in the dorsal regions of Type 2 and 3 cells, and in lateral regions of Type 3 and 5 cells. Local intrinsic circuits have been observed in which both the interneurons and the projective, with early axonic collaterals, are involved. The prevalence of neurons to which an afferent role has been attributed (Type 2 and 3 cells) compared with efferent cells (Type 5), is in agreement with hodological studies which indicate that the PAG receives multiple and numerous afferents in comparison with the relatively scarce efferent fibers. These projections can be intensely and deeply elaborated and modulated by means of local intrinsic circuits.

Histoautoradiographic and electron microscopic studies on short-term explant cultures of the glaucomatous trabecular meshwork.

Explant cultures of 15 trabeculectomy specimens derived from glaucomatous eyes were studied, using electron microscopy and histoautoradiography. The morphology of the trabecular meshwork was monitored for 3-14 days after explanation. In 4 cases the trabeculectomy specimens were cut in 2-3 pieces each and then incubated with 14C-glucosamine at different times during the observation period (2-10 days). The cells of the cribriform layer and the outer corneoscleral trabeculae become "activated" as soon as 3-4 days after explantation, developing a large amount of endoplasmic reticulum, Golgi material, and numerous mitochondria. Histoautoradiographically, intense labeling of the trabecular meshwork, especially the cribriform layer, was found after incubation with 14C-glucosamine. We assume that under tissue culture conditions the "activated" cells of the glaucomatous meshwork produce various types of glycosaminoglycans (GAGs) but, based on the experiments alone, no final conclusions can be made in this respect.

Labelling of cerebellar mossy fiber afferents with intra-axonal horseradish peroxidase

Several unidentified units recorded in the intermediate zone of lobules IV and V of the cerebellar cortex responded to superficial radial nerve stimulation with a distinctive short train of 5 to 11 low amplitude spikes (less than 5 mV) at a frequency of 800 to 1200 Hz. Responses of this type have in the past been attributed to mossy fiber afferents. Following the electrophysiological characterization horseradish peroxidase was successfully injected by iontophoresis into four axon terminals. The labelled axons presented the typical structure of mossy fibers with terminal rosettes and corresponded to the morphology of these axons described in Golgi material.

Variability in the terminations of GABAergic chandelier cell axons on initial segments of pyramidal cell axons in the monkey sensory-motor cortex.

Chandelier cell axons were studied in the sensory-motor cortex of adult monkeys. The axonal fields of Golgi-impregnated chandelier cells in layer II in motor cortex are flattened sagittally. The vertical terminal portions of the axons varied both in length and in the numbers converging to form terminations of greater or lesser complexity. Golgi-impregnated plexuses were embedded in plastic and resectioned serially at 2.5-3.0 micrograms. A single axonal field could have as many as 400 terminal rows. All lie 3-13 micrograms beneath pyramidal cell somata. These terminations are not randomly distributed but instead, form clusters. Further resectioning the plastic sections for electron microscopy revealed that all the terminations are on the initial axon segments of pyramidal cells and all form symmetric synaptic contacts. In immunocytochemical material stained for glutamic acid decarboxylase (GAD), the enzyme involved in the synthesis of GABA, GAD-positive boutons were found to form symmetric synaptic contacts with a variety of postsynaptic elements including the axon hillocks and axon initial segments of pyramidal cells. Serial reconstructions from electron micrographs revealed GAD-positive terminals synapsing with the axon initial segment of pyramidal cells joined by cytoplasmic bridges and forming vertically oriented rows identical to those of chandelier cell terminals identified positively in the resectioned Golgi material. The GAD-positive terminals forming initial segment synapses were never continuous with GAD-positive terminals forming axo hillock synapses. The latter probably arise from basket cell axons. Initial segments of pyramidal cell axons in layers II and III were contacted by more GAD-positive terminals than the initial segments of pyramidal cell axons in layer V. The largest pyramidal cells in layer III received the most synapses. Many larger pyramidal cells, identified as callosally projecting cells by the retrograde transport of horseradish peroxidase (HRP), were shown in serial electron micrographs to possess large numbers of initial segment synapses, comparable to those seen in the immunocytochemical material. Serial reconstructions of pyramidal cell axons from axon hillock to the first myelin internode in resectioned Golgi, immunocytochemical and HRP material showed that the number of synapses varied from 2 to 52 for layers II and III and from 2 to 26 for layer V. The number of synapses on the axon hillocks varied from zero to 12. The variability in these terminations may be an important factor in the shaping of the functional properties of the pyramidal cells.

The Dorsomedial Portion of Trigeminal Nucleus Oralis (Vo) in the Rat: Cytology and Projections to the Cerebellum

Electrophysiological studies have described four major tactile areas in the rat cerebellar cortex. These areas are in crus I, crus II, the paramedian lobule (PML), and the uvula, and a major portion of each is related to the ipsilateral orofacial region. This study demonstrates that neurons in trigeminal nucleus oralis (Vo) that project to the orofacial portions of these four major tactile areas are localized in the dorsomedial (DM) subdivision of the nucleus. The distribution, light-microscopic morphology, and relative densities of trigeminocerebellar neurons within DM, retrogradely labeled with horseradish peroxidase (HRP) following injections into each of the four major tactile areas, were analyzed and compared as well as correlated with the myelo- and cytoarchitecture of DM observed in Nissl sections, 1-micron sections, and Golgi material. On the basis of myelo- and cytoarchitectonic as well as trigeminocerebellar connectional criteria, three portions of DM were identified: caudal DM (CDM), middle DM (MDM), and rostral DM (RDM). The greatest portion of DM is made up of MDM (1.3 mm long), which can be further subdivided into dorsal (MDMd) and ventral (MDMv) zones. CDM forms the caudal 800 microns of DM, while RDM makes up the rostral 280 microns of the subdivision. Longitudinally running deep axon bundles permeate CDM, MDMv, and RDM, but are conspicuously absent from MDMd. The majority of neurons found throughout CDM, MDMv, and RDM have medium-sized (15- to 30-microns) somata and can be divided into two types on the basis of their somatodendritic morphology. CDM, MDMv, and RDM also contain a small neuronal cell type (5- to 15-microns cell body) that is encountered less frequently than either one of the two types of medium-sized cells. A fourth type of neuron with a large (25- to 50-microns) fusiform- to pyramidal-shaped cell body is the least frequently observed neuronal cell type and is located principally in CDM and MDMv. MDMd contains a fifth type of neuron characterized by a small (5- to 15-microns) oval soma. Data from the retrograde HRP experiments show that all five of these neuronal cell types in their respective portions of DM project to one or more of the orofacial portions of the four major tactile areas of the cerebellar cortex. Many medium-sized neurons of both types in CDM, MDMv, and RDM project to crus I, crus II, and/or PML.(ABSTRACT TRUNCATED AT 250 WORDS)