Golgi Material Research Articles

The Toxoplasma gondii dense granule protein TgGRA3 interacts with host Golgi and dysregulates anterograde transport.

ABSTRACTAfter entry into the host cell, the intracellular parasite Toxoplasma gondii resides within a membrane-bound compartment, the parasitophorous vacuole (PV). The PV defines an intracellular, parasite-specific niche surrounded by host organelles, including the Golgi apparatus. The mechanism by which T. gondii hijacks the host Golgi and subverts its functions remains unknown. Here, we present evidence that the dense granule protein TgGRA3 interacts with host Golgi, leading to the formation of tubules and the entry of host Golgi material into the PV. Targeted disruption of the TgGRA3 gene delays this engulfment of host Golgi. We also demonstrate that TgGRA3 oligomerizes and binds directly to host Golgi membranes. In addition, we show that TgGRA3 dysregulates anterograde transport in the host cell, thereby revealing one of the mechanisms employed by T. gondii to recruit host organelles and divert their functions. This article has an associated First Person interview with the first author of the paper.

Self-organization of core Golgi material is independent of COPII-mediated endoplasmic reticulum export.

The Golgi is a highly organized and dynamic organelle that receives and distributes material from and to the endoplasmic reticulum (ER) and the endocytic pathway. One open question about Golgi organization is whether it is solely based on ER-to-Golgi transport. Here, we analyzed the kinetics of Golgi breakdown in the absence of COPII-dependent ER export with high temporal and spatial resolution using quantitative fluorescence microscopy. We found that Golgi breakdown occurred in two phases. While Golgi enzymes continuously redistributed to the ER, we consistently observed extensive Golgi fragmentation at the beginning of the breakdown, followed by microtubule-dependent formation of a Golgi remnant structure (phase 1). Further Golgi disintegration occurred less uniformly (phase 2). Remarkably, cisternal Golgi morphology was lost early in phase 1 and Golgi fragments instead corresponded to variably sized vesicle clusters. These breakdown intermediates were devoid of COPI-dependent recycling material, but contained typical 'core' Golgi components. Furthermore, Golgi breakdown intermediates were able to disassemble and reassemble following cell division, indicating that they retained important regulatory capabilities. Taken together, these findings support the view that Golgi self-organization exists independently of ER-to-Golgi transport.

Long-term evaluation of cytoarchitectonic characteristics of prefrontal cortex pyramidal neurons, following global cerebral ischemia and neuroprotective melatonin treatment, in rats

Global cerebral ischemia induces alterations of working memory, as evidenced in the eight-arm radial maze, in the absence of significant changes of pyramidal neuron population in the prefrontal cortex. These alterations can be prevented by a neuroprotective melatonin treatment. Thus, the cytoarchitectonic characteristics of the pyramidal neurons located at layers III and V in the prefrontal cortex of rats that had been submitted 120 days earlier to acute global cerebral ischemia (15min four-vessel occlusion), and melatonin (10mg/(kgh) for 6h, i.v.) or vehicle administration, starting 30min after the end of cerebral blood flow interruption, were evaluated in order to gain information on the changes of the neural substrate underlying disruption of prefrontocortical functioning. Soma size, rough length and number of bifurcations of basilar and apical dendrites, as well as spine density and proportions of the different types of spines in a 50μm length segment of a secondary dendrite branching from the apical and the basilar dendrites, of pyramidal neurons of the dorsal medial prefrontal cortex, were evaluated in Golgi material. A significant reduction of soma size, apical and basilar dendrite length, number of dendritic bifurcations, and spine density were observed in pyramidal neurons at layers III and V after cerebral ischemia, while these alterations were prevented by melatonin treatment. These cytoarchitectural differences between groups seem to underlie the observed alterations in spatial working memory of ischemic, vehicle-treated rats in the absence of pyramidal neuron loss, as well as the better display of these functions long after ischemia and melatonin neuroprotection.

STOP-like Protein 21 Is a Novel Member of the STOP Family, Revealing a Golgi Localization of STOP Proteins

Neuronal microtubules are stabilized by two calmodulin-regulated microtubule-associated proteins, E-STOP and N-STOP, which when suppressed in mice induce severe synaptic and behavioral deficits. Here we show that mature neurons also contain a 21-kDa STOP-like protein, SL21, which shares calmodulin-binding and microtubule-stabilizing homology domains with STOP proteins. Accordingly, in different biochemical or cellular assays, SL21 has calmodulin binding and microtubule stabilizing activity. However, in cultured hippocampal neurons, SL21 antibodies principally stain the somatic Golgi and punctate Golgi material in neurites. In cycling cells, transfected SL21 decorates microtubules when expressed at high levels but is otherwise principally visible at the Golgi. The Golgi targeting of SL21 depends on the presence of cysteine residues located within the SL21 N-terminal domain, suggesting that Golgi targeting may require SL21 palmitoylation. Accordingly we find that SL21 is palmitoylated in vivo. N-STOP and E-STOP, which contain the Golgi targeting sequences present in SL21, also display distinct Golgi staining when expressed at low level in cycling cells. Thus neuronal proteins of the STOP family have the capacity to associate with Golgi material, which could be important for STOP synaptic functions.

Spine-type densities of hippocampal CA1 neurons vary in proestrus and estrus rats

Spines-mediated synaptic activity has been associated to learning ability. Dendritic spines from hippocampal CA1 pyramidal neurons of proestrus rats have been reported to be more numerous than in estrus animals, but some behavioral studies have reported a better performance during the estrus stage of the estrous cycle. Because spine shape has been shown to be strongly related to the post-synaptic processing of information, a quantitative morphological study related to the proportional density of each type of spine, was conducted in Golgi material of hippocampal CA1 pyramidal cells of proestrus and estrus rats. After three regular estrous cycles had been asserted, seven Sprague–Dawley female adult rats in proestrus and eight in estrus, were used. Mushroom-shaped spines from hippocampal pyramidal cells predominated in proestrous rats, being 15.1% more numerous in this stage than in estrus; while thin spines were the predominant type of spine in estrous animals, being 15.5% more abundant in estrus than in proestrus. The predominance of the mushroom-shaped or thin spines in the hippocampal CA1 pyramidal neurons during the respective stages of the rat estrous cycle, could be related to the organization of the hippocampal activity-dependent mnemonic information.

The shapes and numbers of amacrine cells: Matching of photofilled with Golgi-stained cells in the rabbit retina and comparison with other mammalian species

Amacrine cells of the rabbit retina were studied by "photofilling" a photochemical method in which a fluorescent product is created within an individual cell by focal irradiation of the nucleus; and by Golgi impregnation. The photofilling method is quantitative, allowing an estimate of the frequency of the cells. The Golgi method shows their morphology in better detail. The photofilled sample consisted of 261 cells that were imaged digitally in through-focus series from a previous study (MacNeil and Masland [1998] Neuron 20:971-982). The Golgi material consisted of 49 retinas that were stained as wholemounts. Eleven of these subsequently were cut in vertical section. Of the many hundreds of cells stained, digital through-focus series were recorded for 208 of the Golgi-impregnated cells. The two methods were found to confirm one another: Most cells revealed by photofilling were recognized easily by Golgi staining, and vice versa. The greater resolution of the Golgi method allowed a more precise description of the cells and several types of amacrine cell were redefined. Two new types were identified. The two methods, taken together, provide an essentially complete accounting of the populations of amacrine cells present in the rabbit retina. Many of them correspond to amacrine cells that have been described in other mammalian species, and these homologies are reviewed.

Cytoarchitecture of the tectum opticum in the Japanese quail

The cytoarchitecture of the optic tectum of the Japanese quail, Coturnix coturnix japonica, was studied using the Golgi-Kopsch method, parvalbumin, calbindin and GABA immunohistochemistry and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry. Our results reveal a large number of different types of interneurons in the quail tectum opticum, only part of which are described in the chick or pigeon. Application of parvalbumin and calbindin immunohistochemistry and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry reveals the following lamination pattern: The stratum opticum, stratum griseum centrale and stratum album centrale remain unstained, while the laminae of the stratum griseum et fibrosum superficiale exhibit a roughly complementary staining pattern of calbindin (laminae c, d, e, f, g, i) and parvalbumin (laminae a, h, i). Nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry yields a dense band in lamina i. The Golgi material reveals the following cell types in the stratum griseum et fibrosum superficiale: marginal cells in the stratum opticum and in lamina h and i, horizontal cells in laminae a and c, large and small radial cells in laminae b, d, h and i, multiform cells in lamina b, bitufted cells in lamina d and e, large pear-shaped cells in lamina g, wide-field cells in lamina j, and stellate cells in lamina j and in the stratum griseum centrale. We consider horizontal cells, bitufted cells, multiform cells and small radial cells to be GABAergic interneurons of the stratum griseum et fibrosum superficiale which seem to be more numerous than in the pigeon tectum opticum. Golgi impregnation and injection of Phaseolus vulgaris leucoagglutinin into the pretectal nucleus lentiformis yielded regularly distributed clusters of telodendra of pretectal axons in lamina d of the stratum griseum et fibrosum superficiale, which are identical in shape and position with axon plexus revealed by Golgi staining.

1104 Differential expressions of calcium-binding proteins in the superior colliculi of developing rats

The cytoarchitecture of the optic tectum of the Japanese quail, Coturnix coturnix japonica, was studied using the Golgi-Kopsch method, parvalbumin, calbindin and GABA immunohistochemistry and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry. Our results reveal a large number of different types of interneurons in the quail tectum opticum, only part of which are described in the chick or pigeon. Application of parvalbumin and calbindin immunohistochemistry and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry reveals the following lamination pattern: The stratum opticum, stratum griseum centrale and stratum album centrale remain unstained, while the laminae of the stratum griseum et fibrosum superficiale exhibit a roughly complementary staining pattern of calbindin (laminae c, d, e, f, g, i) and parvalbumin (laminae a, h, i). Nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry yields a dense band in lamina i. The Golgi material reveals the following cell types in the stratum griseum et fibrosum superficiale: marginal cells in the stratum opticum and in lamina h and i, horizontal cells in laminae a and c, large and small radial cells in laminae b, d, h and i, multiform cells in lamina b, bitufted cells in lamina d and e, large pear-shaped cells in lamina g, wide-field cells in lamina j, and stellate cells in lamina j and in the stratum griseum centrale.We consider horizontal cells, bitufted cells, multiform cells and small radial cells to be GABAergic interneurons of the stratum griseum et fibrosum superficiale which seem to be more numerous than in the pigeon tectum opticum. Golgi impregnation and injection of Phaseolus vulgaris leucoagglutinin into the pretectal nucleus lentiformis yielded regularly distributed clusters of telodendra of pretectal axons in lamina d of the stratum griseum et fibrosum superficiale, which are identical in shape and position with axon plexus revealed by Golgi staining.

Cytoarchitecture of the medial geniculate body in the mustached bat (Pteronotus parnellii)

The cytoarchitectonic organization of the medial geniculate body and adjoining thalamic nuclei was analyzed in the mustached bat (Pteronotus parnellii). These subdivisions provide a reference for structural, physiological, connectional, and neurochemical work. Most nuclei recognized in other mammals exist in the mustached bat, although the relative volume of the three divisions was species specific. The ventral division contains medium-sized neurons and a few smaller cells and is well developed. Neurons in the lateral part lie in regularly aligned rows corresponding to the laminae in Golgi material; in the medial part, these laminae are obscured by fibers. The dorsal division has at least four nuclei, each with a unique cytoarchitecture and myeloarchitectonic organization. The suprageniculate nucleus is prominent and has many large radiate neurons. Cells in the superficial dorsal nucleus have weekly laminated dendrites, while dorsal nucleus neurons have spherical dendritic fields. There is a wide range of neuropil patterns within the dorsal division. The suprageniculate nucleus has thick myelinated axons, while the fibers in the superficial and dorsal nuclei are much thinner. The rostral pole nucleus becomes prominent in the anterior one-half of the auditory thalamus; its architectonic affiliation is equivocal, and connectional and immunocytochemical studies suggest that it may belong to the dorsal division. The medial division is one nucleus with many types of neurons, and it has coarse axons without laminar orientation. It is the smallest of the divisions and is present throughout the medial geniculate complex, except at the caudal tip and at the rostral pole. Many features of medial geniculate body organization evident in other mammals are recognized in the mustached bat. These include a prominent ventral division, some of whose neurons have a laminar organization, and a comparatively small medial division that is devoid of fibrodendritic laminae. Other features, such as the presence of a large rostral pole nucleus, whose homologue in other species is uncertain, or the sparse number or small cells that may participate in local circuits, set it apart from carnivores and primates and suggest that there are species specific patterns of medial geniculate body organization.

The neurons of the medial geniculate body in the mustached bat (Pteronotus parnellii)

The neurons in the medial geniculate body were studied in Golgi preparations from adult mustached bats (Pteronotus parnellii). Their somatic and dendritic configurations were compared with those of cells in other, nonecholocating mammals. A second goal was to use the thalamic nuclear subdivisions derived from Golgi material to integrate the findings in parallel studies of cytoarchitecture, immunocytochemistry, and tectothalamic connections. Three primary divisions are defined. The ventral division is large and has a stereotyped neuronal organization. Medium-sized perikarya (about 10 microns in diameter) represent tufted neurons; the fibrodendritic plexus forms laminae in the lateral part along which midbrain axons terminate. A smaller, possibly intrinsic, neuron with thin, sparse dendrites is rarely impregnated. Neurons in the larger, medial part, which represents frequencies of 60 kHz and higher, have more spherical dendritic fields; their branching pattern remains tufted, and the laminar organization was less evident. The dorsal division is about equal in size, and it has many nuclei and a corresponding neuronal diversity. These neurons are medium-sized except in the suprageniculate nucleus, where many cells are larger. Four dorsal division nuclei are recognized. Each has neurons with radiate or weakly tufted dendritic arbors. Superficial dorsal nucleus neurons are oriented from medial to lateral, imparting a slightly laminated appearance to the neuropil. A few smaller, stellate neurons with modest dendritic domains are present. Suprageniculate nucleus neurons have radiating dendritic fields that project spherically; they have fewer branches than dorsal nucleus neurons. The posterior limitans nucleus is dorsomedial to the suprageniculate nucleus; it has small neurons with long, sparsely branched dendrites. The rostral pole nucleus, included in the dorsal division on cytoarchitectonic grounds, had too few neurons impregnated to reveal its neuronal architecture. The medial division, the smallest of the main parts, is one nucleus with at least six types of cells, including the magnocellular, bushy tufted, disc-shaped, medium-sized multipolar, elongated, and small stellate neurons. There is no laminar arrangement. Many of the neurons resemble those in rodent, marsupial, carnivore, and primate auditory thalamic nuclei. Despite such morphological correspondences, functional differences, such as the evolution of combination sensitivity, suggest that structurally comparable auditory thalamic neurons may subserve diverse physiological representations.

Ultrastructural changes in the efferent duct and epididymis of men with obstructive infertility

Ultrastructural changes in the efferent duct and in different regions of the epididymis in men with obstructive azoospermia were compared with corresponding tissues collected from men of proven fertility who underwent castration due to malignancy of the prostate. Major degenerative changes were seen in the efferent duct and the caput epididymidis of men with obstruction at the caput epididymidis which may have been induced by fluid pressure due to defective absorption of testicular fluid in the caput epididymidis. These degenerative changes included decrease in tubular and lumen diameter of the caput and the cauda epididymides, decrease in height of the stereocilia, reduction in rough endoplasmic reticulum and Golgi material, and presence of lipofuscin and osmiophilic dense bodies. The degenerative changes were less when the site of obstruction was in the cauda epididymidis since fluid reabsorption would continue to take place normally in the caput epididymidis. In men who had undergone vasoepididymostomy (VEA), the ejaculated spermatozoa showed a high percentage of morphological abnormalities which may have occurred due to adverse effects of long-term obstruction on spermatogenesis.

Accessory Oculomotor Nuclei of Man

A morphoquantitative study was carried out to clarify the cytoarchitecture of the human nuclear complex of the posterior commissure (NPC), a collection of cell groups which is thought to be involved in the pupillary light reflex and eye movements. On the basis of the topographical features, 5 different parts (rostral, principal, magnocellular, subcommissural and infracommissural) were identified in NPC. In these 5 parts, the neuronal population consisted of small and medium-sized neurons with a structural pattern similar to that of the central reticular formation. The only exception was the magnocellular part in which the neurons, mainly medium-sized and large, did not show the reticular-like features. In the Golgi material, two types of NPC neurons were identified: multipolar and fusiform cells. In the multipolar cells, 2-3 dendrites emerged from the soma and gave off a wide dendritic arborization. Many of the dendrites and axons of the multipolar cells were seen spreading outside NPC. The fusiform cells had 2 dendrites emerging from the opposite poles of their elongated perikaryon. Dendrites and axons of the fusiform cells always lay inside NPC. These observations suggest that the multipolar cells are mainly projective neurons and that the fusiform cells are neurons which mainly perform a local integrative function. Since the present study concludes our investigation of the human accessory oculomotor nuclei, a comparison of their structure was carried out. All the nuclei showed a common reticular-like structure, although each nucleus had its own structural particularities. These results are in agreement with and give an anatomical support to the functional findings.

Electron microscopy of in vivo recorded and intracellularly injected inferior olivary neurons and their GABAergic innervation in the cat

This paper reports on the detailed morphology of inferior olivary neurons in the cat following electrophysiological examination, intracellular injection with horseradish peroxidase, and gamma aminobutyric acid (GABA) immunocytochemistry. The activity of olivary cells was recorded intracellularly in vivo and their response to mesodiencephalic stimulation was tested. In a number of cases their response to stimulation of the contralateral superior cerebellar peduncle was also tested. Mesodiencephalic stimulation resulted in monosynaptic, and superior peduncle stimulation in disynaptic activation of cells in the medial accessory and principal olivary subdivisions. Rebound olivary activity was usually only found after mesodiencephalic stimulation. Light microscopic investigation of osmicated and Araldite embedded Vibratome sections was facilitated considerably when performing the osmication in a glucose solution. Peroxidase labeled olivary cells, like that earlier described for Golgi-impregnated material, possess a complex globular dendritic geometry. Especially, and unlike Golgi material, the abundance of exceptionally long and complex spiny appendages could be appreciated. The axons usually stemmed from first order dendrites and did not give rise to recurrent axon collaterals. The ultrastructural analysis of this material, mainly from serial sections, was combined with postembedding GABA immunohistochemistry. In this way, GABAergic as well as non-GABAergic profiles were studied in conjunction with HRP labeled cellular elements. The GABAergic terminals usually contained pleomorphic vesicles and made symmetrical synapses whereas non-GABAergic terminals nearly always formed asymmetrical synapses and contained round or oval vesicles. Most, if not all, HRP labeled spiny appendages were incorporated in glomeruli. A particular spiny appendage may contribute more than one spine head to a glomerular core, which, on average, consisted of spiny elements of six different neurons. A glomerular core is surrounded by approximately the same amounts of GABAergic and non-GABAergic boutons. Also, all spiny appendages, and most of their individual spine heads, are contacted by GABAergic as well as non-GABAergic boutons. Spiny appendages on the axon hillock may be incorporated in dendritic glomeruli, however, most synapses with the hillock were made by GABAergic boutons. The combined physiological and morphological observations imply that 1) the cerebellar nuclei can exert an excitatory influence on inferior olivary neurons through a mesodiencephalic relay, 2) the GABAergic nucleo-olivary input seems to be capable of diminishing the oscillatory tendencies of olivary neurons, and 3) the mesodiencephalic (non-GABAergic) and cerebellar (GABAergic) input may subserve a timing function since these inputs systematically impinge upon the same olivary spines.

Laminar distribution and morphology of NADPH-diaphorase containing neurons in the superior colliculus and underlying periaqueductal gray of the rat

We have studied the laminar distribution of reduced nicotinamide dinucleotide phosphate diaphorase (NADPH-d) activity and the morphology of positive neurons in the superior colliculus (SC) and the underlying periaqueductal gray (PAG) of the rat. The morphology of NADPH-d-positive neurons has been compared to that of Golgi-impregnated cells. The highest activity occurs in the stratum zonale and stratum griseum superficiale, contrasting with the pale neuropil in the stratum opticum, where only a few positive neurons are found. In the stratum griseum intermedium positive neurons are grouped in patches separated by narrow, NADPH-d-negative bands. In the deeper layers, the neuropil is NADPH-d-negative, and few neurons show enzymatic activity. In contrast, numerous neurons in the dorsolateral part of the PAG are intensely positive. They are continuous with the positive neurons in the stratum album profundum, with no clear border between the two centers. In both SC and PAG, only small and medium sized neurons are NADPH-d-positive. In comparison with Golgi material, all types of small neurons in the superficial layers show NADPH-d activity; NADPH-d histochemistry, however, does not visualize the characteristic dendritic appendages of these neurons. The large neurons of the SC and PAG, probably representing the long-projecting neurons of these centers, do not contain the enzyme.

Morphological taxonomy of the neurons of the primate striatum

A quantitative taxonomy of primate striatal neurons was elaborated on the basis of the morphology of Golgi-impregnated neurons. Dendritic arborizations were reconstructed from serial sections and digitized in three dimensions by means of a video computer system. Topological, metrical, and geometrical parameters were measured for each neuron. Groups of neurons were isolated by using uni- and multidimensional statistical tests. A neuronal species was defined as a group of neurons characterized quantitatively by a series of nonredundant parameters, differing statistically from other groups, and appearing as a separate cluster in principal component analysis. Four neuronal species were isolated: (1) the spiny neuronal species (96% of striatal neurons) characterized by spine-free proximal dendrites (up to 31 microns) and spine-laden distal dendrites, which are more numerous, shorter, and less spiny in the human than in the monkey, (2) the leptodendritic neuronal species (2%) characterized by a small number of long, thick, smooth, and sparsely ramified dendrites, (3) the spidery neuronal species (1%) characterized by very thick dendritic stems and a large number of varicose recurrent distal processes, and (4) the microneuronal species (1%) characterized by numerous short, thin, and beaded axonlike processes. All striatal neurons give off a local axonal arborization. The size and shape of cell bodies were analyzed quantitatively in Golgi material and in materials treated for Nissl-staining, immunohistochemical demonstration of parvalbumin and histochemical demonstration of acetylcholinesterase. Only three types were distinguishable: small, round cell bodies corresponding to either spiny neurons or microneurons, medium-size elongated cell bodies, which were parvalbumin-immunoreactive and corresponded to leptodendritic neurons, and large round cell bodies, which were acetylcholinesterase-positive and corresponded to spidery neurons. Thorough analysis of previously elaborated classifications revealed that spidery neurons do not exist in rats and cats and that large cholinergic neurons in these species correspond to leptodendritic neurons. From this, it can be assumed that the dendritic domain of striatal cholinergic neurons is considerably smaller in primates than in other species. Computer simulations based on both the frequency of each neuronal species and their three-dimensional dendritic morphology revealed that the striatum consists of two intertwined dendritic lattices: a fine-grain lattice (300-600 microns) formed by the dendritic arborizations of spiny, spidery, and microneurons, and a large-grain lattice (1,200 microns) formed by the dendritic arborizations of leptodendritic neurons. This suggests that cortical information can be processed in the striatum through two different systems: a fine-grain system that would conserve the precision of the cortical input, and a large-grain system that would blur it.

Effect of perinatal administration of ethanol on the CA1 pyramidal cell of the hippocampus and Purkinje cell of the cerebellum: an ultrastructural survey

Previous studies of Golgi-impregnated material demonstrated the vulnerability of the dendritic arbors of the CA1 pyramidal cells of the hippocampus and the Purkinje and granule cells of the cerebellum to perinatal exposure to ethanol. The purpose of this investigation was to determine whether the alterations seen in the Golgi material could be corroborated at the ultrastructural level, and to qualitatively survey the neuropil surrounding the affected neurons. Pregnant mice were maintained on either a chocolate-flavoured liquid diet containing 25% of its caloric value as ethanol (5.4% v/v) or a chow and water diet. The treatment period began on gestational day 12 and continued until postnatal day 7, at which time the ethanol-exposed animals were returned to a chow and water diet. Pups were killed on postnatal day 14, and the tissue was processed for ultrastructural and Golgi analysis. The results confirm our Golgi findings that ethanol severely compromises the dendritic arbor, and also indicate that ethanol exposure produces changes in the organization of the neurophil, as well as alterations in the organization of the perikaryal organelles, and glial swelling.

Immunoprobe localization in mammalian embryos

Visualization of preimplantation mammalian embryos by electron microscopy is difficult due to the large size of the ircells, their relative lack of internal structure, and their highly hydrated cytoplasm. For example, the fertilized egg of the mouse is a single cell of approximately 75μ in diameter with little organized cytoskelet on and apaucity ofor ganelles such as endoplasmic reticulum (ER) and Golgi material. Thus, techniques that work well on tissues or cell lines are often not adaptable to embryos at either the LM or EM level.Over several years we have perfected techniques for visualization of mammalian embryos by LM and TEM, SEM and for the pre-embedding localization of antigens. Post-embedding antigenlocalization in thin sections of mouse oocytes and embryos has presented a more difficult challenge and has been explored in LR White, LR Gold, soft EPON (after etching of sections), and Lowicryl K4M. To date, antigen localization has only been achieved in Lowicryl-embedded material, although even with polymerization at-40°C, the small ER vesicles characteristic of embryos are unrecognizable.

Heterogeneity of chandelier neurons in monkey neocortex: Corticotropin‐releasing factor‐and parvalbumin‐immunoreactive populations

Chandelier neurons are a unique subclass of cortical nonpyramidal neurons. The axons of these neurons terminate in distinctive vertically arrayed cartridges that synapse on the axon initial segment of pyramidal neurons. In this study, the rapid Golgi method and immunohistochemical techniques were used to characterize the morphology, regional distribution, laminar location, and biochemical content of chandelier neurons in the prefrontal and occipital cortices of three monkey species. As in our previous studies of visual areas V1 and V2 (Lund: Journal of Comparative Neurology 257:60-92, 1987; Lund et al.: Journal of Comparative Neurology 202:19-45, 1981, 276:1-29, 1988), Golgi impregnations of areas 46 and 9 of macaque prefrontal cortex show chandelier neurons to be present in layers 2 through superficial 5. The vertical arrays of terminal boutons (axon cartridges) typical of this neuron class are also present in layers 2-6 of the prefrontal cortex, but are not found in layer 1 or the subcortical white matter. In immunohistochemical studies, a calcium-binding protein, parvalbumin, and a neuropeptide, corticotropin-releasing factor (CRF), identify rod-like structures that are morphologically similar to the axon cartridges of chandelier neurons seen in the Golgi material. In addition, both parvalbumin- and CRF-immunoreactive cartridges are located below the somata of unlabeled pyramidal neurons and appear to outline the axon initial segment of these neurons. However, we find that parvalbumin and CRF are present in only subpopulations of chandelier axon cartridges. For example, in adult primary visual cortex, parvalbumin-labeled cartridges are present in very low numbers only in layers 2-3, whereas in prefrontal and occipital association cortices these cartridges are a very prominent component of layers 2-superficial 3 and are present in much lower density in the deeper cortical layers. In contrast to these findings in adult macaque monkeys, prefrontal and occipital association cortices of infant macaque monkeys contain a very high density of parvalbumin-labeled cartridges in layer 4 and relatively few in the superficial cortical layers. Furthermore, in adult squirrel monkey prefrontal cortex, CRF-labeled cartridges are predominately present in layer 4, but these CRF-immunoreactive structures have not been observed in the homologous regions of infant or adult macaque monkeys. These findings indicate that even for neurons of such distinctive morphology and presumably constant functional role as chandelier neurons, factors such as regional and laminar location, age, and primate species are associated with differences in the biochemical content of subpopulations of these neurons.

Accessory Oculomotor Nuclei of Man

A morphoquantitative study was carried out to provide detailed information regarding the cytoarchitecture and neuronal morphology of the nucleus of Darkschewitsch (ND) of man. The neuronal population showed heterogeneity of shape and size of the nerve cell bodies. Small and medium-sized neurons appeared scattered in a wide neuropil. In the Golgi material, two types of neurons were identified: multipolar and fusiform cells. Multipolar cells, which were the most numerous (77%), had 3-5 dendrites giving off primary bifurcations at a short distance from the nerve cell body. Sometimes dendrites and axons were seen to spread outside the ND. The fusiform cells had 1-2 dendrites emerging from the opposite poles of the elongated nerve cell bodies. The dendrites tended to run unbranched for long distances in the section plane before dichotomizing. The dendrites and axons of the fusiform cells always lay inside the ND. The cytoarchitectural features of the ND corresponded to the characters of the reticular formation so that the ND of man could be considered to be a typical reticular nucleus inside the central gray matter. The prevailing presence of multipolar neurons whose processes often spread outside the ND could suggest that the ND is a mainly projective nucleus.

Regional differences in the morphology and enzyme distribution of the spiny dogfish ( Squalus acanthias) ciliary epithelium

The ciliary epithelium of spiny dogfish eyes has previously been used for studies of epithelial electrolyte transport and the results are found to be comparable to those obtained in mammals. In this study we report the ultrastructure and enzyme histochemistry of the ciliary epithelium of spiny dogfish in comparison with that of mammals. In contrast to the mammals, the lens in spiny dogfish is connected to the anterior and middle part of the ciliary epithelium by a broad zonula-like suspensory ligament and by a short ligament to the ventral papilla, while the posterior region is mainly free of zonula-like fibers. The non-pigmented epithelium (NPE) of the posterior region shows numerous membrane infoldings, interdigitations and many mitochondria in the cytoplasm as well as histochemical staining for Na+ K(+)-ATPase and carbonic anhydrase (CA). These findings are similar to those seen in the anterior pars plicata of mammals which is mainly involved in aqueous secretion. In the anterior and middle part, the NPE cells have only a few membrane infoldings and few mitochondria in the cytoplasm, but abundant surfaces of rough endoplasmic reticulum, numerous ribosomes and Golgi material, indicating protein synthesis. Histochemically the cells stain for Na+, K(+)-ATPase but not for CA. These findings are comparable to the pars plana in mammals. In contrast to mammals, where the pigmented epithelium (PE) shows nearly no staining for Na+, K(+)-ATPase but an intensive one for CA, the PE cells of spiny dogfish are heavily stained for Na+, K(+)-ATPase in all regions of the eye, but show nearly no staining for CA.