J. J. L. van der Want

The presynaptic grid: A new approach

A new electron microscopic facility is described which enables systematic visualization of E-PTA-stained presynaptic grids in full en face position. This EM-facility is used to analyze the size and the number of dense projections of synaptic grids in different brain areas of the rabbit. The observations support the view that dense projections form an intrinsic part of all central synapses and are organized in a hexagonal or triangular pattern. The observation of annulate and horseshoe-shaped synaptic grids is in agreement with previous observations on synaptic connections with subsynaptic plate perforations. A non-normal frequency distribution of dense projections per synaptic grid with distinct peaks is suggestive for the existence of distinct size classes of synaptic contacts. Analyses of the frequency distribution of dense projections in different areas and at different levels below the pial surface in adult animals point to a specificity of the distinct size classes related to the afferent origin or the target cell they are projecting on. Investigation of grid size and number of dense projections during a period of rapid synaptogenesis reveals that newly formed synapses also have a specific size. The complementarity of dense projections and vesicle attachment sites implicit in the model of Akert et al. has been used to calculate the number of vesicle attachment sites per sq. micrometer on account of the density of projections per grid. The agreement between our values and those of Akert and Peper, based on the analyses of vesicle attachment sites in freeze-etch specimen, is striking. The possible implications of these observations in relation to synaptic efficacy and neuronal microcicuitry are discussed.

The peripheral and central projections of the Edinger-Westphal nucleus in the rat. A light and electron microscopic tracing study

The peripheral and central efferent projections of the rostral part of the Edinger-Westphal nucleus in the rat were investigated at the light and electron microscopic level by means of iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin and retrograde tracer injections of Fast blue and Nuclear yellow into the facial nucleus and into the principal olive. Two pathways leaving the rostral part of the Edinger-Westphal nucleus were studied, a peripheral and a central descending pathway. Fluorescent experiments demonstrated that the central pathway fibers originated from distinct individual Edinger-Westphal neurons. These neurons were mainly distributed throughout the rostral part of the Edinger-Westphal nucleus and had fusiform cell bodies. The neurons rarely form collateral projections. The central descending pathway left the Edinger-Westphal nucleus medially and terminated bilaterally in the principal olive, in the subnuclei A, B and C of the inferior olive and ipsilaterally in the medial accessory olive. The central pathway also terminated contralaterally in the lateral parabrachial nucleus, the facial nucleus, the trigeminal brainstem nuclear complex, the lateral reticular nucleus and the rostroventral reticular nucleus. The projection to the facial nucleus provides evidence for the existence of a polysynaptic loop forming the central part of the corneal blink reflex. Projections from the Edinger-Westphal nucleus to the cerebellar cortex or the deep nuclei, as described in cat and primate, could not be confirmed. The peripheral pathway left the Edinger-Westphal nucleus ventrally and terminated on dendrites of ciliary ganglion cells, along smooth muscle cells of ciliary ganglion associated arterioles and in the proximity of ciliary ganglion associated venules. The central and peripheral terminals that originate in the Edinger-Westphal nucleus all had similar ultrastructural features: clear, round vesicles and electron dense mitochondria. The terminals originating from the central descending pathway were often found to be arranged in glomerular-like structures. The central and peripheral terminals made asymmetric synaptic membrane specializations (Gray type one), except terminals innervating the ciliary ganglion associated vessels, which showed no synaptic contacts.

Lymphatic uptake and biodistribution of liposomes after subcutaneous injection - IV. Fate of liposomes in regional lymph nodes

The ability of clodronate-containing liposomes to deplete lymph nodes of macrophages was used as a tool to investigate the fate of liposomes in regional lymph nodes after subcutaneous (s.c.) administration. Reduced lymph node localization of liposomes in macrophage-depleted lymph nodes confirmed that phagocytosis by macrophages plays an important role in lymph node retention of liposomes. Depletion of macrophages had less effect on lymph node localization of small liposomes than on the lymph node localization of large liposomes. Inclusion of distearoylphosphatidylethanolamine (DSPE)-poly(ethyleneglycol) (PEG-PE) into the liposomes, which is known to oppose macrophage uptake, did not affect lymph node localization in macrophage-depleted or control lymph nodes. We conclude that PEG-liposomes retained by lymph nodes are also taken up by lymph node macrophages. Morphological observations visualizing the uptake of PEG-liposomes by lymph node macrophages support this conclusion.

Efferent projections of the olivary pretectal nucleus in the albino rat subserving the pupillary light reflex and related reflexes a light microscopic tracing study

The olivary pretectal nucleus is a primary visual centre sensitive to luminance changes. It is involved in the pupillary light reflex, the consensual pupillary light reflex and related reflexes, such as the lid closure reflex whereby pupillary constriction takes place. Since the olivary pretectal nucleus is a small nucleus, previous studies using degeneration, horseradish peroxidase and radioactive amino acid tracing were limited regarding to the exclusiveness of the projections from the olivary pretectal nucleus. In the present study the position of the olivary pretectal nucleus in the rat was first localized by physiological recording of the neurons upon luminance stimulation. Subsequently, an anterograde tracer Phaseolus vulgaris leucoagglutinin was injected iontophoretically. This allows a much more precise localization of the olivary pretectal nucleus projections. Ascending and descending pathways originating from the olivary pretectal nucleus were observed. Ascending fibres project bilaterally to the intergeniculate leaflet, the ventral part of the lateral geniculate nucleus and ipsilaterally to the anterior pretectal nucleus. In addition, contralateral projections were observed to the zona incerta and the fields of Forel. Descending fibres project bilaterally to the periaqueductal gray, the nucleus of Darkschewitsch, the interstitial nucleus of Cajal, the Edinger-Westphal nucleus and the intermediate gray layer of the superior colliculus. Also a contralateral projection to the oculomotor nucleus and an ipsilateral projection to the pontine nucleus and the nucleus of the optic tract were found. Furthermore, the contralateral olivary pretectal nucleus received a small projection. Retrograde tracing experiments using two fluorescent dyes revealed that the fibres projecting to the contralateral olivary pretectal nucleus and to the contralateral interstitial nucleus of Cajal are collaterals. The projection from the olivary pretectal nucleus to the facial nucleus which has been described to receive an input in cats could not be confirmed for the rat. The fact that the Edinger-Westphal nucleus, the interstitial nucleus of Cajal and the superior colliculus receive an input from the olivary pretectal nucleus suggests that this primary visual centre is not only involved in the pupillary light reflex, but also in controlling eye and head position and saccadic eye movements. Although visual acuity largely depends on receptive field sizes of retinal ganglion cells and their central connections, the stronger sympathetic influence during the pupillary light reflex in animals with frontally placed eyes compared to animals with laterally placed eyes may also contribute to the higher visual acuity in animals with frontally placed eyes.

The allocation of nerve fibres to the anterior eye segment and peripheral ganglia of rats. II. The sympathetic innervation.

The sympathetic innervation of the peripheral ganglia related to the eye, i.e. the trigeminal ganglion, the ciliary ganglion and the pterygopalatine ganglion, and of the anterior eye segment was studied in rats. Selective labelling of sympathetic nerves was obtained by means of injection of [3H]leucine into the superior cervical ganglion. Bundles of sympathetic nerve fibres were found in the trigeminal ganglion and the pterygopalatine ganglion but were absent in the ciliary ganglion. In addition individual sympathetic nerve fibres, which may have contacts with trigeminal ganglion cells, were found between the ganglion cell bodies all over the trigeminal ganglion indicating a sympathetic innervation of this ganglion. In the anterior eye segment, there appeared to be a sympathetic innervation of the ciliary cleft, the ciliary body and the iris. Within the ciliary body sympathetic nerve fibres innervate the central stroma and the stroma of the ciliary processes. Labelled sympathetic nerve fibres were also observed in the stroma of the iris and were most abundant in its periphery. Most sympathetic fibres reach the iris and ciliary body by way of the base of the ciliary body. Only few sympathetic fibres are present in the ciliary cleft. No sympathetic innervation of the cornea was found.

Retinopretectal projections in albino and pigmented rabbits: An autoradiographic study

The ipsilateral and contralateral retinal projection was studied in pigmented rabbits and in 3 strains of albino rabbits by anterograde transport of [3H]proline and [3H]fucose combined with autoradiographic techniques. Special attention was paid to the terminals in the pretectal area of both the pigmented and albino strains. On the contralateral side terminal labeling was found in both pigmented and albino rabbits in the nucleus of the optic tract (NOT), the anterior pretectal nucleus (PA), the posterior pretectal nucleus (PP) and the pretectal olivary nucleus (PO). Ipsilaterally labeling was found only in the pigmented strain in small patches in the PP. Ipsilateral projection was not found in the albinos in the pretectal area. The results are in agreement with the findings of Scalia in pigmented rabbits. The absence of ipsilateral labeling in the pretectal region in albinos is in contrast with earlier findings of Giolli and Takahashi et al., in pigmented rabbits but is in agreement with the observations of Takahashi and Oyster. Since no radioactively labeled fibers were found to project to the NOT in either pigmented or albino rabbits, these results do not support the hypothesis of Collewijn that the inverted optokinetic nystagmus in albinos is due to misrouting of the ipsilateral retinal fibers to the NOT.

Corticotropin-releasing factor and urocortin differentially modulate rat Purkinje cell dendritic outgrowth and differentiation in vitro

The precise outgrowth and arborization of dendrites is crucial for their function as integrators of signals relayed from axons and, hence, the functioning of the brain. Proper dendritic differentiation is particularly resonant for Purkinje cells as the intrinsic activity of this cell‐type is governed by functionally distinct regions of its dendritic tree. Activity‐dependent mechanisms, driven by electrical signaling and trophic factors, account for the most active period of dendritogenesis. An as yet unexplored trophic modulator of Purkinje cell dendritic development is corticotropin‐releasing factor (CRF) and family member, urocortin, both of which are localized in climbing fibers. Here, we use rat organotypic cerebellar slice cultures to investigate the roles of CRF and urocortin on Purkinje cell dendritic development. Intermittent exposure (12 h per day for 10 days in vitro) of CRF and urocortin induced significantly more dendritic outgrowth (45% and 70%, respectively) and elongation (25% and 15%, respectively) compared with untreated cells. Conversely, constant exposure to CRF and urocortin significantly inhibited dendritic outgrowth. The trophic effects of CRF and urocortin are mediated by the protein kinase A and mitogen‐activating protein kinase pathways. The study shows unequivocally that CRF and urocortin are potent regulators of dendritic development. However, their stimulatory or inhibitory effects are dependent upon the degree of expression of these peptides. Furthermore, the effects of CRF and urocortin on neuronal differentiation and re‐modeling may provide a cellular basis for pathologies such as major depression, which show perturbations in the expression of these stress peptides.

Megakaryocytic dysfunction in myelodysplastic syndromes and idiopathic thrombocytopenic purpura is in part due to different forms of cell death

Platelet production requires compartmentalized caspase activation within megakaryocytes. This eventually results in platelet release in conjunction with apoptosis of the remaining megakaryocyte. Recent studies have indicated that in low-risk myelodysplastic syndromes (MDS) and idiopathic thrombocytopenic purpura (ITP), premature cell death of megakaryocytes may contribute to thrombocytopenia. Different cell death patterns have been identified in megakaryocytes in these disorders. Growing evidence suggests that, besides apoptosis, necrosis and autophagic cell death, may also be programmed. Therefore, programmed cell death (PCD) can be classified in apoptosis, a caspase-dependent process, apoptosis-like, autophagic and necrosis-like PCD, which are predominantly caspase-independent processes. In MDS, megakaryocytes show features of necrosis-like PCD, whereas ITP megakaryocytes demonstrate predominantly characteristics of apoptosis-like PCD (para-apoptosis). Triggers for these death pathways are largely unknown. In MDS, the interaction of Fas/Fas-ligand might be of importance, whereas in ITP antiplatelet autoantibodies recognizing common antigens on megakaryocytes and platelets might be involved. These findings illustrate that cellular death pathways in megakaryocytes are recruited in both physiological and pathological settings, and that different forms of cell death can occur in the same cell depending on the stimulus and the cellular context. Elucidation of the underlying mechanisms might lead to novel therapeutic interventions.

Chapter 14 GABAergic neurons and circuits in the pretectal nuclei and the accessory optic system of mammals

Publisher Summary This chapter discusses the ultrastructural organization of the inhibitory components of the accessory optic system mediated by gamma-aminobutyric acid (GABA). There is a growing body of evidence that the inhibitory neurotransmitter GABA is involved in transmission in many regions of the visual system. In the visual cortex, GABA plays a role in directional sensitivity; at the level of the retinal ganglion cells—properties, such as speed and direction selectivity—are generated in part by inhibitory, presumably GABA-releasing (GABAergic), mechanisms. In the retina of the frog, it has been suggested that GABA might be involved in reducing the slip velocity of images on the retina during the slow component of optokinetic nystagmus. Two classes of GABAergic cell bodies have been described in the chapter. They probably can be divided into GABAergic local interneurons and GABAergic projection neurons. GABAergic cell bodies receive few terminals, which are in contrast to nonGABAergic somata that receive many synaptic contacts. GABAergic dendrites that originate from GABAergic cell bodies, however, receive numerous terminals, both GABAergic and nonGABAergic.

GABA immuno-electron microscopic study of the nucleus of the optic tract in the rabbit

The pretectal nucleus of the optic tract (NOT) was investigated immunocytochemically with an antiserum against gamma aminobutyric acid (GABA) employing the pre‐embedding peroxidase antiperoxidase technique at the light microscopic level and the postembedding colloidal gold technique at the electron microscopic level. GABA immunoreactivity was observed in cell bodies of different sizes and as punctate structures in the neuropil. In the electron microscope, besides immunoreactive dendrites, four different types of terminals were found to be GABA‐immunopositive; three types of terminals with clustered and flattened vesicles (F‐profile) and a fourth type with pleomorphic vesicles, presumably of dendritic origin (P‐profile). Both P‐and F‐profiles formed symmetrical synapses with dendritic profiles arranged in clusters ensheathed by glial elements. GABA‐immunopositive terminals were observed in synaptic contact with somata and retinal terminals (R‐profiles) that were always GABA‐immunonegative. Some GABA‐immunopositive somata showed presynaptic contacts with dendrites. The presence of GABA in numerous distinct elements in the NOT and the diversity in labeled somata and terminals demonstrate the importance of the inhibitor neurotransmitter in the NOT and suggest that its function is not limited to interneurons.