Dexamethasone-induced reactivation of bovine herpesvirus type 5 latent infection in experimentally infected rabbits results in a broader distribution of latent viral DNA in the brain

Bovine herpesvirus type 5 (BHV-5) is a major agent of meningoencephalitis in cattle and establishes latent infections mainly in sensory nerve ganglia. The distribution of latent BHV-5 DNA in the brain of rabbits prior to and after virus reactivation was studied using a nested PCR. Fifteen rabbits inoculated intranasally with BHV-5 were euthanized 60 days post-inoculation (group A, N = 8) or submitted to dexamethasone treatment (2.6 mg kg-1 day-1, im, for 5 days) and euthanized 60 days later (group B, N = 7) for tissue examination. Two groups of BHV-1-infected rabbits (C, N = 3 and D, N = 3) submitted to each treatment were used as controls. Viral DNA of group A rabbits was consistently detected in trigeminal ganglia (8/8), frequently in cerebellum (5/8), anterior cerebral cortex and pons-medulla (3/8) and occasionally in dorsolateral (2/8), ventrolateral and posterior cerebral cortices, midbrain and thalamus (1/8). Viral DNA of group B rabbits showed a broader distribution, being detected at higher frequency in ventrolateral (6/7) and posterior cerebral cortices (5/7), pons-medulla (6/7), thalamus (4/7), and midbrain (3/7). In contrast, rabbits inoculated with BHV-1 harbored viral DNA almost completely restricted to trigeminal ganglia and the distribution did not change post-reactivation. These results demonstrate that latency by BHV-5 is established in several areas of the rabbit's brain and that virus reactivation leads to a broader distribution of latent viral DNA. Spread of virus from trigeminal ganglia and other areas of the brain likely contributes to this dissemination and may contribute to the recrudescence of neurological disease frequently observed upon BHV-5 reactivation.

Dexamethasone-induced reactivation of bovine herpesvirus type 5 latent infection in experimentally infected rabbits results in a broader distribution of latent viral DNA in the brain.

Bovine herpesvirus type 5 (BHV-5) is a major agent of meningoencephalitis in cattle and establishes latent infections mainly in sensory nerve ganglia. The distribution of latent BHV-5 DNA in the brain of rabbits prior to and after virus reactivation was studied using a nested PCR. Fifteen rabbits inoculated intranasally with BHV-5 were euthanized 60 days post-inoculation (group A, N = 8) or submitted to dexamethasone treatment (2.6 mg kg(-1) day(-1), im, for 5 days) and euthanized 60 days later (group B, N = 7) for tissue examination. Two groups of BHV-1-infected rabbits (C, N = 3 and D, N = 3) submitted to each treatment were used as controls. Viral DNA of group A rabbits was consistently detected in trigeminal ganglia (8/8), frequently in cerebellum (5/8), anterior cerebral cortex and pons-medulla (3/8) and occasionally in dorsolateral (2/8), ventrolateral and posterior cerebral cortices, midbrain and thalamus (1/8). Viral DNA of group B rabbits showed a broader distribution, being detected at higher frequency in ventrolateral (6/7) and posterior cerebral cortices (5/7), pons-medulla (6/7), thalamus (4/7), and midbrain (3/7). In contrast, rabbits inoculated with BHV-1 harbored viral DNA almost completely restricted to trigeminal ganglia and the distribution did not change post-reactivation. These results demonstrate that latency by BHV-5 is established in several areas of the rabbit's brain and that virus reactivation leads to a broader distribution of latent viral DNA. Spread of virus from trigeminal ganglia and other areas of the brain likely contributes to this dissemination and may contribute to the recrudescence of neurological disease frequently observed upon BHV-5 reactivation.

Intrapreputial infection of young bulls with bovine herpesvirus type 1.2 (BHV-1.2): acute balanoposthitis, latent infection and detection of viral DNA in regional neural and non-neural tissues 50 days after experimental reactivation.

Venereal infection of bulls with bovine herpesvirus type 1.2 (BHV-1.2) may result in acute balanoposthitis followed by the establishment of latent infection, presumably in dorsal root nerve ganglia. We herein report the characterization of the acute and latent infection of young bulls with a Brazilian BHV-1.2 isolate and the investigation of neural and non-neural sites in which viral DNA persists during latent infection, i.e. 110 days after inoculation and 50 days after experimental reactivation. Intrapreputial inoculation of BHV-1.2 isolate SV-56/90 (10(6.5)pfu per animal) resulted in severe balanoposthitis, characterized by redness of the penis and preputial mucosa, coalescent vesicles and fibrinous exsudate in all four infected bulls. Virus shedding was detected in preputial secretions and semen up to days 14 and 13 pi, respectively. Dexamethasone administration at day 60 pi led to reactivation of the infection in all animals, resulting in virus shedding in preputial secretions and/or in semen. At day 50 post-reactivation (pr), the animals were euthanized and regional tissues were collected for PCR and virus isolation. Viral DNA was consistently detected in the dorsal root ganglia of nerves genito-femoral (4/4) and obturator (4/4); frequently in the pudendal (3/4), sciatic (3/4) and rectal caudal nerve ganglia (2/3). In addition, viral DNA was detected in the pelvic sympathetic plexus of one bull and in regional lymph nodes (deep inguinal (2/4); sacral (1/4); medial iliac (1/4)) of two bulls. No infectious virus could be recovered from homogenates of DNA positive tissues, indicating the absence of actively replicating virus. These results demonstrate that BHV-1.2 DNA may persist in several sacral nerve ganglia and in regional lymph nodes as well during latent infection, i.e. 50 days after experimental reactivation. These findings may help in understanding the pathogenesis of acute and latent genital infection by BHV-1.2.

Regionalization of oviducts in Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) and its potential significance for fertilization.

The structural analysis of oviducts in Boophilus microplus (Canestrini, 1887) in different stages of ingurgitation has indicated that they are constituted primarily of an internal cylinder and an external cylinder with different cell types being found between them. Copulated females in active ingurgitation process show typical variations along the internal cylinder, and three regions can be defined: anterior, ring-like and transitional. Based on such regionalization, hypotheses were raised about where and how fertilization takes place, a process yet to be clarified for the acari.

Corpus callosum axons in the developing visual cortex of the gerbil revealed by DiI and Fluorogold.

The distribution of commissural neurons and terminals was revealed by DiI and Fluorogold injections in gerbils. The distribution of corpus callosum projections in visual cortical areas changes significantly during postnatal development of the gerbil. On postnatal days 5, 8 and 10, the animals do not show callosal projections. Widespread callosal endings are developed by postnatal day 15. This diffuse projection concentrates in strips at the border of area 17/18A in adult animals (postnatal day 20 and older).

Autoradiographic and electron microscopic studies of retinal rosettes in genetically microphthalmic mice.

We report results obtained in the microphthalmic strain of mice 944. Heterozygotes appear normal, but they produce litters in which typically between 2 and 5 offspring exhibit microphthalmia and rosettes in the retina and the optic nerve. As a result these animals are blind. Our investigations using 3H-thymidine for autoradiographic estimation of postnatal cell proliferation and electron microscopy for detailed observation of the morphology of the rosettes show an increased proliferation of neuronal cells (more than 200%), especially in rosettes. In the rosettes a centrally located lumen exists in which cytoplasmatic processes can be found. In ultrastructural investigations these processes were identified as residues of the outer segments of photoreceptor cells.

Distribution patterns of Wisteria floribunda agglutinin binding sites and parvalbumin-immunoreactive neurons in the human visual cortex: a double-labelling study.

Extracellular lattic-like coatings--known as perineuronal nets (PNs)--ensheath certain types of neurons in the mammalian neocortex. PNs and densely stippled zones in some cortical layers contain proteoglycans stainable e.g. with several plant lectins. The present study is focused on the binding sites of the plant lectin Wisteria floribunda agglutinin (WFA) and on its relationship to parvalbumin-immunoreactive (PARV-ir) neuronal constituents in the human visual cortical fields 17 and 18. Size and numbers of PNs varied not only between these two areas, but also between its layers. In area 17 the PNs appeared accumulated in two conspicuous bands which included layer IV, particularly layer IVb, and layer VI. Layer IVc of area 17 contained numerous small faintly stained PNs. In area 18 a two-tiered organization of PNs in layers IIIb-c and layer V was emphasized. The neuropil staining by WFA was much more intense in layers IIIb-c of area 18 than of area 17. In areas 17 and 18 the vast majority of PNs was associated with sub-populations of PARV-ir cells resembling several classes of GABAergic cortical interneurons. Among PARV-negative cells surrounded by PNs, only exceptionally pyramide-like neurons were detected. The patterns of lectin-labelling in the human visual primary and association cortex resembled those of macaque monkeys and differed from those in the visual cortex of rat and gerbil. These differences between mammals representing divergent modes of visual specialization may support the view that such extracellular matrix components in these occipital cortical areas are involved in visual information processing subserving fast inhibitory interneuronal actions.

Postnatal development of NADPH-diaphorase/nitric oxide synthase positive nerve cells in the visual cortex of the rat.

The postnatal development of NADPH-diaphorase (NADPH-d)/nitric oxide synthase (NOS) positive nerve cells was studied in the visual cortex of rats on postnatal day 1, 5, 10, 15, 20, 30 and at the age of 1 year. NADPH-d was demonstrated enzymhistochemically and NOS immunohistochemically using a polyclonal antibody. NADPH-d is localized in nerve cell somata, dendrites, axons and blood vessels, whereas NOS immunoreactivity is only detectable in nerve cells. The identity of NADPH-d cells with those which contain NOS was proved in double labelling experiments in the cortex of rats on postnatal day 5, 15 and at the age of 1 year. The results of these experiments have shown that in the cortex of rats NADPH-d positive cells are identical with NOS-positive cells in the different stages. Therefore we have used NADPH-d histochemistry in all other postnatal stages as a marker for neurons which contain NOS. NOS positive nerve cells appear very early on postnatal day 1 in the intermediate (white matter) and subplate (layers V and VI) region as small undifferentiated neurons. During the following postnatal differentiation these neurons reached their typical morphology in the second week and appeared in all layers. Neurons in layers V and VI preceded those in the superficial layers. Nerve cells in the white matter seem to have their own differentiation pattern because they showed characteristic features of immaturated varicose dendrites for a longer time. The investigation of soma size with the computerized "Kontron Videoplan" system (Zeiss, Germany) showed the largest cell bodies on postnatal day 20 which then decreased towards adulthood. Between postnatal day 10 and 20 some NOS-positive neurons especially in the deep layers displayed symptoms of degeneration, like shrunken cell bodies, corkscrew and twisted dendrites. Furthermore, NOS-positive neurons in layer I are not detectable in adult neocortex. These observations could suggest that some NOS-positive cells in the cerebral cortex of rats may occur only transiently. Also in the neuropil some alterations in the localization of NOS positive axonal boutons were observed. On postnatal day 10 NOS negative cell somata were shadowy surrounded by boutons. During the further development from postnatal day 20 until adulthood this particular position was no longer visible. Beside the NOS cells which played a transient role, the majority of these cells survived to adulthood and are a morphological (Martinotti-cells with ascending axons) and chemical (GABAergic, NADPH-d/NOS positive, peptide containing cells) defined cell type in the neuronal network of the cortex of the rat.

On the structure of the pretectal nuclei of the rat: an immunocytochemical and tracer study.

We have studied the distribution of the calcium-binding proteins parvalbumin, calbindin and calretinin, the NADPH diaphorase activity and the morphology of the commissural neurons, revealed by the stereotactic applications of fluorogold in the pretectal complex of the rat. The histochemical differentiation of the pretectal complex shows a complementary pattern of parvalbumin and calbindin containing cells. Only a few of the neurons in the pretectal complex contain calbindin. Calretinin immunoreactivity is scant and diffuse. The NADPH-diaphorase activity is restricted to neurons and terminals in the nucleus of the optic tract and the dorsal terminal nucleus. Due to numerous active fibers which traverse these nuclei they display a reticular appearance. Commissural neurons constitute 20% of the cell number of the pretectal complex and are restricted to the dorsal and lateral terminal nuclei.

Morphological analyses of NADPH-diaphorase/nitric oxide synthase positive structures in human visual cortex.

Human visual cortex was studied using NADPH-diaphorase histochemistry and nitric oxide synthase immunohistochemistry. Large, strongly stained, sparsely spined non-pyramidal cells (average soma diameter: 16 x 16 microns) occur in layers II-VI, but are commonest in layers II-III. Small weakly stained multipolar cells (average soma diameter 3.6 x 4 microns, stellate like cells) in layers II-VI are concentrated in layer IV of areas 17 and 18. The density of these cells, measured with a computer assisted microscopy system is less in area 18 than 17. Large, strongly stained, predominantly horizontal cells (average soma diameter 12 x 19 microns) are localized in the underlying white matter. Axons of the large, strongly NADPH-diaphorase positive cells are thin and unbranched with fine boutons. These axons ascend to layer I. The large, strongly stained cells in layers II-VI we identify as Martinotti neurons. In layer I parallel unbranched positive fibres with some fine boutons run horizontally and build dense axonal plexuses together with the axons of Martinotti neurons. Axons of presumed extrinsic origin are morphologically different from NADPH-diaphorase positive intrinsic fibres. They show thick varicosities running in different directions and forming a network in layers III-VI. Basket like formations of these fibres were frequently observed in layers IV, V and VI. Other fibres seem to innervate blood vessels. Nitric oxide synthase was also demonstrated immunohistochemically by a polyclonal rabbit nitric oxide synthase antiserum. The morphology and distribution of the immunostained cells correspond with those seen with NADPH-diaphorase histochemistry. Double labelling experiments confirm the colocalization of NADPH-diaphorase and nitric oxide synthase in all demonstrated cells. Immunohistochemical demonstration of glial fibrillary acidic protein has shown that astrocytes are not involved in the NADPH-diaphorase/NOS system in the human visual cortex.

Aberrant visual pathways in microphthalmic mice.

We report results obtained in the microphthalmic strain of mice 944. Heterozygotes appear normal, but they produce litters in which typically between 2 and 5 offspring exhibit microphthalmia. As a result these animals are blind. Our investigations using the fluorescent tracer DiI show that in microphthalmic mice there is a small compensation for the missing retinal input by terminals or axon collaterals originating in the somatosensory thalamus. These morphological findings agree with somatosensory responses recorded in the visual cortex (EEG recordings).

Light- and electron microscopic localization of parvalbumin, calbindin D-28k and calretinin in the dorsal lateral geniculate nucleus of the rat.

The localization of parvalbumin, calbindin D-28k and calretinin have been investigated in the dorsal lateral geniculate nucleus (d lgn) of the rat at the light and electron microscopical level. Parvalbumin and calretinin positive sites are restricted to nerve fibres, whereas calbindin is present in fibres as well as in nerve cells showing morphological characteristics of interneurons. Ultrastructurally parvalbumin immunoreactivity is found in large terminals surrounded by glial lamellae containing round vesicles and making asymmetric contacts on dendrites. These morphological characteristics are typical for retinal endings of type 2a. Another kind of parvalbumin positive presynaptic terminals are seen on the surface of unstained nerve cell bodies and features symmetrical contacts. We conclude that this type represents axonal terminals of GABAergic neurons of the thalamic reticular nucleus. Calbindin positive nerve cells in the d lgn occur in the latero-dorsal part and according to morphological characteristics, belong to interneurons. Calbindin positive nerve cells receive synaptic terminals deriving from different kind of unstained presynaptic profiles. Calretinin immunoreactivity is localized in small to medium sized presynaptic endings with round vesicles, pale mitochondria and Gray-type 1 contacts on dendrites of relay- and interneurons. Some calretinin positive terminals are located in triads or in complex encapsulated regions. Therefore we identify calretinin positive terminals as the retinal inputs of type 2b. Our results demonstrate the expression of the three calcium binding proteins in morphologically, physiologically and biochemically different structures within the d lgn of the rat. The distribution differs from that found in the d lgn of the cat or monkey.

The calcium-binding protein calretinin is localized in a subset of interneurons in the rat cerebral cortex: a light and electron immunohistochemical study.

The distribution of the calcium binding protein, calretinin (CR) has been investigated immunohistochemically in the cerebral cortex of albino rats by light- and electron microscopy. At the light microscopical level the pattern of CR-immunoreactivity (ir) appeared very similar in all regions of the rat cerebral cortex. CR-immunoreactive cells were found sparsely in layer I to layer VI, and frequently also in the white matter of the corpus callosum. All CR-ir neurons revealed morphological characteristics of local interneurons. The calretinin positive interneurons could be grouped according to their laminar occurrence, dendritic arborization and the soma size into 5 cell type classes. Quantitative measurements were performed only in the visual cortex. CR-ir neurons were more frequent in the superficial layers II and III. In all other layers, CR-ir cells are sparsely distributed with no preferential laminar localization. At the electron microscopical level, CR-ir axonal boutons formed frequently symmetrical axo-dendritic contacts. In all animals we observed CR-ir axons forming also synaptses of asymmetrical type. In summary calretinin labelled an interneuronal subpopulation of the rat cerebral cortex, which seemed not to overlap in its distribution and labelled structures to those, containing the related calcium binding proteins parvalbumin and calbindin D-28k.

[Neuronal structure abnormality in the orbito-frontal cortex of schizophrenics]. / Neuronale Strukturanormalität im orbito-frontalen Cortex bei Schizophrenien.

Changes were described in the neuronal structure of neurones, impregnated after the method of Golgi-Bubenaite in schizophrenic patients. Investigations were done in area 11 (Brodmann 1909) of patients with paranoid-hallucinatory schizophrenia. Control cases were patients without any clinical or morphological brain diseases and patients with catatonic schizophrenia. 1.1 We investigated numerous typical triangle cells in lamina VI of patients with paranoid-hallucinatory schizophrenia. Only few were seen in the controls. 1.2 In the paranoid-hallucinatory schizophrenia brains there are lamina VI pyramidal cells with ramified apical dendrites reaching lamina II. 2. In lamina V there are many pyramidal cells with two to five apical dendrites, such cells were never seen in controls, these pyramids have only one main apical dendrite. 3. A small group of L III pyramids have atypical long spines. A lot of these spines have splitted spine heads. The spine number is significantly increased. 4. There is an atypical simplified angioarchitecture in paranoid hallucinatory schizophrenia-patients and no normal arborization of the brain vessels in three planes, like in controls. We think that the abnormal findings in paranoid-hallucinatory schizophrenia are not the consequence of specific therapeutics. We interpret our findings as possible variabilities of a normal brain structure.

[Neurons in the visual cortex of Microtus brandti]. / Neuronen im visuellen Cortex von Microtus brandti.

Neurons were described in the visual Cortex of Microtus brandti, a Mongolian harmful rodent living in day-activity. We find following types of neurons in our Golgi-material: 1. spiny neurons: pyramidal and stellate neurons. 2. smooth or sparsely spined neurons: smooth, large neurons, sparsely spined small neurons with descending axons, sparsely spined neurons with ascending axons. Double-bouquet-, chandelier and neuroglioforme cells are not impregnated. There are no bipolare neurons (Martinotti cells) among the neurons with ascending axons. The small, sparsely spined neurons are not only in lamina IV - like in other species - but they can also be found in laminae II to IV. Their distribution of spines on the distal parts of dendrites seems to be characteristical for rodents. The lamination of the visual cortex of Microtus brandti is the same like in the rat. All cells are of large size in relation to the body mass of the animal.

Morphology of neurons in the rat basal forebrain nuclei: comparison between NADPH-diaphorase histochemistry and immunohistochemistry of glutamic acid decarboxylase, choline acetyltransferase, somatostatin and parvalbumin.

Transversal sections through the basal forebrain of 11 adult male rats were immunostained for glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT), somatostatin (SOM) and parvalbumin (PARV). Immunohistochemistry of ChAT, PARV, and SOM was combined with histochemistry of NADPH-diaphorase (NADPH-d) to obtain information on the colocalization of various neuroactive substances and this enzyme and to facilitate the recognition of morphological details of double-stained neurons. The distribution patterns of GAD- and PARV-immunoreactive cells were only in part congruent in basal forebrain nuclei in the rat. In the medial septal nucleus (MS) and the vertical limb of the diagonal band (vDB) PARV-immunopositive neurons were homogeneously scattered inside the nucleus, whereas the GAD-immunoreactive cells were much more numerous in the lateral part of this nuclear complex. In the horizontal limb of the diagonal band (hDB) and the nucleus preopticus magnocellularis (NPM), where GAD-immunoreactive cells occurred in high number, only very few cells contained PARV-immunoreaction product. In the substantia innominata-nucleus basalis Meynert complex (SI-NB) and in the ventral pallidum (VP) the neuropil was heavily stained with the GAD-immunoreaction product. The number of GAD-positive cells appeared low in the SI-NB, but much higher in the VP. In this nucleus GAD- and PARV-immunoreactive cells seem to be identical. PARV-positive neurons are very sparse in the SI-NB. Double-staining of PARV-immunoreactivity and NADPH-d was not registered. These nuclei were the only ones in which some cells with SOM-like immunoreactivity were observed. Among ChAT-positive neurons those double-stained with NADPH-d occurred in moderate number, but with obvious regional differences. In MS-vDB and the marginal zone of hDB the two neuron groups were intermingled, but only in the innermost part of the hDB ChAT-single-immunostained cells form aggregates, which were also typical of the zone in the SI-NB that surrounds and infiltrates the globus pallidus (GP). Double-labelled cells were more frequent in the lateral aspect of the NPM and SI-NB. Cells single-stained for NADPH-d were frequent in the MS-vDB along the border toward the lateral septal nuclei, but low in number in the NPM, VP and SI-NB. The functional aspects of the occurrence of GAD-immunoreactive cell aggregates in the lateral preoptic area (LP) and the lateral hypothalamic area (LH) were discussed with special regards to extrinsic GABAergic input in the dorsal SI-NB.(ABSTRACT TRUNCATED AT 400 WORDS)

[Expansion and thickness of the visual cortex of microphthalmic mice and healthy Wistar animals]. / Ausdehnung und Dicke des visuellen Kortex von Microphthalmus-Mäusen und gesunden Geschwistertieren.

In connection with our studies about the development and the structure of the visual system of the brain of normal and genetically microphthalmic mice in this paper we have investigated the extend of the visual cortex and the thickness of the primary visual cortex (area Oc1). The most important results are: The size of the surface of the neocortex and of the areas Oc1 and Oc2m shows no significant differences between the normal and the microphthalmic mice. The whole thickness of area Oc1 is at day 20 in the microphthalmic mice significantly greater than in normal animals. In the microphthalmic mice lamina I, III and V are thicker than in normal mice. Lamina IV is in microphthalmic mice significantly thinner than in normal animals. The interpretation of these results is difficult because at PD 20 the cortex and his laminae are not full developed. But there are in the microphthalmic mice not such clear changes (reduction) on the cortical level as in the subcortical structures of the visual system. The reduction of the thickness of lamina IV maybe caused by a reduced projection from the lateral geniculate nucleus.