Enhancer-deficient amphotropic murine leukemia virus and recombinants with heterologous transcription elements can be efficiently amplified and detected in Mus dunni fibroblasts.

Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species, including humans, and is a potential contaminant in MLV vector preparations for human gene transfer studies. Mus dunni fibroblasts are routinely used for amplification and detection of contaminating virus. We have recently characterized an amphotropic MLV mutant lacking the 75-bp viral enhancer elements and spontaneous MLV-(RCMV) recombinants that have acquired cytomegalovirus (CMV) transcription elements. Both of these viruses replicate in specific human cell types. To test whether the formation of such viruses can be detected and controlled with current routine procedures, we have analyzed the replication of these amphotropic MLV mutants in Mus dunni fibroblasts. We find that M. dunni cells are permissive for enhancer-deficient and CMV promoter-recombinant MLV from several human cell lines. Thus, M. dunni fibroblasts are suitable for the amplification and subsequent detection of enhancer-deficient and enhancer-recombinant MLV in vector preparations.

Replication of enhancer-deficient amphotropic murine leukemia virus in human cells.

Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species, including humans, and is a potential contaminant in MLV vector preparations for human gene transfer studies. The generation of replication-competent virus is considered less likely with vectors that delete the viral transcription elements. This conclusion is based on data obtained in rodents, where MLV replication depends on the expression of viral genes under the control of 75-bp enhancer elements in the long terminal repeat. We demonstrate here that in some human cells replication of amphotropic MLV is possible in the absence of these enhancer elements. Replication of the enhancer-deficient virus MLV-(MOA)Delta E is observed in selected human sarcoma and B lymphoma lines and proceeds at a lower rate than that of the intact virus. No insertion of a foreign promoter or enhancer into the long terminal repeat was detected. Our data suggest the presence of a secondary enhancer element within the MLV provirus that can in selected human cells mediate virus transcription and replication in the absence of the 75-bp U3 enhancers.

Amphotropic murine leukemia virus replication in human mammary epithelial cells and the formation of cytomegalovirus-promoter recombinants.

Amphotropic murine leukemia virus (MLV) can replicate in human cells and is a potential contaminant in vector preparations for human gene transfer studies. We have recently shown that replication of amphotropic MLV in specific human sarcoma and lymphoma lines is possible in the absence of the viral 75-bp transcription enhancer elements. Here, we have tested the replication of an amphotropic MLV, MLV-(MOA), and an enhancer-deficient mutant of this virus in human breast carcinoma-derived cell lines. The proviral expression plasmids use a cytomegalovirus (CMV) promoter for the initial transcription of virus RNA. We found that all cells analyzed are permissive for replication of MLV-(MOA). Enhancer-deficient virus is unable to replicate. However, in two lines the replication defect can be rescued by the spontaneous insertion of a CMV promoter and enhancer into the U3 region. This recombinant virus MLV-(RCMV) replicates with kinetics similar to that of MLV-(MOA) but is restricted to specific cell lines. The potential formation of RCMV recombinants during MLV vector preparation must be considered.

Developmental changes of MAP2 immunoreactivity in the hippocampus proper and dentate gyrus of the rat.

Microtubules are present in high concentration in the nervous system and are a prominent component of the neuronal cytoskeleton. Microtubules are composed of tubulin and variety of microtubule-associated proteins (MAPs), which have been implicated in the regulation of microtubule assembly and function. MAP2 is the most abundant of these proteins, and it has been extensively characterized in various functional and pathological conditions. In the present study the distribution of MAP2 was examined in each layer of the CA1 and CA3 regions of the hippocampus proper and dentate gyrus in rat development. A total of 40 brains at various ages starting from postnatal day (P) 0 to P90 were examined. After perfusional fixation the brains were frozen and cut on the coronal plane and stained with either cresyl violet or standard immunohistochemical methods using the anti-MAP2 antibody. MAP2 exhibited a somatodendritic pattern of localization in cells of the hippocampus. Staining was most prominent in dendrites and perikarya as well as granules surrounding cell bodies. In a newborn rat's brain immunostaining was intense in granules and faint in perikarya. Between P4 and P21 immunostaining density for MAP2 was stronger and appeared in perikarya, granules, and dendritic trees. After P21 the perikarya and dendrites of the pyramidal layer and stratum radiatum of the hippocampus proper, as well as the molecular and granular layer of dentate gyrus, showed reduced immunoreactivity. In the stratum oriens of the hippocampus and polymorphic layer of the dentate gyrus immunoreactivity was still strong until P90.

Mouse mammary tumor virus superantigen expression is reduced by glucocorticoid treatment.

Expression of mouse mammary tumor virus (MMTV)-encoded superantigens in B lymphocytes are required for viral transmission and pathogenesis. Due to problems with detection and quantification of the superantigen protein, most reports about the mechanism of superantigen expression from the viral sag gene rely on the quantitative analysis of putative sag mRNAs. The description of multiple promoters as a source of putative sag mRNA has complicated the situation even further. All conclusions about the level of superantigen protein expression based on these data remain circumstantial. To test the effect of the glucocorticoid hormone dexamethasone on the total superantigen expression from an infectious MMTV provirus we used a quantitative assay that is based on a superantigen-luciferase fusion protein. MMTV gene expression from the major promoter in the 5' long terminal repeat (LTR) is strongly induced in the presence of glucocorticoid hormones. We now demonstrate that, in the presence of dexamethasone, sag gene expression is reduced despite increased transcription from the MMTV 5' LTR and increased amounts of putative sag mRNA initiated at the LTR promoter. These data show that the expression of the MMTV sag gene does not correlate with the activity of the major LTR promoter and thus differs from all other MMTV genes.

Expression of calbindin-D28k and parvalbumin during development of rat's basolateral amygdaloid complex.

Parvalbumin and calbindin-D28k are calcium-binding proteins, which are considered to be markers for certain populations of GABAergic neurons. Their correct development in the basolateral amygdaloid complex is critical for the proper emotional functioning in adult live of human and animals. Therefore, in this paper we describe the pattern of the morphological differentiation and distribution of immunoreactive elements of the parvalbumin and calbindin-D28k in this complex on the basis of immunohistochemically stained material obtained from embryonic (E20) and postnatal (P0-P90) rat brains. Calbindin-D28k appeared early in the development, already in the prenatal life. At this time immunopositive reaction was visible only in cell bodies. However, during development the population of immunopositive neurons was divided into four types: (1) polygonal; (2) piriform-like; (3) bipolar; and (4) pyramidal-like. Two weeks after birth calbindin-D28k immunoreactivity also appeared in neuropil. First, there were visible calbindin-D28k positive fibers and granules that encircled unstained cell bodies and formed basket-like structures. Subsequently, these granules appeared along proximal parts of unstained dendrites forming, so called 'cartridges'. The distribution of calbindin-D28k positive cells during postnatal life was rather homogenous throughout whole basolateral complex. Intensity of calbindin-D28k immunoreactivity reached mature level on the 21st day after birth.The maturation pattern of parvalbumin immunopositive elements followed the same sequence as calbindin-D28k, but it started much later - since the 17th day after birth and reached mature appearance on the 30th day of life. Contrary to calbindin-D28k, parvalbumin was not homogeneously distributed in the basolateral complex. Originally, parvalbumin was restricted to the magnocellular part of basolateral nucleus but it was finally expressed also in the parvicellular part of basolateral nucleus and the dorsolateral part of lateral nucleus. The differences in development of these two calcium-binding proteins indicate that parvalbumin and calbindin-D28k play diverse roles during development and maturation of the basolateral amygdala.

Tactile experience induces c-fos expression in rat barrel cortex.

Understanding gene expression that is responsive to sensory stimulation is central to elucidate molecular mechanisms underlying neuronal plasticity. In this study we demonstrate two new methods of stimulating whiskers that provide major sensory input to rat neocortex. In the first paradigm, animals were placed on the top of a cylinder and their vibrissae were brushed by hand. In the second paradigm, animals were placed for a brief period of time into a new, wired cage resulting in vibrissae stimulation when they explored the new environment. Both approaches induced c-Fos expression in barrel cortex corresponding to the stimulated vibrissae, especially in layer IV. Layers II/III and V/VI also showed c-Fos induction, but there were no detectable changes in layer VIb. The majority of c-Fos-expressing cells are probably not inhibitory neurons, because they do not show parvalbumin staining. Both paradigms, in contrast to the previous methods, are simple to use and do not require anesthesia, restraint of animals, or elaborate experimental setups.

The developmental changes of the "paraclaustral reservoir" of migrating cells in the rat brain: a study using morphometric and in situ DNA end labeling techniques.

A distinct group of small cells lying in the ventral part of the external capsule in the rat brain is clearly visible at birth. On the basis of its location (medially to the prepiriform claustrum) and probably its function (as a source of neurons for adjacent structures), we define this nucleus as the "paraclaustral reservoir". The present study reveals the cellular changes of the paraclaustral reservoir during postnatal development of the rat brain using unbiased morphometry and in situ DNA end labeling. During the first 4 days after birth the density and total number of cells in the paraclaustral reservoir were stable; after this period a decrease of these parameters was observed until the complete disappearance of this structure at the end of first postnatal week. The rather low number of TUNEL (TdT mediated dUTP nick end labeling of fragmented DNA) positive nuclei in the paraclaustral reservoir suggests that apoptosis is not a crucial mechanism leading to decay of this structure.

Relationship of calcium-binding protein containing neurons and projection neurons in the rat basolateral amygdala.

Two-laser and two-color approaches were used to observe the colocalization of the calcium-binding proteins, calbindin D28k and parvalbumin, and the retrograde tracer, Fluoro-Gold (FG) in the basolateral amygdala of the rat. The study was performed on five adult rats into which FG was injected to the frontal association cortex. Then, the localization of the retrogradely labeled neurons in the basolateral amygdala was compared with the localization of the neurons labeled by calcium-binding proteins. The present study showed that most of the retrogradely labeled neurons in the posterior part of the basolateral amygdala are also calbindin-positive. Even though a lot of parvalbumin-positive endings were present at the surface of the retrogradely labeled cells, we did not observe the colocalization of the parvalbumin and projective neurons.

The basolateral amygdaloid complex--its development, morphology and functions.

For many years the amygdaloid body has been an object of numerous investigations on different species, because the basolateral complex, being the main part of the amygdaloid body, is regarded as "sensory input" to this structure. It plays a very important role in so called emotional memory and learning, what is particularly important in early developmental stages. Impairment at this time may cause psychiatric problems in later life, like neurosis, phobia, unconscious fear or panic attacks. Complicated functions of the basolateral complex require precise control and modulation especially in early development. In this review the morphological changes during the development and maturation will be discussed and compared with neurotransmitter as well as with the expression of the calcium binding proteins at various stages of the development.

The corticoclaustral connections in the rat studied by means of the fluorescent retrograde axonal transport method.

The corticoclaustral connections in the rat were investigated by means of the method of the retrograde axonal transport of the fluorescent tracer (Fluoro-Gold; FG). The material consisted of 20 adult Wistar rats. The fluorescent tracer was injected into the anterior, middle or posterior parts of the claustrum. The retrogradely labeled neurons were detected in the layer VI of the neocortex. Injections of the tracer into the anterior part of the claustrum resulted in labeling of the neurons in the motor cortex. After administration of the tracer into the middle part of the insular claustrum, labeled neurons were present both in the motor and somatosensory cortices, while the injections of the tracer into its posterior part resulted in labeling of neurons in the visual cortex. Administration of the fluorescent tracer into the insular claustrum of the rat resulted in labeling of the cortical neurons of the corresponding areas of both hemispheres, however, the contralateral projections seem to be less numerous than the ipsilateral. Our results confirm the existence of reciprocal connections of the claustrum with the neocortex and suggest its role in integration and modification of information reaching the neocortex.

The amygdaloid body of the rabbit--a morphometric study using image analyser.

The amygdaloid body is a telencephalic structure belonging to the limbic system. The amygdaloid body consists of the two main nuclear groups: corticomedial and basolateral. The former-phylogenetically older group is composed of the central, medial, and cortical nuclei, while the latter, phylogenetically younger one, of the lateral, basolateral and basomedial ones. The results presented in our paper indicate differences in the structure and topography of the specific amygdaloid nuclei. Their subdivisions in the rabbit are not as evident as in the rat. Apart from structural differences, the cellular composition of specific nuclei does not differ distinctly. It can suggest that their intrinsic and extrinsic connections might be similar and the role and function of them is maintained (with few exceptions) through the phylogeny.

Parvalbumin immunoreactivity changes in the thalamic reticular nucleus during the maturation of the rat's brain.

The thalamic reticular nucleus (Rt) is a thin lamina of cells, through which thalamocortical and corticothalamic fibers pass. It is interposed between the thalamic nuclei and the internal capsule and it is composed of GABA-ergic cells with synapses that receive impulses from both kinds of fibers. Rt takes part in the negative feed-back system of controlling the information transfer from the thalamus to the cerebral cortex and it is focused in the sleep-waking cycle. The pattern of parvalbumin reactivity during maturation of Rt becomes the main aim of our study. The study was performed on 36 rats on various postnatal days (P0, P1, P2, P4, P5, P7, P10, P14, P17, P21, P30 and P90). The animals were anesthetized, transcardially perfused, cut on cryostat into 30-microns-thick frontal sections, stained immunocytochemically using standard ABC method and a mouse monoclonal antibody against parvalbumin. A small amount of round and oval, parvalbumin immunopositive cells was detected at stage PO, predominantly in the intermediate part of Rt, whereas the cells in ventral and lateral part at the same time were only slightly immunopositive. At P10 the cells in the intermediate part became more fusiform or oval because of the appearance of dendrites. At P14 we were able to observe separate, punctuated structures interpreted as the axonal endings. There were plenty of them at the time of full maturation of the intermediate portion of reticular nucleus (stage P21). At this time, the dorsal and ventral parts had their first synapses, too, but their maturation ended a week later. At P30 multipolar neurons, with round and fusiform somata were distributed relatively homogeneously throughout Rt. We compared the stage with the parvalbumin reactivity of the adult rat and found no difference in the morphological pattern of PV neurons.

The pattern of synaptophysin changes during the maturation of the amygdaloid body and hippocampal hilus in the rat.

Synaptophysin is an integral membrane protein associated with small, electron-lucent synaptic vesicles. Immunohistochemistry for this protein is a sensitive method to study subtle changes in synaptic density and distribution in various brain regions. In the present study, the synaptogenesis was examined in the rat basolateral amygdala in comparison with the hippocampal hilus, from the day of birth to adulthood. A total of 41 brains at various ages starting from P0 to P90 (P--postnatal day) were examined. After perfusional fixation the brains were frozen and cut in the coronal plane and stained either with cresyl violet or standard immunohistochemical methods using the anti-synaptophysin antibody. Synaptophysin positive granules appeared just after birth in both structures, but their number was very low (about 0.28 x 10(6) and 0.13 x 10(6) per mm3 in the amygdala and hippocampus, respectively). In the basolateral amygdala the number of synapses increased rapidly reaching the maximum at P14 (1.6 x 10(6) per mm3) followed by about 45% decrease in number up to P30 and later being stabile. In the hippocampus two increases of the synaptogenesis were observed. The first at P7 (about 1.7 x 10(6) of synapses per 1 mm3) which was followed by dramatic decrease up to 0.7 x 10(6) per mm3 at P14. The second increase appeared later (about P90) and reached 1.7 x 10(6) per mm3. After that time the density of synapses was stabile. It may be supposed that the first characteristic wave of synaptogenesis observed in the hippocampus and amygdaloid body is due to the overproduction of synapses observed at that time in other cortical regions. The late wave of synaptogenesis found in the hippocampus is related to the great plasticity of the interneuronal connections in this period of development.

Neuronal changes in the basolateral complex during development of the amygdala of the rat.

Neuronal changes in the amygdala basolateral complex were studied during development and maturation in fetal and postnatal rat brains using morphometrical methods. Forty brains of animals of various ages were fixed in formalin, frozen and cut into 25 microm thick sections and stained with cresyl violet or haematoxylin and eosin (H&E). In cresyl violet preparations, the complex appeared for the first time on embryonic day (E)17 and was composed of two homogeneous nuclei lateral and basolateral. On about the seventh postnatal day, each of these nuclei was divided into two parts the first one into the dorsolateral and ventromedial and the second one into the anterior and posterior. Morphometric investigations showed a different increase of the neuronal and nuclear size in various parts of the basolateral complex up to postnatal day (P)14; after that time these parameters did not change significantly. The neuronal density and the total number of neurons stabilized at P7 in all parts of this complex, except for the dorsolateral part of the lateral nucleus in which a 30% decrease of the total number of cells was observed. From P14, in all nuclei under study, the total number of neurons did not change significantly.

Volume and topographical changes of the basolateral complex during the development of the rat's amygdaloid body.

Volume and topography of basolateral complex during development and maturation in fetal and postnatal rat brains were studied using morphometrical methods. 39 rat brains of various ages were fixed in formalin, frozen and cut into 25-micron-thick sections and stained with cresyl violet. In cresyl violet preparation the basolateral complex appeared first on 17th prenatal day and it was composed of two homogenous parts--lateral and basolateral nuclei. On about 7th postnatal day each of these nuclei divided into two parts--the first one into the dorsolateral and ventromedial part, while the second one--into the anterior and posterior parts. Morphometric investigations showed that volume of the basolateral complex and its parts underwent the biggest changes up to 14th postnatal day, and we suspected that during this time it was more sensitive to pathological changes. After that day the volume of the basolateral complex had changed only a little.

Acetylcholinesterase activity as a marker of maturation of the basolateral complex of the amygdaloid body in the rat.

Development and maturation of the basolateral complex of the amygdaloid body were studied in fetal and postnatal rat brains. In cresyl violet-stained sections the basolateral complex was distinguishable at the 17th prenatal day. On about the 14th postnatal day, it showed all the features of the adult structure. The acetylcholinesterase activity appeared in neuropil of the basolateral complex on the seventh postnatal day; it increased slowly up to the 60th day. After that age, the acetylcholinesterase activity was stable in all parts of the basolateral complex and corresponded to its cytoarchitectonic differentiation. Our results suggest that the process of maturation of the amygdaloid basolateral complex, being related to the ingrowing of the cholinergic fibers from the basal forebrain, lasts at least up to the end of the second month of postnatal life.

Loss of neurons in the claustrum of aging brain.

The study was performed on 19 brains of nondemented patients with age ranging from 36 to 89 years. After embedding in paraffin, coronal 8-microns-thick serial sections were cut and stained either with cresyl violet or with immunocytochemical methods for amyloid and tangles. Morphometrical studies were performed in all parts of the claustrum along its whole extension. Changes related to aging (neuronal loss and decrease in volume) were found in all parts of the claustrum, but the time of origin of these changes is different in various parts of this structure. We did not observe neurofibrillary pathology in any parts of the claustrum. In the oldest subjects a small number of amyloid plaques was found in the paraamygdalar part of the claustrum. We suggest that the neurons of the claustroneocortical loop are affected severely and earlier than those in the claustroentorhinal loop.

Division of the human claustrum according to its architectonics and morphometric parameters.

The topography and cytoarchitectonics of the claustrum as well as morphometric parameters of its neurons were studied in 10 human brains obtained from patients without any detectable neuropathological changes. We distinguished four parts of the claustrum: dorsal, orbital, temporal and paraamygdalar. The dorsal and orbital parts contain larger cells, than those of the temporal and paraamygdalar parts, although these differences were statistically non significant. The highest neuronal density was observed in the paraamygdalar part. The nucleus and nucleus@cell body area ratio was significantly smaller in the dorsal part than in other parts of the claustrum. We described three types of neurons in the claustrum: (1) medium-sized either fusiform or triangular cells with darkly stained cytoplasm; they predominate in the dorsal and temporal parts, (2) medium-sized as well as large cells, either multipolar or pyramidal-like with lightly stained cytoplasm; they are most numerous in the orbital and paraamygdalar parts, (3) small, multipolar or oval neurons with darkly stained ring of cytoplasm; these types of neurons are uniformly distributed throughout all parts of the claustrum. The subdivision of the human claustrum is in accordance with our observations that each of these parts possesses connections with different cortical regions.