A lectin horseradish peroxidase study of the origin of ascending fibers in the medial forebrain bundle of the rat. The upper brainstem

The origins of projections within the medial forebrain bundle from the upper brainstem were examined with the horseradish peroxidase technique. Labeled cells were found in approximately 15 upper brainstem nuclei following injections of a conjugate of horseradish peroxidase and wheat germ agglutinin at various levels of the medial forebrain bundle. Labeled nuclei included (from caudal to rostral): dorsal and ventral parabrachial nuclei; Kolliker-Fuse nucleus; dorsolateral tegmental nucleus; A7 (lateral pontine tegmentum medial to lateral lemniscus); median and dorsal raphe nuclei; distinct group of cells oriented mediolaterally in the dorsal pontine tegmentum below the central gray; B9 (ventral midbrain tegmentum dorsal to medial lemniscus); retrorubral nucleus; nucleus of Darkschewitsch, interfascicular nucleus; rostral and caudal linear nuclei; ventral tegmental area; medial part of substantia nigra, pars compacta; and the supramammillary nucleus. With the exception of the ventral parabrachial nucleus, Kolliker-Fuse, A7, B9 and substantia nigra, pars compacta, each of the nuclei mentioned above sent strong projections along the medial forebrain bundle to the rostral forebrain. Sparse labeling was observed throughout the pontine and midbrain reticular formation. With the exception of the dorsal raphe nucleus, projections to the most anterior regions of the medial forebrain bundle (level of the anterior commissure) essentially only arose from presumed dopamine-containing nuclei-retrorubral nucleus (A8 area), interfascicular nucleus, rostral and caudal linear nuclei, substantia nigra pars compacta, and ventral tegmental area. Evidence was reviewed indicating that major forebrain sites of termination for these dopaminergic nuclei are structures that have been collectively referred to as the 'ventral striatum'. It is concluded from the present findings that several pontine and mesencephalic cell groups are in a position to exert a strong, direct effect on structures in the anterior forebrain and that the medial forebrain bundle is the main communication route between the upper brainstem and the forebrain.     

Morphological variability, lectin binding and Na+,K+-activated adenosine triphosphatase activity of isolated Müller (glial) cells from the rabbit retina

Rabbit retinal Müller cells were isolated by means of papaine and mechanical dissociation. These cells were shown to have a well preserved morphology and to preserve viability for many hours. Intense wheat germ agglutinin binding occurs on the photoreceptor side of Müller cells, especially in the microvillous region. Rabbit retinal Müller cells have a Na+,K+-activated adenosine triphosphatase activity in the same order of magnitude as brain astroglial cells.     

A quantitative study of the brainstem cholinergic projections to the ventral part of the oral pontine reticular nucleus (REM sleep induction site) in the cat

The ventral part of the cat oral pontine reticular nucleus (vRPO) is the site in which microinjections of small dose and volume of cholinergic agonists produce long-lasting rapid eye movement sleep with short latency. The present study determined the precise location and proportions of the cholinergic brainstem neuronal population that projects to the vRPO using a double-labeling method that combines the neuronal tracer horseradish peroxidase–wheat germ agglutinin with choline acetyltransferase immunocytochemistry in cats. Our results show that 88.9% of the double-labeled neurons in the brainstem were located, noticeably bilaterally, in the cholinergic structures of the pontine tegmentum. These neurons occupied not only the pedunculopontine and laterodorsal tegmental nuclei, which have been described to project to other pontine tegmentum structures, but also the locus ceruleus complex principally the locus ceruleus and peri-, and the parabrachial nuclei. Most double-labeled neurons were found in the pedunculopontine tegmental nucleus and locus ceruleus complex and, much less abundantly, in the laterodorsal tegmental nucleus and the parabrachial nuclei. The proportions of these neurons among all choline acetyltransferase positive neurons within each structure were highest in the locus ceruleus complex, followed in descending order by the pedunculopontine and laterodorsal tegmental nuclei and then, the parabrachial nuclei. The remaining 11.1% of double-labeled neurons were found bilaterally in other cholinergic brainstem structures: around the oculomotor, facial and masticatory nuclei, the caudal pontine tegmentum and the praepositus hypoglossi nucleus. The disperse origins of the cholinergic neurons projecting to the vRPO, in addition to the abundant noncholinergic afferents to this nucleus may indicate that cholinergic stimulation is not the only or even the most decisive event in the generation of REM sleep.     

Extra-hypothalamic afferent inputs to the supraoptic nucleus area of the rat as determined by retrograde and anterograde tracing techniques

To detect neuronal cell bodies whose axon projects to the hypothalamic supraoptic nucleus, small volumes (10-50 nl) of 30% horseradish peroxidase or 2% fast blue solutions were pressure-injected into the area of one supraoptic nucleus of rats. Both dorsal and ventral approaches to the nucleus were used. In animals where the injection site extended beyond the limits of the supraoptic nucleus, retrogradely labelled cell bodies were found in many areas of the brain, mainly in the septum, the nucleus of the diagonal band of Broca and ventral subiculum in the limbic system; the dorsal raphe nucleus, the locus coeruleus, the nucleus of the dorsal tegmentum, the dorsal parabrachial nucleus, the nucleus of the solitary tract and the catecholaminergic A1 region in the brain stem; in the subfornical organ and the organum vasculosum of the lamina terminalis, as well as in the median preoptic nucleus. In contrast, when the site of injection was apparently restricted to the supraoptic nucleus, labelling was only clearcut in the two circumventricular organs, the median preoptic nucleus, the nucleus of the solitary tract and the A1 region. Injections of wheat germ agglutinin coupled with horseradish peroxidase (60-80 nl of a 2.5% solution) made in the septum and in the ventral subiculum anterogradely labelled fibers coursing in an area immediately adjacent to the supraoptic nucleus but not within it. In contrast, labelling within the nucleus was found following anterograde transport of tracer deposited in the A1 region and in an area that includes the nucleus of the solitary tract. Neurones located in the perinuclear area were densely labelled by small injections into the supraoptic nucleus; they may represent a relay station for some afferent inputs to the supraoptic nucleus. These results suggest that the supraoptic nucleus is influenced by the same brain areas which project to its companion within the magnocellular system, the paraventricular nucleus.     

Production of a monoclonal antibody for evaluation of hard red winter wheat cultivars to wheat streak mosaic virus

The monoclonal antibody technology has provided a means to produce a supply of highly specific uniform antibody which is useful in the detection of plant viruses and which facilitates disease resistance screening. Because of the specificity of a monoclonal antibody to an epitope, a monoclonal antibody may not react to a partially degraded protein. Wheat streak mosaic virus (WSMV) is a member of the potyvirus group and is transmitted by the wheat curl mite Eriophyes tulipae Keifer. The capsid protein of WSMV, like many potyviruses, is degraded in planta. Monoclonal antibodies produced to WSMV reacted to native as well as trypsin treated virions. The antibodies were also useful for evaluation of hard red winter wheat cultivars inoculated with WSMV in the fall or in the spring under field conditions.     

Characterization of the translocated chromosome using fluorescencein situ hybridization and random amplified polymorphic DNA on twoTriticum aestivum—Thinopyrum intermedium translocation lines resistant to wheat streak mosaic or barley yellow dwarf virus

Fluorescencein situ hybridization (FISH) was used to determine the breakpoint of the translocation chromosome in two bread wheat (Triticum aestivum) germplasm lines withThinopyrum intermedium chromatin carrying resistance to either wheat streak mosaic virus (WSMV) or barley yellow dwarf virus (BYDV). In addition, genome-specific random amplified polymorphic DNA (RAPD) markers were used to ascertain the genomic sources of theTh. intermedium chromosomes carrying the WSMV or BYDV resistance. CI17766, a WSMV-resistant wheat germplasm line derived from induced homoeologous pairing by using theph1b mutant, had a translocation chromosome composed of the complete 4AL and about 45% of proximal 4AS from wheat, and the entire 4ES ofTh. intermedium. The BYDV-resistant translocation line, TC14, derived from tissue culture, had a very short distal segment of 7StL fromTh. intermedium terminally attached to 56% of the proximal 7DL. These observations indicate that translocations in these wheat germplasm lines did not involve centromeric breaks and fusion but were a result of homoeologous chromosome recombination.accepted for publication by J. S. (Pat) Heslop-Harrison     

Isolation and partial characterization of the tegumental outer membrane of schistosomula of Schistosoma mansoni

Separation of the external membranes from freshly converted mechanical schistosomula of Schistosoma mansoni was achieved by osmotic shock under hypertonic conditions, followed by mechanical shearing and ultracentrifugation. Prior to treatment, the schistosomula were surface labeled by introduction of N-DNP-epsilon-aminocaproylphosphatidylethanolamine molecules into their lipid bilayer followed by anti-DNP antibodies and stained with either 125I-protein-A or ferritin labeled secondary anti-DNP antibodies. This label provided a membrane marker by which the purity of the preparation could be assessed at each stage. Fluorescence staining with FITC-conjugated secondary antibodies prior to treatment revealed that the homogeneously stained membrane of the intact schistosomula became swollen and ruptured after the osmotic shock. The isolated membrane pellet was intensely fluorescent. Electron microscopical examination revealed mostly vesicles, some of them with organized multilayer assembly. The vesicles were ferritin labeled, indicating that they originated from the outer surface membrane of the schistosomula. A 100 fold enrichment in the alkaline phosphatase activity and about 300 fold enrichment in acetylcholinesterase activity in the membrane preparations, as compared to the intact schistosomula, was found. The isolated tegument was analyzed by SDS-polyacrylamide gel electrophoresis. The pattern obtained showed three major bands, of molecular weights 69 000, 45 000 and 12 000 alongside with a large number of minor bands. Immunoprecipitation of the isolated 125I-labeled membrane antigens with antisera from chronically infected mice revealed these three major bands together with three other bands of molecular weight 38 000, 23 000 and 16 000.     

The laminar distribution of cortical connections with the tecto- and cortico-recipient zones in the cat's lateral posterior nucleus

The anterograde and retrograde transport of wheat germ agglutinin congugated to horseradish peroxidase was used to examine the laminar organization of cortical connections with the two visual zones that comprise the cat's lateral posterior nucleus. Microelectrophoretic deposits of the tracer into the principal tecto-recipient zone in the medial division of the lateral posterior nucleus revealed reciprocal connections with the following cortical fields: areas 19 and 21a, the medial and lateral banks of the middle suprasylvian sulcus, and the dorsal and ventral banks of the lateral suprasylvian sulcus, which correspond to the dorsal lateral suprasylvian and ventral lateral suprasylvian visual areas of Palmer et al. [(1978) Brain Res. 177, 237-256] and an area in the fundus of the posterior suprasylvian sulcus. In each of these cortical areas two distinct populations of cells were labeled, small pyramidal neurons in layer VI and large pyramidal cells in layer V. Overlying these backfilled cells were two bands of anterograde label, a narrow strip in layer I and a wide band centered in layer IV. Deposits of wheat germ agglutinin conjugated to horseradish peroxidase confined to the striate-recipient zone in the lateral portion of the lateral posterior nucleus resulted in cortical label in areas 17, 18, 19, 20a and b, 21a, the medial and lateral banks of the middle suprasylvian sulcus, the posterior suprasylvian sulcus and in the fundus of the splenial sulcus. In all cortical areas other than 17 and 18, the laminar distribution of label was the same as that found after deposits of the tracer into the medial division of the lateral posterior nucleus. In contrast, areas 17 and 18 contained backfilled cells that were confined to layer V and anterograde label that was restricted to layer I. These findings indicate that the cortical areas that receive a direct projection from the A laminae of the dorsal lateral geniculate nucleus maintain a distinct laminar organization of reciprocal connections with the extrageniculate visual thalamus. Conversely, all other visual areas of the cortex share a common pattern of reciprocal connections with both the tecto- and striate-recipient zones of the lateral posterior nucleus.     

Projections from the paratrigeminal nucleus and the medullary and spinal dorsal horns to the peribrachial area in the cat

The projections from the medullary and spinal dorsal horns to the dorsolateral pons were investigated in the cat utilizing both the retrograde and anterograde transport of a wheat germ agglutinin-horseradish peroxidase complex and the retrograde transport of the fluorescent dyes Fast Blue and Nuclear Yellow. After injections of wheat germ agglutinin-horseradish peroxidase into the area surrounding the brachium conjunctivum, numerous neurons were labeled ipsilaterally near levels of the obex in the paratrigeminal nucleus. Such neurons were located in connected pockets of neuropil located within the spinal trigeminal tract and along its medial edge. Most of the neurons labeled in the dorsal horns after such injections were found in lamina I. Those found in the medullary dorsal horn were mostly ipsilateral to the injection while those in the spinal dorsal horn were found bilaterally. Some labeled neurons were also found in lamina V of both the medullary and spinal dorsal horns bilaterally. When the injection was centered in either the medial parabrachial nucleus or the Kolliker-Fuse nucleus, a greater number of neurons were labeled ipsilaterally in lamina V of the medullary dorsal horn. Since neurons in lamina I of the medullary dorsal horn also project to the medial thalamus, fluorescent dyes were used to determine if the same neuron might project to both targets. Fast Blue was first injected into either the peribrachial area or the medial thalamus. After an appropriate period, Nuclear Yellow was injected into that target not injected first with Fast Blue. The injection of Nuclear Yellow was always placed on the side of the brain opposite to the first injection. Both dyes were transported retrogradely and were found in neurons located in lamina I of the medullary dorsal horn. However, no double-labeled neurons were seen. In general those labeled after injections of the medial thalamus were more superficial than those labeled after injections of the dorsolateral pons. The anterograde transport of wheat germ agglutinin-horseradish peroxidase was used to determine the termination of the projections from neurons in the medulary dorsal horn and the cervical spinal cord to the peribrachial area. After injections into these areas a moderate to sparse labeling of the lateral parabrachial nucleus and the Kolliker-Fuse nucleus was seen. It was mostly ipsilateral in cases with injections of the medullary dorsal horn but was bilateral following injections into the cervical enlargement.(ABSTRACT TRUNCATED AT 400 WORDS)     

A lectin horseradish peroxidase study of the origin of ascending fibers in the medial forebrain bundle of the rat. The lower brainstem

The origins of projections within the medial forebrain bundle from the lower brainstem were examined with the horseradish peroxidase technique. Labeled cells were found in at least 15 lower brainstem nuclei following injections of a conjugate or horseradish peroxidase and wheat germ agglutinin at various levels of the medial forebrain bundle. Dense labeling was observed in the following cell groups (from caudal to rostral): A1 (above the lateral reticular nucleus); A2 (mainly within the nucleus of the solitary tract); a distinct group of cell trailing ventrolaterally from the medial longitudinal fasciculus at the level of the rostral pole of the inferior olive; raphe magnus; nucleus incertus; dorsolateral tegmental nucleus (of Castaldi); locus coeruleus; nucleus subcoeruleus; caudal part of the dorsal (lateral) parabrachial nucleus; and raphe pontis. Distinct but light labeling was seen in raphe pallidus and obscurus, nucleus prepositus hypoglossi, nucleus gigantocellularis pars ventralis, and the ventral (medial) parabrachial nucleus. Sparse labeling was observed throughout the medullary and caudal pontine reticular formation. Several lower brainstem nuclei were found to send strong projections along the medial forebrain bundle to very anterior levels of the forebrain. They were: A1, A2, raphe magnus (rostral part), nucleus incertus, dorsolateral tegmental nucleus, raphe pontis and locus coeruleus. With the exception of the locus coeruleus, attention has only recently been directed to the ascending projections of most of the nuclei mentioned above. Evidence was reviewed indicating that fibers from lower brainstem nuclei with ascending medial forebrain bundle projections distribute to widespread regions of the forebrain.It is concluded from the present findings that several medullary cell groups are capable of exerting a direct effect on the forebrain and that the medial forebrain bundle is the major ascending link between the lower brainstem and the forebrain.