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PubMed1996

Non-mirror-symmetric patterns of callosal linkages in areas 17 and 18 in cat visual cortex.

In the cat, callosal connections in area 17 are largely confined to a 5-6-mm-wide strip at the 17/18 border. It is commonly thought that callosal fibers extending from between the 17/18 border regions interconnect loci that are mirror-symmetric with respect to the midline of the brain, but this idea has not been tested experimentally. The present study examined the organization of callosal linkages in the 17/18 border region of normal adult cats by analyzing the patterns of connections revealed in one hemisphere after small injections of different fluorescent tracers into the opposite 17/18 callosal region. The location of the injection sites within areas 17 and 18 was assessed by examining architectonic data and by inspecting the labeling pattern in the ipsilateral visual thalamus. Area 17 and 18 were separated by a 1-1.5-mm-wide zone of cytoarchitectonic transition rather than by a sharp border. The results show that, in general, callosal fibers interconnect loci that are not mirror-symmetric with respect to the midline. Thus, area 17 injections placed nearly 3 mm away from the 17/18 transition zone produced discrete labeled areas located preferentially within the contralateral 17/18 transition zone. However, when the injection site was within the 17/18 transition zone, labeled cells were found primarily medial and lateral to, but not within, the 17/18 transition zone in the contralateral hemisphere. Previous studies have indicated that the 17/18 transition zone contains a representation of a strip of the ipsilateral visual field. Comparison of the retinotopy of the 17/18 border region with the mirror-reversed pattern of callosal linkages found in the present study suggests that callosal fibers link points that are in retinotopic correspondence in both hemispheres.

Animals↗
PubMed2000

Organization of callosal linkages in visual area V2 of macaque monkey.

In visual area V2 of the macaque monkey callosal cells accumulate in finger-like bands that extend 7-8 mm from the V1/V2 border, or approximately half the width of area V2. The present study investigated whether or not callosal connections in area V2 link loci that are located at the same distance from the V1/V2 border in both hemispheres. We analyzed the patterns of retrograde labeling in V2 resulting from restricted injections of fluorescent tracers placed at different distances from the V1/V2 border in contralateral area V2. The results show that varying the distance of V2 tracer injections from the V1/V2 border led to a corresponding variation in the location of labeled callosal cells in contralateral V2. Injections into V2 placed on or close to the V1 border produced labeled cells that accumulated on or close to the V1 border in contralateral V2, whereas injections into V2 placed away from the V1 border produced labeled cells that accumulated mainly away from the V1 border. These results provide evidence that callosal fibers in V2 preferentially link loci that are located at similar distances from the V1/V2 border in both hemispheres. Relating this connectivity pattern to the topographic map of V2 suggests that callosal fibers link topographically mirror-symmetrical regions of V2, i.e., callosal fibers near the V1/V2 border interconnect areas representing visual fields on, or close to, the vertical meridian, whereas callosal connections from regions away from the V1/V2 border interconnect visuotopically mismatched visual fields that extend onto opposite hemifields.

Animals↗
PubMed2002

Origins of callosal projections to the supplementary motor area (SMA): a direct comparison between pre-SMA and SMA-proper in macaque monkeys.

The two subdivisions of the supplementary motor area (SMA), the pre-SMA (rostrally) and SMA-proper (caudally), exhibit distinct functional properties and clear differences with respect to their connectivity with the spinal cord, the thalamus, and other homolateral motor cortical areas. The goal of the present study was to establish in monkeys whether these subdivisions also differ with regard to their callosal connectivity. Two fluorescent retrograde tracers (Fast Blue and Diamidino Yellow) were injected in each animal, one in the pre-SMA and the second in the SMA-proper. Tracer injections in the pre-SMA or in SMA-proper resulted in significant numbers of labeled neurons in the opposite SMA, premotor cortex (PM), cingulate motor areas (CMA), and cingulate gyrus. Labeled neurons in M1 were rare, being observed only after injection in the SMA-proper. The two subdivisions of the SMA differed in the proportion of labeled neurons found across areas providing their callosal inputs. The SMA-proper receives about half of its callosal inputs from its counterpart in the other hemisphere (42-65% across monkeys). A comparable proportion of neurons was found in the pre-SMA after injection in the opposite pre-SMA (32-47%). The pre-SMA receives more callosal inputs from the rostral halves of the dorsal PM, the ventral PM, and the CMA than from their caudal halves. In addition, the pre-SMA, but not the SMA-proper, receives callosal inputs from the prefrontal cortex. The SMA-proper receives more callosal inputs from the caudal halves of the dorsal PM and ventral PM than from their rostral halves. The two subdivisions of the SMA receive callosal inputs from the same cortical areas (except the prefrontal cortex and M1), but they differ with respect to the quantitative contribution of each area of origin. In conclusion, quantitative data now support the notion that pre-SMA receives more transcallosal inputs than the SMA-proper.

Amidines↗
PubMed1981

Synaptic termination of thalamic and callosal afferents in cingulate cortex of the rat.

The distribution of degenerating thalamic and callosal afferents to cingulate cortex in the rat is analyzed. Both light microscopic silver impregnation and quantitative electron microscopic techniques demonstrate differences in the form, number, and laminar distribution of these two afferents in anterior and posterior cingulate cortices. Afferents from the mediodorsal thalamic nucleus terminate in area 24. Most terminals are in layer IIIb, fewer in layer Ia-b, and least in layers V and VI. In contrast, callosal afferents terminate mainly in layers Ib-c, II, IIIa, V, and VI. Thus, thalamic and callosal afferents terminate in a complementary pattern except in layers Ib and IIIb where they overlap. Quantitative analysis of degenerating axon terminals in area 24 indicates that there may be as many as seven times more callosal than mediodorsal thalamic terminals in this cortex. Projections of the anterior thalamic nuclei terminate in areas 29b and 29c, primarily in layer Ia, with fewer in layers Ib-IV and least in layers V and VI. Callosal afferents end mainly in layers V and VI and less densely in layers I-IV, which results in some overlap of thalamic and callosal afferents in layers Ic, IV, and V. In addition, patterns of termination of callosal afferents in posterior cingulate cortex change at borders between previously defined cytoarchitectural areas. Anterior thalamic terminals in area 29c differ from other thalamocortical afferents described previously in that they form two types of terminals. One is large (2-4 micrometer in diameter) and occurs mainly in layer Ia, whereas the second type is smaller and is present in layers Ib-V. Both types of terminals form asymmetric synapses mainly with dendritic spines.

Afferent Pathways↗
PubMed1984

The organization of immature callosal connections.

In newborn kittens, the anterograde transport of horseradish peroxidase, alone or bound to wheat-germ agglutinin, indicates that callosal axons have entered selectively the restricted portions of the neocortical gray matter (e.g., the area 17/18 border) which receive callosal afferents in adults. The callosal axons do also reach regions where they lack in the adult, but there they seem not to penetrate far into the gray matter. Neonatal injections of retrograde fluorescent tracers restricted to the gray matter in areas 17, 18, and posteromedial lateral suprasylvian area (PMLS) label neurons in the contralateral hemisphere only when the tracers were directed into regions known to receive callosal axons. In particular, injections near the 17/18 border label neurons in the contralateral hemisphere at the homologous site and at restricted, retinotopically corresponding locations in other visual areas: a pattern similar to the adult one. In contrast, an injection reaching the white matter of areas 17 or 18 labels a wider, continuous territory extending mediolaterally over most visual areas from 17 to posterolateral lateral suprasylvian area (PLLS) and including regions which later become acallosal; in addition, labeled neurons are found in the limbic cortex medial to area 17 and in the auditory cortex lateral to PLLS, none of which is known to project to either 17 or 18 in the adult. In flattened reconstructions of the cortex, the shape of the territory labeled by each of these injections is characteristically, although somewhat irregularly, crescent shaped; its rostrocaudal position varies with that of the injection. An injection extending into the white matter of more lateral visual areas (19, 21a, PMLS) labels callosal neurons over a similar territory, which extensively overlaps that labeled by the 17/18 border injections and likewise includes regions which are acallosal in the adult. In spite of the overlapping distribution of labeling obtained from separate injection sites, as in adults, each cytoarchitectonically (or retinotopically) defined area seems to receive from a different set of neurons, although a few neurons send bifurcating axons to more than one area. In conclusion, injections restricted to the cortical gray matter reveal a topographic organization of juvenile callosal connections similar to that of the adult. In contrast, injections extending into the white matter and adequate to reach the transitory callosal axons which appear to be confined there reveal what appears to be an earlier organization. These two organizations probably reflect different morphogenetic factors.

Animals↗
PubMed1986

Organization of the callosal connections of visual areas V1 and V2 in the macaque monkey.

The interhemispheric efferent and afferent connections of the V1/V2 border have been examined in the adult macaque monkey with the tracers horseradish peroxidase and horseradish peroxidase conjugated to wheat germ agglutinin. The V1/V2 border was found to have reciprocal connections with the contralateral visual area V1, as well as with three other cortical sites situated in the posterior bank of the lunate sulcus, the anterior bank of the lunate sulcus, and the posterior bank of the superior temporal sulcus. Within V1, callosal projecting cells were found mainly in layer 4B with a few cells in layer 3. Anterograde labeled terminals were restricted to layers 2, 3, 4B, and 5. In extrastriate cortex, retrograde labeled cells were in layers 2 and 3 and only very rarely in infragranular layers. In the posterior bank of the lunate sulcus, labeled terminals were scattered throughout all cortical layers except layers 1 and 4. In the anterior bank of the lunate sulcus and in the superior temporal sulcus, anterograde labeled terminals were largely focused in layer 4. Callosal connections in all contralateral regions were organized in a columnar fashion. Columnar organization of callosal connections was more apparent for anterograde labeled terminals than for retrograde labeled neurons. In the posterior bank of the lunate sulcus, columns of callosal connections were superimposed on regions of high cytochrome activity. The tangential extent of callosal connections in V1 and V2 was found to be influenced by eccentricity in the visual field. Callosal connections were denser in the region of V1 subserving foveal visual field than in cortex representing the periphery. In V1 subserving the fovea, callosal connections extended up to 2 mm from the V1/V2 border and only up to 1 mm in more peripheral located cortex. In area V2 subserving the fovea, cortical connections extended up to 8 mm from the V1/V2 border and only up to 3 mm in peripheral cortex.

Animals↗
PubMed1986

Callosal connectivity of areas V1 and V2 in the newborn monkey.

The callosal connectivity of areas V1 and V2 in the newborn monkey has been investigated with the neuroanatomical tracers wheat germ agglutinin conjugated to horseradish peroxidase and free horseradish peroxidase. In the adult, callosal projecting neurons in cortex subserving the lower parafoveal visual field were found to extend from the V1/V2 border for a distance of 1-2.5 mm into V1 and 8 mm into V2. In the newborn, the tangential extent and total number of callosal projecting neurons were the same as in the adult. Within area V1, callosal projecting neurons in the adult and newborn were limited to supragranular layers. In the adult, axon terminals of callosal projections were located in layers 4B and 5 and were excluded from layer 4C. In the newborn, axon terminals were more extensively distributed than in the adult and invaded layer 4C. In area V2, the laminar distribution and the patchy location of callosal connections in regions of high cytochrome oxidase activity were similar in the newborn and adult animals. In both newborns and adults, the patchy distribution of callosal projections persisted when the neuroanatomical tracers were injected over extensive regions of the contralateral striate and extrastriate cortex. In the adult, area V1 and V2 project contralaterally to two heterotopic sites located in the fundus of the lunate sulcus and the superior temporal sulcus. This was also found to be the case in the newborn. In the adult the terminals of these heterotopic projections were focused in layer 4. This was not the case in the newborn, where after injection limited to the contralateral V1/V2 border they were more evenly distributed among the different cortical layers. Following extensive contralateral injection of tracer, terminals in cortex anterior to V2 were focused over layer 5 and the bottom of layer 4.

Animals↗
PubMed1991

Synapses made by axons of callosal projection neurons in mouse somatosensory cortex: emphasis on intrinsic connections.

This is one of a series of papers aimed at identifying the synaptic output patterns of the local and distant projections of subgroups of pyramidal neurons. The subgroups are defined by the target site to which their main axon projects. Pyramidal neurons in areas 1 and 40 of mouse cerebral cortex were labeled by the retrograde transport of horseradish peroxidase (HRP) transported from severed callosal axons in the contralateral hemisphere. Terminals of the local axon collaterals of these neurons ("intrinsic" terminals) were identified in somatosensory areas 1 and 40, and their distribution and synaptic connectivity were examined. Also examined were the synaptic connections of "extrinsic" callosal axon terminals labeled by lesion induced degeneration consequent to the severing of callosal fibers. A post-lesion survival time of 3 days was chosen because by this time the extrinsic terminals were all degenerating, whereas the intrinsic terminals were labeled by HRP. Both intrinsic and extrinsic callosal axon terminals occurred in all layers of the cortex where they formed only asymmetrical synapses. Layers II and III contained the highest concentrations of both types of callosal axon terminal. Analyses of serial thin sections through layers II and III in both areas 1 and 40 yielded similar results: 97% of the extrinsic (277 total sample) and of the intrinsic (1215 total sample) callosal axon terminals synapsed onto dendritic spines, likely those of pyramidal neurons; the remainder synapsed onto dendritic shafts of both spiny and nonspiny neurons. Thus the synaptic output patterns of intrinsic vs. extrinsic callosal axon terminals are strikingly similar. Moreover, the high proportion of axospinous synapses formed by both types of terminal contrasts with the proportion of asymmetrical, axospinous synapses that occur in the surrounding neuropil where only about 80% of the asymmetrical synapses are onto spines. This result is in accord with previous quantitative studies of the synaptic connectivities of both extrinsic and intrinsic axonal pathways in the cortex (White and Keller, 1989: Cortical Circuits; Boston: Birkhauser): in all instances, axonal pathways are highly selective for the types of elements with which they synapse.

Animals↗
PubMed1994

Pyramidal and nonpyramidal callosal cells in the striate cortex of the adult rat.

The aim of this study has been to determine the neuronal types (pyramidal and nonpyramidal) within the rat's visual cortex, which project through the corpus callosum. To this end, the morphology and laminar distribution of callosal cells have been investigated by combining Diamidino Yellow retrograde tracing with intracellular injection of Lucifer Yellow in slightly fixed tissue slices. The visual callosal projection arises from pyramidal cells of diverse morphology in layers II to VIb, as well as from several modified pyramids located mainly in layers II, IV (star pyramids) and VIb (horizontal or inverted pyramids and related forms of spiny stellate cells). Our results indicate that in rats, as in other mammals, several types of nonpyramidal neurons also contribute to the contralateral projection. Bitufted cells in layers II-III and V were found to project contralaterally. Moreover, a spine-free layer V cell and a sparsely spiny multipolar neuron of layer IV were also labeled. In both stellate cells, partial axonal labeling reveals that these callosal cells display a local axonal arborization. Finally, our results of retrograde transport with Diamidino Yellow and with another sensitive retrograde tracer, the beta subunit of the cholera toxin, demonstrate for the first time that the two main neuronal types of layer I participate in the callosal projection. In layer I, several small horizontal cells of the inner half of layer I and a large subpial cell displaying long radiating dendrites were also injected. The latter cell may correspond to the Cajal-Retzius cell of the adult rat. In spite of the important differences in the organization of the visual system between rodents and cats, the callosal projection in both mammals is composed of a large variety of pyramidal cells and several nonpyramidal neurons. This high morphological diversity suggests that the callosal projection is much more physiologically complex than the extracortical efferents of the visual cortex, resembling other cortico-cortical connections. The roles that the different callosal cells may play in the processing of visual information are discussed in relation to the known functions of the corpus callosum.

Amidines↗
PubMed1990

Morphological evidence for callosally projecting nonpyramidal neurons in rat visual cortex.

This investigation shows that some of the callosally projecting neurons in rat visual cortex are nonpyramidal cells. Callosally projecting neurons were labeled by injections of horseradish peroxidase (HRP) into the area 17/18a border zone of the contralateral hemisphere. The retrogradely transported HRP was visualized with diaminobenzidine or with tetramethylbenzidine. In some of the labeled neurons the reaction product was diffuse, so that the neurons had a Golgi-like appearance, but in others the reaction product was granular, or punctate. The majority of neurons with a Golgi-like appearance were pyramidal cells, but one callosally projecting neuron from layer V area 18a was confirmed by electron microscopy to be a nonpyramidal neuron. This dearth of well-filled nonpyramidal cells suggested that callosally projecting nonpyramidal neurons may not transport sufficient HRP to show Golgi-like filling, and so punctately labeled neurons from areas 17, 18a and 18b were examined. Reacted sections from areas 17, 18a and 18b of control animals, into which no tracer had been injected, were also examined, but in these control preparations no granules similar to the HRP granules within the neuronal profiles of the experimental animals were encountered. In methylene blue-stained 1-microns sections, neuronal profiles from the control animals possessed only blue staining lysosomes, while neuronal profiles from the experimental animals exhibited both lysosomes and HRP granules. It was determined, from the counts of HRP granules in neurons from the experimental animals, that in selected regions of areas 17, 18a, and 18b similar percentages of the pyramidal and nonpyramidal neuronal populations (ranging from 100% to 34%) contained HRP granules, and so had callosally projecting axons. However, most callosally projecting nonpyramidal neurons had far fewer HRP granules than the pyramidal neurons, again indicating that they transport less HRP. This could account for the fact that callosally projecting nonpyramidal neurons only rarely show a Golgi-like filling, and this could be one reason why such cells have been overlooked in most previous studies.

Animals↗
PubMed1984

A comparison of visual callosal organization in normal, bilaterally enucleated and congenitally anophthalmic mice.

Visual callosal connections were examined using the horseradish peroxidase (HRP) technique in normal, neonatal and adult C57BL mice, and in adults of this strain which were bilaterally enucleated within 12 h of birth. In addition, callosal connections were also delineated in two strains of congenitally anophthalmic mice, ZRDCT-an and orJ. Material from 129/J mice served as controls for the latter strain. In normal adults anterograde labelling and HRP labelled cells were visible primarily at the borders of area 17. In the 17-18a border region, labelled neurons were located primarily in layers II-III and V. In the medial striate cortex, a small number of labelled cells were present, primarily in lamina VI. Anterograde HRP labelling in the normal adults was also located primarily at the borders of area 17. At the 17-18a border, it was very heavy in layers V and VI, somewhat lighter in layer IV, and fairly dense in layers II-III and the lower half of lamina I. Labelling indicative of anterograde HRP transport was also visible in lowermost lamina V and layer VI across the entire mediolateral extent of area 17. In normals injected with HRP on postnatal day 2 and perfused 24 h later, callosal neurons were distributed throughout the dorsal posterior neocortex, primarily in layers V and VI. Only a very few labelled cells were visible in the supragranular laminae. In adult mice blinded at birth, the zone of callosal cells and terminals extended much further into area 17 than in normals, but aside from the anterograde labelling in layer VI and lowermost lamina V, the medial one-third of the striate cortex was still for the most part devoid of callosal cells and fibers. The laminar distributions of the labelled cells and anterograde transport in the blinded animals were the same as in the normal mice. In both strains of anophthalmic mice the pattern of callosal connections was unlike that in either the normals or neonatal enucleates. In the caudal "visual" cortex, callosal cells and anterograde transport indicative of terminal labelling were visible primarily in the 17-18a border area. Rostrally, however, they were both distributed in multiple (two-three) patches within area 17. Serial reconstructions demonstrated that these patches tended to be aligned in stripes which ran parallel to the 17-18a border. One of these was always located at the 17-18a border, and here the laminar distribution of labelled cells and anterograde labelling was the same as in the normals.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Distribution of visual callosal projection neurons in hamsters subjected to transection of the optic radiations on the day of birth.

The optic radiations of hamsters were transected on the day of birth and visual callosal projections in these animals were traced using retrograde transport of either horseradish peroxidase (HRP) or the fluorescent tracers True blue (TB) or Diamidino yellow (DY) when the animals reached maturity (greater than 45 days of age). In the hemisphere ipsilateral to the neonatal lesion, the distribution of callosal cells was markedly altered. These neurons were almost completely restricted to a continuous band in lower lamina V and the upper portion of layer VI. Anterograde HRP transport to the deafferented hemisphere also revealed an abnormal distribution of callosal terminals. The band of labelling that is located along the 17-18a border in the normals was much broader than is normally the case. In the hemisphere contralateral to the lesion, the distributions of callosal cells and terminals were essentially normal. Labelled neurons were located in the infragranular layers (primarily lower layer V and the upper part of lamina VI) throughout area 17 and also in layers II-IV in the 17-18a border region. Anterograde labelling was visible in layers V and VI throughout the mediolateral extent of the dorsal posterior neocortex and supragranular labelling was restricted to the lateral portion of area 17 and medial 18a. These results suggest that the normal thalamic projection to the visual cortex is necessary for the establishment of the strip of supragranular callosal projection neurons which is normally located in the 17-18a border region, but not for the establishment (or maintenance) of callosal projections by large numbers of neurons in the infragranular laminae. They show further that neonatal transection of the optic radiations results in reduction in the correspondence between the distributions of callosal cells and terminals in the deafferented hemisphere.

Animals↗
PubMed1988

The maturational status of thalamocortical and callosal connections of visual areas V1 and V2 in the newborn monkey.

Cytochrome oxidase (CytOx) is known to preferentially stain those regions of the visual cortex which receive direct projections from the thalamus. The pattern of CytOx stain has been used to investigate the maturation of thalamic input to areas V1 and V2 in the newborn monkey. In both areas, the intensity of CytOx activity was similar in newborns and adults. The distribution of CytOx in area V2 was not found to vary with age. In area V1, the only difference in CytOx activity in newborns was a relative immaturity of staining in layer 4C. The callosal connections of visual areas V1 and V2 were investigated by the axonal transport of wheat germ agglutinin conjugated to horseradish peroxidase and free horseradish peroxidase. In the adult, V1 was found to be reciprocally callosally connected for a distance of 1-2.5 mm from the V1/V2 border, whilst V2 was connected for a distance of 3-8 mm from the border. In both areas, callosal connections showed a certain degree of clustering, particularly in V2 which contained 97-98% of the total number of callosal connections of these two areas. In the newborn, the number, tangential extent and clustered distribution of callosal connections were as in the adult. In the newborn, as in the adult, callosal connections coincided with regions of high CytOx activity in area V2. The results showing a relative maturity of the tangential distribution of callosal projecting neurons on the one hand, and an immaturity of thalamic projections on the other, are discussed in terms of: (1) the maturational status of the newborn monkey compared to other mammals at the moment of birth and (2) the possible role of visual experience in shaping cortical connections.

Aging↗
PubMed1990

Neurogenesis and development of callosal and intracortical connections in the hamster.

The developmental time-course of callosal and ipsilateral corticocortical projections was studied in embryonic and postnatal hamsters, from the time of neurogenesis until the appearance of adult patterns. Callosal neurogenesis was determined by combining the incorporation of [3H]thymidine injected on specific embryonic days with retrograde labelling of callosal neurons in the adult animal. The development of both callosal and corticocortical projections was studied by the transport of wheat germ agglutinin conjugated to horseradish peroxidase. Despite a significant radial disperson of postmigratory neurons born on the same day, it was found that the birthdates of callosally-projecting neurons in the frontal cortex were not restricted to a short period of time, but extended between embryonic days 13 and 15. This period covers the neurogenesis of cells in cortical layers III-V. Elongation of callosal axons (and possibly also of corticocortical fibres) started a couple of days before birth in the frontal cortex, and continued through the first postnatal days. After a "waiting period" of a few days, axons from both sets of projections were seen innervating restricted target sectors of the cortex. The zones of origin of these projections were initially exuberant, but were subsequently trimmed to overlap completely with the corresponding terminal fields. It is concluded that callosal and ipsilateral corticocortical projections undergo similar sequences of ontogenetic stages, suggesting that the development of neocortical connectivity as a whole may be governed by one and the same set of rules.

Animals↗
PubMed2005

Caffeine inhibits callose deposition in the cell plate and the depolymerization of microtubules in the central region of the phragmoplast.

Treatment of tobacco BY-2 cells with 10 mM caffeine that was started after the cells had entered the mitotic phase did not completely inhibit the deposition of callose in the cell plate and allowed the centrifugal redistribution of phragmoplast microtubules. On the other hand, when treatment with caffeine was started before the cells entered the mitotic phase, the deposition of callose was completely inhibited and the redistribution of phragmoplast microtubules was also inhibited. As the inhibition of redistribution of phragmoplast microtubules seems to be caused by the inhibition of depolymerization of microtubules at the central region of the phragmoplast, these results strongly suggest that the deposition of callose in the cell plate is tightly linked with the depolymerization of phragmoplast microtubules. Callose deposition was observed in phragmoplasts isolated from caffeine-treated cells as well as in those isolated from non-caffeine-treated cells, and caffeine did not inhibit callose synthesis in isolated phragmoplast, indicating that caffeine neither inhibits the accumulation of callose synthase at the equatorial regions of the phragmoplast nor arrests callose synthase itself.

Aphidicolin↗
PubMed1991

Rapid Enrichment of CHAPS-Solubilized UDP-Glucose: (1,3)-beta-Glucan (Callose) Synthase from Beta vulgaris L. by Product Entrapment : Entrapment Mechanisms and Polypeptide Characterization.

Rapid enrichment of CHAPS-solubilized UDP-glucose:(1,3)-beta-glucan (callose) synthase from storage tissue of red beet (Beta vulgaris L.) is obtained when the preparation is incubated with an enzyme assay mixture, then centrifuged and the enzyme released from the callose pellet with a buffer containing EDTA and CHAPS (20-fold purification relative to microsomes). When centrifuged at high speed (80,000g), the enzyme can also be pelleted in the absence of substrate (UDP-Glc) or synthesis of callose, due to nonspecific aggregation of proteins caused by excess cations and insufficient detergent in the assay buffer. True time-dependent and substrate-dependent product-entrapment of callose synthase is obtained by low-speed centrifugation (7,000-11,000g) of enzyme incubated in reaction mixtures containing low levels of cations (0.5 millimolar Mg(2+), 1 millimolar Ca(2+)) and sufficient detergent (0.02% digitonin, 0.12% CHAPS), together with cellobiose, buffer, and UDP-Glc. Entrapment conditions, therefore, are a compromise between preventing nonspecific precipitation of proteins and permitting sufficient enzyme activity for callose synthesis. Further enrichment of the enzyme released from the callose pellet was not obtained by rate-zonal glycerol gradient centrifugation, although its sedimentation rate was greatly enhanced by inclusion of divalent cations in the gradient. Preparations were markedly cleaner when product-entrapment was conducted on enzyme solubilized from plasma membranes isolated by aqueous two-phase partitioning rather than by gradient centrifugation. Product-entrapped preparations consistently contained polypeptides or groups of closely-migrating polypeptides at molecular masses of 92, 83, 70, 57, 43, 35, 31/29, and 27 kilodaltons. This polypeptide profile is in accordance with the findings of other callose synthase enrichment studies using a variety of tissue sources, and is consistent with the existence of a multi-subunit enzyme complex.

Journal Article↗
PubMed2005

Callose synthase (CalS5) is required for exine formation during microgametogenesis and for pollen viability in Arabidopsis.

Callose (beta-1,3-glucan) is produced at different locations in response to biotic and abiotic cues. Arabidopsis contains 12 genes encoding callose synthase (CalS). We demonstrate that one of these genes, CalS5, encodes a callose synthase which is responsible for the synthesis of callose deposited at the primary cell wall of meiocytes, tetrads and microspores, and the expression of this gene is essential for exine formation in pollen wall. CalS5 encodes a transmembrane protein of 1923 amino acid residues with a molecular mass of 220 kDa. Knockout mutations of the CalS5 gene by T-DNA insertion resulted in a severe reduction in fertility. The reduced fertility in the cals5 mutants is attributed to the degeneration of microspores. However, megagametogenesis is not affected and the female gametes are completely fertile in cals5 mutants. The CalS5 gene is also expressed in other organs with the highest expression in meiocytes, tetrads, microspores and mature pollen. Callose deposition in the cals5 mutant was nearly completely lacking, suggesting that this gene is essential for the synthesis of callose in these tissues. As a result, the pollen exine wall was not formed properly, affecting the baculae and tectum structure and tryphine was deposited randomly as globular structures. These data suggest that callose synthesis has a vital function in building a properly sculpted exine, the integrity of which is essential for pollen viability.

Arabidopsis↗
PubMed2005

Callose (beta-1,3 glucan) is essential for Arabidopsis pollen wall patterning, but not tube growth.

BACKGROUND: Callose (beta-1,3 glucan) separates developing pollen grains, preventing their underlying walls (exine) from fusing. The pollen tubes that transport sperm to female gametes also contain callose, both in their walls as well as in the plugs that segment growing tubes. Mutations in CalS5, one of several Arabidopsis beta-1,3 glucan synthases, were previously shown to disrupt callose formation around developing microspores, causing aberrations in exine patterning, degeneration of developing microspores, and pollen sterility. RESULTS: Here, we describe three additional cals5 alleles that similarly alter exine patterns, but instead produce fertile pollen. Moreover, one of these alleles (cals5-3) resulted in the formation of pollen tubes that lacked callose walls and plugs. In self-pollinated plants, these tubes led to successful fertilization, but they were at a slight disadvantage when competing with wild type. CONCLUSION: Contrary to a previous report, these results demonstrate that a structured exine layer is not required for pollen development, viability or fertility. In addition, despite the presence of callose-enriched walls and callose plugs in pollen tubes, the results presented here indicate that callose is not required for pollen tube functions.

Alleles↗