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PubMed1999

Callosal axon guidance defects in p35(-/-) mice.

Mice lacking p35, an activator of cdk5 in the central nervous system (CNS), exhibit defects in a variety of CNS structures, most prominently characterized by a disruption in the laminar structure of the neocortex (Chae et al., 1997). In addition, alterations of certain axonal fiber tracts are found in the cortex of p35 mutant mice. Notably, the corpus callosum appears bundled at the midline, but dispersed lateral to the midline. Tracer injection experiments in adult p35 mutant mice reveal that projecting cortical axons fail to assimilate into the corpus callosum, and take oblique paths to the midline. After crossing the midline, cortical axons defasciculate prematurely from the corpus callosum and take similarly oblique paths through the cortex. This callosal phenotype is not detected in reeler mice, which also exhibit defects in cortical lamination, suggesting that the lack of fasciculation of callosal axons is not an inherent manifestation of a disruption of cortical lamination. The embryonic callosal axon tract is defasciculated before crossing the midline, suggesting that axon guidance may be affected during embryonic development of the corpus callosum. In addition, embryonic thalamocortical afferents also exhibit a defasciculated phenotype. These results suggest that defective axonal fasciculation and guidance may be primary responses to the loss of p35 in the cortex. Furthermore, this study postulates a role for the p35/cdk5 kinase in molecular signaling pathways necessary for proper guidance of selective axons during embryonic development.

Agenesis of Corpus Callosum↗

PubMed2000

Dopamine terminals synapse on callosal projection neurons in the rat prefrontal cortex.

Dopamine (DA) afferents to the prefrontal cortex (PFC) play an important role in the cognitive functions subserved by this cortical area. Within the PFC, DA terminals synapse onto the distal dendrites of both local circuit neurons and pyramidal projection cells. We have previously demonstrated in the rat PFC that some of the dendrites and spines postsynaptic to DA terminals arise from pyramidal neurons that project to the nucleus accumbens. However, it is not known whether the pyramidal cells that give rise to callosal intercortical connections of the PFC also receive DA synaptic input. To address this question, retrograde tract tracing using an attenuated strain of pseudorabies virus (PRV-Bartha) was combined with immunocytochemistry for tyrosine hydroxylase (TH) to identify DA terminals in the PFC. Thirty-six to 40 hours following injection of PRV into the contralateral PFC, numerous callosal projection neurons were extensively labeled throughout their dendritic trees, with no evidence of PRV trans-synaptic passage. In tissue prepared for electron microscopy, labeling for PRV was distributed throughout pyramidal cell somata and extended into distal dendrites and dendritic spines. Some PRV-labeled dendrites and spines received symmetric synaptic input from terminals containing peroxidase labeling for TH. These results demonstrate that DA terminals synapse onto the distal dendrites of callosally projecting PFC neurons and suggest substrates through which DA may modulate interhemispheric cortical communication.

Animals↗

PubMed2006

Imaging, anatomical, and molecular analysis of callosal formation in the developing human fetal brain.

A complex set of axonal guidance mechanisms are utilized by axons to locate and innervate their targets. In the developing mouse forebrain, we previously described several midline glial populations as well as various guidance molecules that regulate the formation of the corpus callosum. Since agenesis of the corpus callosum is associated with over 50 different human congenital syndromes, we wanted to investigate whether these same mechanisms also operate during human callosal development. Here we analyze midline glial and commissural development in human fetal brains ranging from 13 to 20 weeks of gestation using both diffusion tensor magnetic resonance imaging and immunohistochemistry. Through our combined radiological and histological studies, we demonstrate the morphological development of multiple forebrain commissures/decussations, including the corpus callosum, anterior commissure, hippocampal commissure, and the optic chiasm. Histological analyses demonstrated that all the midline glial populations previously described in mouse, as well as structures analogous to the subcallosal sling and cingulate pioneering axons, that mediate callosal axon guidance in mouse, are also present during human brain development. Finally, by Northern blot analysis, we have identified that molecules involved in mouse callosal development, including Slit, Robo, Netrin1, DCC, Nfia, Emx1, and GAP-43, are all expressed in human fetal brain. These data suggest that similar mechanisms and molecules required for midline commissure formation operate during both mouse and human brain development. Thus, the mouse is an excellent model system for studying normal and pathological commissural formation in human brain development.

Axons↗

PubMed2006

Cortical, callosal, and thalamic connections from primary somatosensory cortex in the naked mole-rat (Heterocephalus glaber), with special emphasis on the connectivity of the incisor representation.

We investigated the distribution of cortical, callosal, and thalamic connections from the primary somatosensory area (S1) in naked mole-rats, concentrating on lower incisor and forelimb representations. A neuronal tracer (WGA-HRP) was injected into the center of each respective representation under guidance from microelectrode recordings of neuronal activity. The locations of cells and terminals were determined by aligning plots of labeled cells with flattened cortical sections reacted for cytochrome oxidase. The S1 lower incisor area was found to have locally confined intrahemispheric connections and longer connections to a small cluster of cells in the presumptive secondary somatosensory (S2) and parietal ventral (PV) incisor fields. The S1 incisor area also had sparse connections with anterior cortex, in presumptive primary motor cortex. Homotopic callosal projections were identified between the S1 lower incisor areas in each hemisphere. Thalamocortical connections related to the incisor were confined to ventromedial portions of the ventral posterior medial subnucleus (VPM) and posterior medial nucleus (Po). Injections into the S1 forelimb area revealed reciprocal intrahemispheric connections to S2 and PV, to two areas in frontal cortex, and to two areas posterior to S1 that appear homologous to posterior lateral area and posterior medial area in rats. The S1 forelimb representation also had callosal projections to the contralateral S1 limb area and to contralateral S2 and PV. Thalamic distribution of label from forelimb injections included ventral portions of the ventral posterior lateral subnucleus (VPL), dorsolateral Po, the ventral lateral nucleus, and the ventral medial nucleus and neighboring intralaminar nuclei.

Animals↗

PubMed1984

Callosal and intrahemispheric connectivity of the prefrontal association cortex in rhesus monkey: relation between intraparietal and principal sulcal cortex.

Horseradish peroxidase (HRP) histochemistry and double labeling with the fluorescent dyes nuclear yellow (NY) and fast blue (FB) were used to examine and compare the laminar and tangential arrangement of ipsilateral (associational) and contralateral (callosal) neurons and their relative density in three regions of prefrontal granular cortex: Walker's area 46 (principal sulcus), area 8A (superior limb of the arcuate sulcus), and area 11 (lateral orbital sulcus). In all three prefrontal regions, neurons with ipsilateral projections were labeled following injections of tracers into the intraparietal sulcus (IPS) and neurons with callosal projections were sequentially or simultaneously labeled with injections into the contralateral principal sulcus (PS). Quantitative analysis indicates that associational and callosal neurons in prefrontal cortex are distinct cell populations with strikingly similar organization including (1) common topography; (2) common laminar positions in layers III, IV, and V; (3) two- to three-fold higher densities in supragranular than infragranular layers; (4) common morphologies including a high proportion of nonpyramidal soma in the deeper cortical layers; (5) common uneven tangential distribution reminiscent of the interdigitation of their terminal fields; and (6) common subpopulations differing on the basis of terminal arbors. These findings indicate that the posterior parietal cortex and the prefrontal cortex form part of an integrated neural system important for spatiotemporal behaviors.

Animals↗

PubMed1987

D-[3H]aspartate retrograde labelling of callosal and association neurones of somatosensory areas I and II of cats.

Experiments were carried out on cats to ascertain whether corticocortical neurones of somatosensory areas I (SI) and II (SII) could be labelled by retrograde axonal transport of D-[3H]aspartate (D-[3H]Asp). This tritiated enantiomer of the amino acid aspartate is (1) taken up selectively by axon terminals of neurones releasing aspartate and/or glutamate as excitatory neurotransmitter, (2) retrogradely transported and accumulated in perikarya, (3) not metabolized, and (4) visualized by autoradiography. A solution of D-[3H]Asp was injected in eight cats in the trunk and forelimb zones of SI (two cats) or in the forelimb zone of SII (six cats). In order to compare the labelling patterns obtained with D-[3H]Asp with those resulting after injection of a nonselective neuronal tracer, horseradish peroxidase (HRP) was delivered mixed with the radioactive tracer in seven of the eight cats. Furthermore, six additional animals received HRP injections in SI (three cats; trunk and forelimb zones) or SII (three cats; forelimb zone). D-[3H]Asp retrograde labelling of perikarya was absent from the ipsilateral thalamus of all cats injected with the radioactive tracer but a dense terminal plexus of anterogradely labelled corticothalamic fibres from SI and SII was observed, overlapping the distribution area of thalamocortical neurones retrogradely labelled with HRP from the same areas. D-[3H]Asp-labelled neurones were present in ipsilateral SII (SII-SI association neurones) in cats injected in SI. In these animals a bundle of radioactive fibres was observed in the rostral portion of the corpus callosum entering the contralateral hemisphere. There, neurones retrogradely labelled with silver grains were present in SI (SI-SI callosal neurones). Association and callosal neurones labelled from SI showed a topographical distribution similar to that of neurones retrogradely labelled with HRP. The laminar patterns of corticocortical neurones labelled with D-[3H]Asp or with HRP were also similar, with one exception. In the inner half of layer II, SII-SI association neurones and SI-SI callosal neurones labelled with the radioactive marker were much less numerous than those labelled with HRP. In cats injected in SII, D-[3H]Asp retrogradely labelled cells were present in ipsilateral SI (SI-SII association neurones). Their topographical and laminar distribution overlapped that of neurones labelled with HRP but, as in cats injected in SI, association neurones labelled with silver grains were unusually rare in the inner layer III.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Topographic organization of the middle temporal visual area in the macaque monkey: representational biases and the relationship to callosal connections and myeloarchitectonic boundaries.

We have used physiological and anatomical techniques to address three general issues concerning the topographic organization of the middle temporal visual area (MT) of the macaque monkey. First, we carried out a quantitative analysis of irregularities and asymmetries in the visual representation in MT. This analysis revealed a striking overemphasis on a restricted portion of the visual field that runs obliquely through the inferior contralateral quadrant and largely avoids both the horizontal meridian and the inferior vertical meridian. This corresponds to the portion of the visual field that would be maximally stimulated during visually guided hand movements. Second, the physiologically determined topographic organization of MT was compared to the pattern of callosal inputs in the same hemisphere, which are known to be distributed irregularly within MT. Callosal inputs tended to be densest near the representation of the vertical meridian, but there were numerous exceptions to this trend. Thus, topographic irregularities account for only part of the irregularities in callosal inputs to MT. Finally, comparison of these data with previous reports shows a strong correlation between body weight and the average size of MT. The representation in myeloarchitectonically defined MT was found to include much of the visual periphery, although it is unclear from our data whether this representation is invariably complete.

Animals↗

PubMed2008

Corpus callosal connection mapping using cortical gray matter parcellation and DT-MRI.

Population maps of the corpus callosum (CC) and cortical lobe connections were generated by combining cortical gray matter parcellation with the diffusion tensor fiber tractography of individual subjects. This method is based on the fact that the cortical lobes of both hemispheres are interconnected by the corpus callosal fibers. T1-weighted structural MRIs and diffusion tensor MRIs (DT-MRI) of 22 right-handed, healthy subjects were used. Forty-seven cortical parcellations in the dorsal prefrontal cortex, ventral prefrontal cortex, sensory-motor cortex, parietal cortex, temporal cortex, and occipital cortex were semi-automatically derived from structural MRIs, registered to DT-MRI, and used to identify callosal fibers. The probabilistic connections to each cortex were mapped on entire mid-sagittal CC voxels that had anatomical homology between subjects as determined by spatial registration. According to the population maps of the callosal connections, the ventral prefrontal cortex and parts of the dorsal prefrontal cortex both project fibers through the genu and rostrum. The CC regions through which the superior frontal cortex passes extend into the posterior body. Fibers arising from the parietal lobe and occipital lobe run mainly through the splenium, while fibers arising from the sensory-motor cortex pass through the isthmus. In general, dorsal or medial cortical lobes project fibers through the dorsal region of the CC, while lateral cortical lobes project fibers through the ventral region of the CC. The probabilistic subdivision of the CC by connecting cortical gray matter provides a more precise understanding of the CC.

Brain Mapping↗

PubMed1993

Consequences of reduced cerebral blood flow in brain development. I. Gross morphology, histology, and callosal connectivity.

Reduced blood flow produced by bilateral carotid artery occlusion (BCAO) caused multiple histopathological alterations in the cerebral cortex of developing cats. BCAO was performed in the second postnatal week. At 1 and 2 months, global structural observations were made using magnetic resonance imaging (MRI). At 3 months, neuron and glial density, extent of myelination, blood vessel distribution, and the distribution of visual callosal projecting neurons (as visualized with the retrogradely transported tracer horseradish peroxidase) were assessed by light microscopy. MRI showed lateral ventricular dilatation at 1 month in five of eight subjects, with wide-ranging severity, although at 2 months only two animals still had enlarged ventricles. Histological observations at 3 months showed that neuron density in the motor cortex, but not the occipital cortex, of BACO animals was significantly lower than that in controls. BCAO animals had more dilated small vessels, again more evident in the motor cortex than in the occipital cortex. From frontal to occipital cortex, the corpus callosum was thinned and the subcortical white matter was reduced. Even with the reduction of white matter, the number of neurons in visual areas 17 and 18 contributing a callosal projection was much higher than normal. BCAO thus altered cerebral vascularization, caused neuronal death, and reduced myelinization over an area much greater than the direct area of carotid perfusion. The excess callosal projection in these animals suggests that neonatal ischemia interferes with the normal process of axon retraction during development.

Aging↗

PubMed1992

Types of callosally projecting nonpyramidal neurons in rat visual cortex identified by lysosomal HRP retrograde labeling.

Callosally projecting neurons, labeled following injection of horseradish peroxidase (HRP) into the 17/18a border of the contralateral hemisphere, have been examined by light and electron microscopy. These neurons exhibit two types of horseradish peroxidase labeling: either a diffuse, Golgi-like labeling, or a granular, punctate labeling. The punctate type of HRP-labeling is the predominant form in nonpyramidal neurons, while pyramidal neurons frequently display either diffuse or punctate labeling. Only punctately labeled neurons have been examined in this study. Light microscopic analyses of 1-microns sections show that in the heavily labeled zone at the area 17/18a border approximately 9% of all of the cells in layer II/III are callosally projecting nonpyramidal cells, and 70% of them are callosally projecting pyramidal cells. Light and electron microscopic examinations indicate that the nonpyramidal neurons are a heterogeneous group which consists of small multipolar neurons, large multipolar neurons, small bipolar neurons, and large bipolar neurons. To investigate the ultrastructural appearance of the punctate HRP labeling, selected neurons have been examined in thin sections. In the electron microscope, the tetramethylbenzidine (TMB) reaction product appears as electron-dense crystals, while the diaminobenzidine (DAB) reaction product appears as dark, electron-dense material which fills the lysosomes. These lysosomes occasionally have a halo of reaction product, but often they are not morphologically distinguishable from dark lysosomes present within neurons from control animals in which the darkening results from staining the thin sections with lead citrate and uranyl acetate. However, labeled neurons possess more dark lysosomes than neurons from control animals. These additional dark lysosomes presumably contain the HRP reaction product visible by light microscopy.

Animals↗

PubMed1995

Cell wall synthesis in cotton roots after infection with Fusarium oxysporum. The deposition of callose, arabinogalactans, xyloglucans, and pectic components into walls, wall appositions, cell plates and plasmodesmata.

Fusarium oxysporum f. sp. vasinfectum penetration hyphae infect living cells in the meristematic zone of cotton (Gossypium barbadense L.) roots. We characterized wall modifications induced by the fungus during infection of the protodermis using antibodies against callose, arabinogalactan-proteins, xyloglucan, pectin, polygalacturonic acid and rhamnogalacturonan I in high-pressure frozen, freeze-substituted root tissue. Using quantitative immunogold labelling we compared the cell walls before and after hyphal contact, cell plates with plasmodesmata during cytokinesis, and wall appositions induced by fungal contact. In the already-existing wall, fungal contact induced only minor modifications such as an increase of xyloglucan epitopes. Wall appositions mostly exhibited epitopes similar to the cell plate except that wall appositions had a much higher callose content. This study shows that wall appositions induced by Fusarium oxysporum hyphae are the result of normal cell wall synthesis and the addition of large amounts of callose. The appositions do not stop fungal growth.

Cell Wall↗

PubMed1993

Hemispheric control of unilateral and bilateral responses to lateralized light stimuli after callosotomy and in callosal agenesis.

Normally, simple digital or manual responses to a light stimulus in the right or left visual hemifields are performed faster with uncrossed hand-field combinations than with crossed hand-field combinations. Because of the organization of visual and motor pathways, the integration of uncrossed responses is assumed to occur within a single hemisphere, whereas a time-consuming interhemispheric transfer via the corpus callosum is considered to be necessary for the integration of crossed responses. However, callosal transfer may be dispensable for those crossed responses which can be controlled through ipsilaterally descending motor pathways by the hemisphere receiving the visual stimulus. We investigated crossed-uncrossed differences (CUDs) in speed of simple visuomotor responses to lateralized flashes in one subject with total section of the corpus callosum and two subjects with complete callosal agenesis. We recorded the reaction times as well as the premotor times, as indicated by the electromyographic latencies of the prime movers, of three types of responses: a distal response involving a thumb flexion, a proximal response chiefly involving a forearm flexion and an axial response involving a shoulder elevation. Further, the three types of responses to a single lateralised flash were performed both unilaterally and bilaterally. The three acallosal subjects showed CUDs greatly exceeding normal values on distal responses, either unilateral or bilateral, and on unilateral proximal responses. These abnormally long CUDs stood in sharp contrast to the insignificant CUDs exhibited by the same subjects on bilateral proximal responses and on unilateral and bilateral axial responses in agreement with correspondingly insignificant CUDs reported for normal subjects. These results confirm that a callosal contribution is important for the execution of fast distal and unilateral proximal responses to a visual stimulus directed to the hemisphere ipsilateral to the responding hand. By contrast, the other types of crossed responses appear to be efficiently coordinated across the midline without the aid of the corpus callosum. This is in keeping with the hypothesis that they are governed by a bilaterally distributed motor system which is preferentially activated for the execution of symmetrical bilateral movements, employing axial and proximal limb muscles.

Accidents, Traffic↗

PubMed1995

Binaural noise stimulation of auditory callosal fibers of the cat: responses to interaural time delays.

The corpus callosum, the principal neocortical commissure, allows for the interhemispheric transfer of lateralized information between the hemispheres. The aim of the present experiment was to study callosal transfer of auditory information in the cat, with particular reference to its contribution to sound localization. The corpus callosum was approached under direct visual control, and axonic responses were recorded under light anesthesia using glass micro-pipettes. Results showed that auditory information is transmitted in the posterior portion of the callosum. Diotic presentations, in which interaural time delay was manipulated, indicated that, for a large number of fibers, the largest excitatory or inhibitory interactions were obtained at null interaural time delay, a condition which supports the notion of a callosal contribution to auditory midline fusion. However, an important number of callosal fibers was also found to be excited maximally at specific, non-zero interaural time delays, suggesting that they preferred sounds situated at spatial locations other than the midline. The results are discussed in relation to those obtained electrophysiologically for the visual and somesthesic modalities and in terms of results obtained in human and animal behavioral experiments.

Acoustic Stimulation↗

PubMed1990

Reciprocal heterotopic callosal connections between the two striate areas in Tupaia.

WGA-HRP injections were placed into area 17 close to the border with area 18 of Tupaia belangeri in order to study the callosal connections of the striate area in this animal. Most callosal neurons were found in the striate cortex (57.6-86.9%), some in the extrastriate area 18 (10.6-28.1%), and a few in even more temporal regions (2.5-14.3%). Concerning only the area 17, reciprocal homotopic connections could be observed as a strip along the area 17/18 border. Additionally, heterotopic callosal connections could be seen in regions representing the binocular visual field, especially the lower part. The area 17 cells were mostly located in the supragranular layers II and III (94.1-97.2%). But neurons could also be found in the infragranular layers, especially layer VI (2.6-5.2%) and in layer IV (0.2-1.1%). Homotopic projections were mostly seen in layers IIIc and V. The majority of the supragranular and infragranular neurons are pyramidal cells. However, a newly defined subpopulation of neurons, most probably stellate cells, were discovered forming a band in sublayer IIIc, very close to the layer III/IV border.

Animals↗

PubMed1980

Absence of callosal collaterals derived from rat corticospinal neurons. A study using fluorescent retrograde tracing and electrophysiological techniques.

In rat the presence of axon collaterals from corticospinal neurons to the contralateral hemisphere has been investigated by means of anatomical and electrophysiological techniques. Anatomical Experiments. Several combinations of fluorescent retrograde tracers were used. In eight rats injections of Evans Blue, "True Blue", "Fast Blue" or DAPI-Primuline were made in areas 10, 6, and 4 and in the most medial part of the S1 granular cortex of one hemisphere, 1.5 mm below cortical surface. These injections were combined with injections of "Fast Blue", DAPI-Primuline, "Granular Blue", "Nuclear Yellow", or Bisbenzimide in the ipsilateral corticospinal tract in the C2 segment. Survival times of the animals varied according to the tracers used. In the non-injected hemisphere the retrogradely labeled corticospinal neurons were present in layer V of especially areas 10, 6, 4 and the medial portion of the S1 granular cortex. However, the retrogradely labeled callosal neurons in these areas were present in all layers except layer I. The labeled callosal and corticospinal neurons in layer V were intermingled and frequently situated very close to one another. However, with none of the tracer combinations were double labeled neurons observed. Electrophysioloogical Experiments. In six rats, layer V neurons of hindlimb-sensorimotor cortex were tested for antidromic responses to stimulation of contralateral corticospinal tract (CST) and corpus callosum (CC). Eighty-five CST neurons were identified, none of which responded antidromically to CC shocks. Eighty-two layer V neurons were identified which responded antidromically to CC shocks, but none of them responded antidromically to CST shocks. CC shocks elicited strong synaptic responses in CST neurons and vice versa. Depth measures indicated extensive intermingling of CST and CC neurons. From both sets of findings it was concluded that, in rat, CST neurons do not give rise to callosal collaterals.

Animals↗

PubMed1981

Ultrastructure of visual callosal neurons in cat identified by retrograde axonal transport of horseradish peroxidase.

The ultrastructure of neurons at the border of areas 17 and 18 of the visual cortex of the cat was studied by the combined use of the retrograde transport of horseradish peroxidase (HRP) and electron microscopy. Callosal neurons were retrogradely labelled by injecting HRP at the 17/18 border region of the contralateral hemisphere. They were found mainly in layer III but also in IV and VI. They were most commonly pyramidal cells and less often large, spiny stellate cells. Pyramidal callosal neurons received only symmetrical synapses on their soma and mainly symmetrical (but a few asymmetrical) synapses on their dendritic shafts. Their abundant spines received asymmetrical synapses. The stellate cells were contacted by moderate numbers of symmetrical and asymmetrical axodendritic and axosomatic synapses and also had asymmetrical axospinous contacts. We propose that the callosal stellate neurons consist of a class of large spiny stellates, recognizable by light and electron microscopic criteria.

Animals↗

PubMed1997

What is the role of the corpus callosum in intermanual transfer of motor skills? A study of three cases with callosal pathology.

Intermanual transfer for a skilled motor task was studied in two patients with total callosal agenesis, and one with an acquired partial callosal lesion and clinical evidence for disturbed transfer of motor signals. Patients had to draw meaningless figures with one upper extremity (original learning, OL) and to reproduce their mirror-reversals thereafter with the other side (transfer learning, TL). Both directions of intermanual transfer were tested in two conditions, that is, between either proximal or distal muscle groups. Transfer was evaluated by comparing OL and TL performance at the same effector. The main variable of interest was movement time during the first eight trials of OL and TL. All three patients displayed a significant benefit for transfer from the dominant to the non-dominant hand but not vice versa during proximal motor activity. When compared with the performance of healthy subjects tested in almost identical conditions in a previously reported study, the proximal transfer behavior was found to be similar for all patients and the normal group. Although patients exhibited no significant benefit for distal transfer, their non-dominant-to-dominant distal transfer was above the normal range. The similar transfer pattern of the patients and healthy subjects when using proximal musculature suggests that proximal transfer may be subserved by identical extracallosal pathways, most probably by the ipsilaterally descending motor systems. Since non-dominant-to-dominant distal transfer was found to be disadvantageous in healthy subjects, the patients' relative superiority in this condition may reflect missing callosal influences of an inhibitory nature.

Adult↗

PubMed2001

Effects of tissue-preparation-induced callose synthesis on estimates of plasmodesma size exclusion limits.

Plasmodesmata are often characterised by their size exclusion limit (SEL), which is the molecular weight of the largest dye, introduced by microinjection, that will move from cell to cell. In this study, we investigated whether commonly used techniques for isolation and manipulation of tissues, and microinjection of fluorescent dyes, affected the SEL, and whether any such effects could be ameliorated by inhibiting callose deposition. We examined young root epidermal cells of Arabidopsis thaliana and staminal hair cells of Tradescantia virginiana, two tissues often used in experiments on symplastic transport. Transport in root tips dissected from the main plant body and in stamen hairs removed from the base of the stamen filament was compared with transport in undissected roots and stamen hairs attached to the base of the filament, respectively. Tissues were microinjected with fluorescent dyes (457 Da to > 3 kDa) with or without prior incubation in the callose deposition inhibitors 2-deoxy-D-glucose or aniline blue fluorochrome. In both tissues, dissection reduced the SEL, which was largely prevented by prior incubation in 2-deoxy-D-glucose but not by incubation in aniline blue fluorochrome. Thus, standard methods for tissue preparation can cause sufficient callose deposition to reduce cell-to-cell transport, and this needs to be considered in studies employing microinjection. Introduction of the dyes by pressure injection rather than iontophoresis decreased the SEL in A. thaliana but increased it in T. virginiana, showing that these two injection techniques do not necessarily give identical results and that plasmodesmata in different tissues may respond differently to similar experimental procedures.

Antimetabolites↗