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PubMed2001

Intranasal inoculation with the olfactory bulb line variant of mouse hepatitis virus causes extensive destruction of the olfactory bulb and accelerated turnover of neurons in the olfactory epithelium of mice.

Viral upper respiratory infections are the most common cause of clinical olfactory dysfunction, but the pathogenesis of dysosmia after viral infection is poorly understood. Biopsies of the olfactory mucosa in patients that complain of dysosmia after viral infection fall into two categories: one in which no olfactory epithelium is seen and another in which the epithelium is disordered and populated mainly by immature neurons. We have used intranasal inoculation with an olfactory bulb line variant of MHV to study the consequences of viral infection on peripheral olfactory structures. MHV OBLV has little direct effect on the olfactory epithelium, but causes extensive spongiotic degeneration and destruction of mitral cells and interneurons in the olfactory bulb such that the axonal projection from the bulb via the lateral olfactory tract is markedly reduced. Moreover, surviving mitral cells apparently remain disconnected from the sensory neuron input to the glomerular layer, judging from retrograde labeling studies using Dil. The damage to the bulb indirectly causes a persistent, long-term increase in the turnover of sensory neurons in the epithelium, i.e. the relative proportion of immature to mature sensory neurons and the rate of basal cell proliferation both increase. The changes that develop after inoculation with MHV OBLV closely resemble the disordering of the olfactory epithelium in some patient biopsies. Thus, damage to the olfactory nerve or bulb may contribute to a form of post-viral olfactory dysfunction and MHV OBLV is a useful model for studying the pathogenesis of this form of dysosmia.

Administration, Intranasal↗

PubMed2001

Projection of sensory neurons with microvilli to the lateral olfactory tract indicates their participation in feeding behaviour in crucian carp.

In the olfactory system of vertebrates, a large number of primary sensory neurons terminate in glomeruli in the olfactory bulb, where they make synapses with a significantly smaller number of secondary neurons. We applied small amounts of a lipophilic neural tracer (Dil) in the glomerular regions of the lateral olfactory bulb in crucian carp, and investigated the centrifugal migration of this stain through the secondary neurons towards the brain and peripherally to the sensory neurons of the olfactory epithelium. In preparations where only the secondary neurons of the lateral olfactory tract (LOT) were stained, the majority (76%) of sensory neurons had cell bodies in the intermediate layer of the olfactory epithelium. Scanning electron microscopy revealed that most of the sensory neurons with cell bodies in the intermediate layers of the olfactory epithelium feature microvilli. Based on observations that the secondary neurons of the LOT mediate feeding behaviour, we feel that there is strong evidence to indicate that the sensory neurons that exhibit microvilli are responsible for mediating the behavioural patterns related to feeding. These results are discussed in relation to physiological experiments on the properties of the sensory neurons and to studies of the innervation pattern of sensory neurons.

Animals↗

PubMed2002

The alarm reaction in crucian carp is mediated by olfactory neurons with long dendrites.

In the present study, we applied a lipophilic tracer, Dil (1,1-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), to the synaptic region of the medial olfactory bulb in formaldehyde-fixed preparations from the crucian carp. We observed staining both in the axons of secondary neurons leading to the brain and in the olfactory receptor neurons (ORNs) of the olfactory epithelium. In those preparations, where staining of the tract was restricted to axons of the medial part of the medial olfactory tract, the majority (86-98%) of the somata of the sensory neurons were found in the deep layers of olfactory epithelium. Since the medial bundle of the medial olfactory tract mediates alarm behaviour in the crucian carp, we conclude that the sensory neurons with long dendrites participate in the reception of alarm pheromones.

Animals↗

PubMed1989

Isolation, culture, and characterization of endothelial cells derived from the post-capillary venules of sheep mesenteric lymph nodes, Peyer's patches, and associated small bowel.

Lymphocyte recirculation is mediated principally by specialized endothelial cells which line the post-capillary venules of lymph nodes and other secondary lymphoid tissues. The ontogeny and physiology of this process have been characterized in sheep in considerable detail. To further enhance the analytical potential of this experimental system we have isolated endothelial cells from the post-capillary venules of ovine mesenteric lymph nodes by perfusion with small (37-74 microns diameter), sulfonated microcarrier beads. Cells isolated in this manner have been maintained in vitro for greater than 12 months through greater than 30 passages. The endothelial nature of these cells has been conclusively established on the basis of morphologic, metabolic, and immunologic criteria. Virtually all (greater than 99%) cells in primary and passaged cultures metabolized Dil-AC-LDL, a known marker for endothelial cells. Furthermore, nearly all (greater than 95%) cells expressed cell-surface von Willebrand factor and antithrombin III, which are known endothelial antigens. All cells expressed major histocompatibility class I antigens but no cells expressed class II antigens. In vitro lymphocyte-binding studies revealed that these cells bound lymphocytes in a dose-dependent fashion. The microcarrier perfusion technique was also used to isolate endothelial cells from the post-capillary venules of ileal Peyer's patches and associated small bowel in sheep. The majority (70%) of cells isolated in this manner resembled the cells isolated from mesenteric lymph nodes both morphologically and metabolically.

Animals↗

PubMed2004

Identification and molecular characterization of myosin gene family in Oryza sativa genome.

Myosins play an important role in various developmental processes in plants. We have identified 14 myosin genes in rice (Oryza sativa cv. Nipponbare) genome using sequence information available in public databases. Phylogenetic analysis of these sequences with other plant and non-plant myosins revealed that two of the predicted sequences belonged to class VIII and the others to class XI. All of these genes were distributed on seven chromosomes in the rice genome. Domain searches on these sequences indicated that a typical rice myosin consisted of Myosin_N, head domain, neck (IQ motifs), tail, and dilute (DIL) domain. Based on the sequence information obtained from predicted myosins, we isolated and sequenced two full-length cDNAs, OsMyoVIIIA and OsMyoXIE, representing each of the two classes of myosins. These two cDNAs isolated from different organs existed in isoforms due to differential splicing and showed minor differences from the predicted myosin in exon organization. Out of 14 myosin genes 11 were expressed in three major organs: leaves, panicles, and roots, among which three myosins exhibited different expression levels. On the other hand, three of the total myosin sequences showed organ-specific expression. The existence of different myosin genes and their isoforms in different organs or tissues indicates the diversity of myosin functions in rice.

Chromosome Mapping↗

PubMed2004

Transplantation of bone marrow stromal cells into the cochlea of chinchillas.

This study aimed to evaluate the potential of bone marrow stromal cells for treatment of inner ear diseases. Autologous marrow cells labeled with Dil were implanted into the inner ear of five gentamicin-treated chinchillas. Histological analysis 3 weeks later revealed robust survival of grafted marrow cells in multiple regions within the cochlea. Marrow cells implanted in the basal turn of the cochlea migrated as far as the apical end or into the spiral ligament of the cochlea. Some grafted cells expressed a neuronal or glial cell marker, indicating their ability to differentiate into neuronal or glial cells. Survival, migrational mobility and differentiation of autologous marrow cells in damaged cochlea suggest their potential as transplants for treatment of various degenerative inner ear diseases.

Animals↗

PubMed1991

Postmortem preservation of ganglion cells in the human retina. A morphometric investigation with the carbocyanine dye DiI.

Postmortem morphometric investigation of autopsy material is helpful for understanding the alterations cells undergo during life. The present work was undertaken to determine the morphologic features of human retinal ganglion cells during the first 2 days after death. Ganglion cells of 16 retinas were obtained from eyes, the corneas of which had been retrieved for keratoplasties. The ganglion cells were stained with the fluorescent carbocyanine dye Dil and investigated 12 to 36 hours postmortem. All labeled cells identified as ganglion cells had well preserved, type-specific dendritic profiles that made it possible to catalog them according to the established classes of ganglion cells. The various types of cells have in common some typical morphologic changes that occur during the postmortem period of observation: swelling and varicosity in the cell bodies and along the dendritic branches and the axons. These swellings were consistent throughout the postmortem period and did not hinder the identification of particular types of ganglion cells. The two major types (parasol and midget) and various subtypes of ganglion cells were identified morphologically. There are two distinct morphologic types of midget cells which differ from each other in the pattern of dendritic branching. Particular emphasis was given to analyze the dendritic geometry of the large parasol cells. A new finding in the human retina is the frequent presence of large parasol ganglion cells, the axons of which course along aberrant intraretinal paths, especially in the extreme periphery of the retina.

Axons↗

PubMed1997

Establishment and characterization of a line of adipose-derived human microvascular endothelial cells (HADMEC-5) transformed by simian virus 40 large T antigen expression: application to endotoxin research.

This paper reports the establishment and initial characterization of an immortalized line of human, adipose tissue-derived microvascular endothelial cells. Transfection of primary endothelial cell cultures was accomplished by the introduction of a plasmid, which contained simian virus 40 large T antigen DNA as well as a Rous sarcoma viral promoter region. One emergent colony, termed HADMEC-5, was isolated and has been passaged 45 times to date. The cells express simian virus 40 large T antigen protein, are immunohistochemically positive for factor VIII-related antigen, bind Ulex europaeus lectin, and accumulate Dil-labeled acetylated low density lipoprotein. The HADMEC-5 line demonstrates a highly proliferative growth rate in the absence of supplemental growth factors, when compared with primary cultures of nontransformed endothelial cells. HADMEC-5 growth remains serum dependent, but exhibits a lower serum requirement than nontransformed cells. The transformed cells grow well upon a variety of matrix compounds and in a variety of growth media. When grown upon Matrigel, the HADMEC-5 cells form three dimensional tube-like structures. The HADMEC-5 line was also tested for its ability to produce eicosanoids in response to bacterial lipopolysaccharide. The transformed cells, tested at passages 7 and 45, displayed a dose-dependent production of prostaglandin E2 in response to lipopolysaccharide in a manner similar to that seen in primary cell cultures. Threshold sensitivity to lipopolysaccharide was 10 pg/mL of media. The HADMEC-5 cell line represents a unique model in which to investigate lipopolysaccharide interactions with microvessel-derived endothelial cells and is of potential value in the study of other aspects of endothelial cell physiology.

Adipose Tissue↗

PubMed1997

Calcium antagonists inhibit oxidative burst and nitrite formation in lipopolysaccharide-stimulated rat peritoneal macrophages.

Activated macrophages are important cell effectors in sepsis/endotoxemia. Superoxide (SO) and nitric oxide (NO) are produced by activated macrophages and are responsible for host defense against microorganisms. Using laser scanning confocal microscopy, we investigated the role of intracellular free calcium ([Ca2+]i) on SO and NO production by rat peritoneal macrophages activated by lipopolysaccharide (LPS). Calcium influx from the extracellular space versus release of calcium from intracellular stores was determined using calcium channel blockers (diltiazem [DIL], verapamil [VER], and nicardipine [NIC]) and dantrolene (DAN), respectively. Cells incubated with LPS had a 30-50 nM increase in [Ca2+]i, (p < .05) compared with non-LPS-treated cells. When stimulated with phorbol myristate acetate, both control and LPS-treated cells sustained a comparable increase in [Ca2+]i, but [Ca2+]i, remained elevated 30 min later in LPS-treated cells. Calcium channel blockers and DAN reduced phorbol myristate acetate-stimulated SO and LPS-stimulated NO production at all concentrations tested (p < .05). Although increased extracellular calcium influx and calcium from intracellular stores are important regulators of SO and NO production in macrophages, extracellular calcium influx seems to have the predominant effect. Calcium antagonists may modulate the inflammatory response via their effects on macrophages.

Animals↗

PubMed2005

Drug-induced lupus after treatment with infliximab in rheumatoid arthritis.

We report a case of a 45-year-old man with an 8-month history of rheumatoid arthritis, who was treated with hydroxychloroquine 400 mg per day and 15 mg intramuscular methotrexate per week without reaching a good control of the disease. The patient was successfully treated with 3 mg/kg infliximab for 20 weeks. Before the last infusion, drug-induced lupus (DIL) was diagnosed based on the clinical features of fever > 37.5 degrees C, recurrence of active synovitis, myalgia, erythematosus rash, pericardial and pleural effusion, and of some laboratory findings (antinuclear antibodies 1:160 and anti double-strand DNA positive by DNA recombinant plasmid assay dsDNA). After infliximab discontinuation and the beginning of therapy with methylprednisolone, lupus symptoms resolved within 6 weeks. A new rheumatoid arthritis flare, occurring after 8 weeks, was controlled by methotrexate plus leflunomide. We also review the development of antinuclear and antidouble-strand DNA antibodies and drug-induced lupus in patients treated with anti-TNFalpha agents (infliximab, etanercept, and adalimumab).

Antibodies, Monoclonal↗

PubMed2004

Serum derived from multiple trauma patients promotes the differentiation of endothelial progenitor cells in vitro: possible role of transforming growth factor-beta1 and vascular endothelial growth factor165.

Ischemia in various organs and tissues takes place during and as a direct result of multiple trauma (MT). Bone marrow-derived endothelial progenitor cells (EPCs) are involved in neovascularization after ischemic incidences. Here, we report that serum derived from patients with MT stimulates differentiation of EPCs in vitro from peripheral blood mononuclear cells (PBMCs). EPCs were identified by DiL-Acetyl-LDL-uptake with concomitant UEA-I-lectin binding. A significant increase in EPC numbers was noted when PBMCs were cultivated for 72 h with the serum of MT patients (n = 25) obtained at 5 days. Furthermore, serum from MT patients enhanced the functional acting of EPCs to form prevascular structures in matrigel. Reverse transcription polymerase chain reaction analysis revealed gene expression of transforming growth factor (TGF)-beta1- and vascular endothelial growth factor (VEGF) receptors 1 and 2. Reverse transcription polymerase chain reaction analysis was based on further cultivated cell preparations, which contained at least 80% EPCs. Moreover, the addition of recombinant VEGF or low concentrations of TGF-beta increased EPC differentiation. In addition, neutralization of TGF-beta1 and of VEGF165 in MT serum using specific antibodies resulted in a significant decrease in EPC differentiation. Our data indicate that TGF-beta1 and VEGF165 play a pivotal role for EPC differentiation induced by serum of polytrauma patients.

Blotting, Western↗

PubMed1990

Climbing Fibre Responses in Olivo-cerebellar Slice Cultures. I. Microelectrode Recordings from Purkinje Cells.

Cerebellar slices prepared from newborn rats were co-cultured with slices derived from the inferior olive of 4-day-old rats. After several weeks in vitro olivary fibres projecting into the cerebellar tissue could be assessed by anterograde labelling with the fluorescent dye 1,1-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (Dil). Following electrical field stimulation of the olivary tissue, all-or-nothing complex spikes were generated in Purkinje cells, which closely resembled climbing fibre responses as seen in situ. These responses were completely and reversibly abolished by 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX, 5 microM), an antagonist of non-N-methyl-d-aspartate excitatory amino acid receptors. Wash in of smaller concentrations of CNQX (0.5 - 2 microM) resulted in a graded dose-dependent depression of the climbing fibre-induced postsynaptic potentials and in a consecutive failure of distinct active components of the complex spikes. With climbing fibre synaptic transmission blocked by CNQX, complex spike-like potentials could, however, still be evoked by intrasomatic injection of depolarizing current pulses. Increasing the concentration of Mg2+ in the bathing solution from 0.5 to up to 8 mM depressed regenerative complex-spike components. Olivary stimulation elicited only monophasic postsynaptic potentials in Purkinje cells under these conditions. These observations indicate that voltage-gated conductances which are substantially involved in the generation of the complex spike, are gated by the climbing fibre synaptic depolarization rather than directly by the climbing fibre transmitter.

Journal Article↗

PubMed1991

The Relationship of Microglial Cells to Dying Neurons During Natural Neuronal Cell Death and Axotomy-induced Degeneration of the Rat Retina.

The interactions between dying neurons and phagocytotic cells within the developing and injured retina remain controversial. The present work explored the role of microglia and investigated whether so-called resident microglial cells are permanently responsible for removing cell debris whenever it is produced. As a first goal, I characterized some quantitative and morphometric features of the small ipsilateral retinocollicular projections and analysed the permanent function of phagocytosing microglia with these projections as a paradigm. To achieve this, I combined the fluorescent dyes Dil and 4Di-10ASP, both of which persist in the labelled ganglion cells after injection into the superior colliculus (SC), and retrograde labelling. After neuronal degradation, the dyes accompany the degradation products, become interiorized and then persist within the phagocytosing microglia. Consequently, early labelling of microglial cells can be assessed by injecting one dye into the SC during the first postnatal day of life, that is, prior to advanced natural neuronal cell death. Labelling of the remaining ipsilaterally projecting neurons with the second dye following intraorbital axotomy in adulthood and during subsequent neuronal death would therefore result in double labelling of some microglial cells, if these were involved in phagocytosis during both the natural and the induced phases of neuronal degradation. The ganglion cells which survived natural neuronal cell death remained fluorescent for 3 months after labelling with either dye on the day of the animal's birth, indicating that both fluorescent probes persisted within neurons. Quantitatively, 1770+/-220 ganglion cells/mm2 were labelled within the contralateral retina and a total population of 1442+/-120 cells/retina were observed within the periphery of the inferior/temporal quadrant of the ipsilateral retina. A smaller, ipsilateral projection of 150+/-24 cells/retina was uniformly scattered throughout the rest of the retinal surface. Transient projections of ganglion cells to either the contralateral or the ipsilateral colliculi and death of labelled ganglion cells during the first postnatal days resulted in labelling of 210+/-36 microglial cells/mm2 within the contralateral retina and a total number of 800+/-120 cells/retina within the inferior/temporal and 200+/-22 cells/retina within the rest of the retina. These labelled microglial cells were observed in adulthood and indicated that after taking away the neuronal cell debris they persisted within the retinal tissue. The small number of prelabelled ganglion cells which formed persistent ipsilateral projections until adulthood were axotomized by transecting the optic nerve, and resulted in additional labelling of microglial cells with the second fluorescent dye as well. Double-labelled microglia were observed within the inferior/temporal quadrant (3500+/-240 cells/retina) and to a lesser extent (340+/-40 cells/retina) scattered over the entire retinal surface. The chronotopological sequence of microglial labelling paralleled that of ganglion cell degeneration. Injection of protease inhibitors into the vitreous body during optic nerve transection retarded retrograde glial cell degeneration, probably by blocking microglial proteases. The results directly proved that the same microglial cells which remove neuronal cell debris in the postnatal retina were reactivated later in life to proteolytically degrade and then phagocytose neurons which had altered because of the axotomy.

Journal Article↗

PubMed1995

A critical period for axon regrowth through a lesion in the developing mammalian retina.

Although the central nervous system of mature mammals is incapable of regeneration, certain elements present in the developing system must permit and promote the growth of new axons to their initial targets. We investigate whether the environment of a developing visual system is capable of supporting regeneration in the Brazilian opossum Monodelphis domestica, in which the retinofugal system develops postnatally. Retinae were lesioned up to the 16th postnatal day and analysed for regeneration after a further 7-10 days. Anterograde tracing with Dil showed axons to have regrown from the axotomized area of retina directly through the lesion. Retrograde tracing with horseradish peroxidase injected into the superior colliculus confirmed that axons from the lesioned area of retina had grown to an appropriate position in the midbrain. The proportion of retinae in which axonal continuity was restored across the lesion decreased as the visual system matured, falling to zero after the 12th postnatal day. Thus a critical period exists in the postnatal opossum in which a retinal lesion permits axon passage. Correlating these results to the known pattern of retinofugal pathway development provides an insight into factors that may restrict this critical period to the 12th postnatal day, and suggests that at least some of the axotomized neurons are regenerating.

Animals↗

PubMed1995

The critical period for repair of CNS of neonatal opossum (Monodelphis domestica) in culture: correlation with development of glial cells, myelin and growth-inhibitory molecules.

A comparison was made of neurite growth across spinal cord lesions in the isolated central nervous system (CNS) of newborn opossums (Monodelphis domestica) at various stages of development. The aim was to define the critical period at which growth after injury ceases to occur, with emphasis on growth-inhibitory proteins, myelin and glial cells. In postnatal opossums 3-6 days old (P3-6), repair was observed 5 days after lesions were made in culture at the cervical level (C7) by crushing with forceps. Through-conduction of action potentials was re-established and axons stained by Dil grew into and beyond the crush. In a series of 66 animals 29 showed repair. In 28 animals at P11-12 with comparable lesions repair was observed in five preparations. At P13-14, the CNS was still viable in culture, but none of the 25 preparations examined showed any axonal growth into the crush or conduction through it. The rostro-caudal gradient of development permitted lesions to be made in mature cervical and immature lumbar regions of P11-12 spinal cord. Growth across crushes occurred in lumbar but not in cervical segments of the same preparation. The development of glial cells and myelin was assessed by electron microscopy and by staining with specific antibodies (Rip-1 and myelin-associated glycoprotein) in cervical segments of neonatal P6-14 opossums. At P8, oligodendrocytes and thin myelin sheaths started to appear followed at P9 by astrocytes stained with antibody against glial fibrillary acidic protein. By P14, astrocytes, oligodendrocytes and well-developed myelin sheaths were abundant. The cervical crush sites of P12 cords contained occasional astrocytes but no oligodendrocytes. Specific antibodies (IN-1) to neurite growth-inhibiting proteins (NI-35/250) associated with oligodendrocytes and myelin in the rat CNS cross-reacted with opossum proteins. Assays using the spreading of 3T3 fibroblasts and IN-1 showed that by P7 inhibitory proteins became apparent, particularly in the hindbrain and cervical spinal cord. The concentrations of NI-35/250 thereafter increased and became abundant in the adult opossum. Our finding of a well-defined critical period, encompassing only 5 days, in CNS preparations that can be maintained in culture offers advantages for analysing mechanisms that promote or prevent CNS repair.

Age Factors↗

PubMed1995

The corticotectal projection of the rat in vitro: development, anatomy and physiological characteristics.

In this study, the formation of the corticotectal projection of the rat in organotypic slice culture was investigated, using both anatomical and physiological approaches. The establishment of fibre connections from visual cortex to superior colliculus explants was monitored after 3, 6, 14, 20 and 30 days in vitro by cortical injections of Dil. As in cortical cultures without cocultured colliculus, fibres anterogradely labelled by this procedure spread radially from the injection site into the surroundings of the explant, without displaying any directional preference. Especially, layer V pyramidal cells could be seen to extend processes not only to the collicular target, but also in the opposite direction, suggesting that no axonal guidance was exerted by the projection target. The total number of fibres projecting in the direction of the colliculus was not higher than of those projecting in the opposite direction. However, fibres projecting into the colliculus were significantly longer. This was also the case when the colliculus was placed next to the pial side of the cortical explant, indicating that outgrowth direction was not related to this observation. We therefore assume a chemotrophic rather than a chemotactic influence of the projection target on cortical axons, which is based on direct contact of axons to the target tissue. It cannot be excluded, however, that the failure to detect chemotactic guidance was caused by the lack of diffusion gradients in our culture system. Innervation of the collicular slice exclusively originated from layer V pyramidal cells, irrespective of the position of the collicular target. Fibre courses suggested that discrimination of the projection target was achieved upon encounter with the collicular surface by direct membrane contact. Inside the collicular tissue, fibre arborizations occurred preferredly in up to three layers perpendicular to the surface. Even after the smallest tracer injections, termination fields were diffusely distributed over the collicular slice. Also, the spatial distribution of retrogradely stained projection neurons did not differ statistically from an equal distribution. Thus, a high degree of convergence and divergence was observed anatomically in the corticotectal projection formed in vitro, corresponding to the immature state in vivo. The functionality of the corticotectal projection was assessed by intracellular recordings from collicular neurons. Electrophysiological properties, such as membrane potential (-68 +/- 11 mV), membrane resistance (35.4 +/- 27.7 M omega) and the time constant (3.0 +/- 2.1 ms) were comparable to reference values, confirming the viability of our culture preparation. The functionality of corticotectal transmission was revealed by intracellularly recorded responses of collicular cells to extracellular cortical stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1996

Developmentally regulated neurite outgrowth response from dorsal root ganglion neurons to heparin-binding growth-associated molecule (HB-GAM) and the expression of HB-GAM in the targets of the developing dorsal root ganglion neurites.

Heparin-binding growth-associated molecule (HB-GAM) is a highly conserved cell surface- and extracellular matrix-associated protein that enhances neurite outgrowth in brain neurons in vitro. To study the possible response of peripheral neurons, we cultured chicken dorsal root ganglion neurons from different developmental stages from embryonic day 4.5 (E4.5; St 25) to E9 (St 35) on recombinant HB-GAM. We discovered that the neurite outgrowth response to HB-GAM is maximal at E5.5-6.5 (St 28-30). In order to correlate this in vitro phenomenon with in vivo phenomena, immunohistochemical staining and in situ hybridization were performed on cryosections. The protein expression of HB-GAM peaked at E6 (St 29) and was most extensive on the dorsal spinal cord and dorsal roots. Using Dil labelling, we confirmed that at the time when sensory afferents travel longitudinally in the bundle of His of the spinal cord, HB-GAM protein expression there is at its peak. Though HB-GAM is a secreted protein, at the RNA level the timing of HB-GAM appearance and existence in the spinal cord and sensory ganglia is in accordance with its protein expression. Our results demonstrate that peripheral neurons are responsive to substrate-bound HB-GAM in a developmentally regulated manner, and that the expression of both HB-GAM mRNA and protein in vivo is spatially and temporally matched to this in vitro phenomenon. HB-GAM is therefore a putative cue for the growth of sensory afferents to and within the dorsal spinal cord.

Animals↗

PubMed1996

Effects of NMDA receptor blockade in the developing rat somatosensory cortex on the expression of the glia-derived extracellular matrix glycoprotein tenascin-C.

The patterning of synaptic connections during development is thought to be influenced by the correlation of neuronal impulse activity. N-methyl-D-aspartate (NMDA) receptors have been implicated in the reorganization of thalamocortical afferents in the visual system. The topographic mapping of the periphery of sensory systems onto the somatosensory cortex in the whisker-barrel field of rodents has served as another important paradigm in the study of extrinsic influences on synaptic rearrangements. In a search for the molecular cues that may contribute to synaptic plasticity, we have investigated the distribution of the glia-derived extracellular matrix glycoprotein tenascin-C, which is highly expressed during the formation of the barrel field map around birth and delineates the boundaries between barrel fields after segregation of afferent inputs. Here we show that systemic and local application of NMDA receptor antagonists at postnatal day 2 inhibited the down-regulation of tenascin mRNA and protein by postnatal day 6 and prevented the appearance of tenascin-positive barrel field boundaries. Furthermore, barrels were not distinguishable by Nissl staining, and segregation of thalamocortical afferents as monitored by anterograde Dil tracing and acetylcholinesterase histochemistry was not complete. These observations indicate that expression of tenascin-C and segregation of afferent inputs are modified by NMDA receptor-dependent neuronal activity.

2-Amino-5-phosphonovalerate↗