Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “TMB”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 883 records · Page 49Linked to original sources
PubMed1983

Phospholipid-induced human platelet activation: effects of calcium channel blockers and calcium chelators.

Human platelet activation (aggregation, [14C]-5HT release and TxB2 production) induced by the phospholipids, PAF and lysophosphatidic acid (LPA) was inhibited by EGTA, TMB-8 (an intracellular calcium antagonist) and by phenylalkylamine (Class II) but not 1,4-dihydropyridine (Class I) calcium channel blockers. Primary aggregation induced by PAF was selectively inhibited by phenylalkylamine (verapamil, methoxyverapamil) calcium channel blockers. Phospholipid-induced human platelet activation depends predominantly on the influx of extracellular calcium, possibly via specific receptor-operated calcium channels.

Biological Transport↗
PubMed1994

Activation of guinea pig platelets by a monoclonal antibody.

We herein describe the characterization of a monoclonal anti-guinea pig platelet antibody, termed GT2-12, which causes aggregation, ATP and [3H]serotonin release, and platelet protein phosphorylation. GT2-12 can bind to platelets and megakaryocytes in guinea pig bone marrow cells. A protein of 34,000 daltons was detected by immunoprecipitation of platelet lysates with GT2-12. Incubation of 32P-labeled guinea pig platelets with GT2-12 resulted in rapid phosphorylation of proteins of 40,000 and 20,000 daltons. GT2-12-induced aggregation and protein phosphorylation of platelets were inhibited by diltiazem, TMB-8 and dibutyryl cAMP and partially inhibited by indomethacin. The F(ab')2 fragment of GT2-12 IgG also induced platelet aggregation, indicating that activation is not mediated by Fc receptors. This type of antibody should be useful for studying platelet function in the guinea pig model.

Adenosine Triphosphate↗
PubMed1996

Calcium, calmodulin and protein kinase C dependence of platelet shape change.

Platelet shape change (PSC) represents the initial phase of platelet activation and is normally investigated in ethylene diamine tetraacetic acid (EDTA) containing platelet rich plasma (PRP); EDTA is a potent chelator of calcium and therefore reduces ionized calcium to negligible levels. It is therefore assumed that it is a process independent of calcium. To test the hypothesis that PSC may be dependent upon intracellular calcium, we examined the effect of 8-(N,N-Diethylamino) octyl 3,4,5-Trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of intercellular calcium mobilization on PSC. It produced a dose dependent inhibition of PSC. We then examined whether PSC was dependent upon calmodulin and protein kinase C, a calcium dependent enzyme which is cardinal to platelet aggregation. Both calmidazolium, a specific inhibitor of calmodulin, and H-9, a specific inhibitor of protein kinase C, produced dose dependent inhibition of PSC. Finally, we investigated whether GP IIb/IIIa receptor which binds fibrinogen was involved in PSC; DMP 728 [(cyclic [D-2-amino-butyryl-N2-methyl-L-arginyl-glycyl-L-aspartyl-3- (a min o-methyl-benzoic acid], methanesulfonic acid salt] a potent GP IIb/IIIa receptor antagonist was without any effect on PSC. We conclude that PSC is a calcium, calmodulin and protein kinase C dependent process like platelet aggregation but that it does not require extracellular calcium or the participation of platelet GP IIb/IIIa complex.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1994

Angiogenesis and the blood-brain barrier in intracerebral solid and cell suspension grafts.

Solid and suspension grafts of fetal central nervous system (CNS) tissue rapidly reform an intact blood-brain barrier (BBB), whereas solid grafts of peripheral nervous system (PNS) tissue fail to establish a BBB as detected by horseradish peroxide (HRP) leakage, administrated intravenously. We examined the acute changes in the BBB after grafting of fetal CNS tissue in solid and suspension form and superior cervical ganglion (SCG) and PNS tissue in the same manner. Adult rats (n = 20) received fetal (day 14-15) forebrain grafts (either solid or cell suspension) to their rostral corpus callosum bilaterally. A second group (n = 20) received SCG solid and cell suspension grafts at the same coordinates with the same technique. The animals were killed on first, third, seventh, and tenth days after grafting. Intravenous HRP (Sigma, type VI, 75 mg/5-g rat) was given 1 hour before perfusion with mixed aldehydes. Fifty-micron coronal sections were examined for the presence and location of the graft by cresyl violet and AChE staining and Mesulam's TMB method to detect HRP leakage. HRP leakage was detected in the parenchyma in all groups on the first and the third days post-transplantation indicating a disrupted BBB. No HRP reaction was seen at days 7 and 10 in groups receiving fetal forebrain tissue whether solid or cell suspension. Solid grafts of SCG consistently demonstrated HRP leakage from the first through the tenth day. However, cell suspension of SCG established a BBB by 7 days. These results suggest that within the solid grafts of CNS and PNS tissue, the permeability of the vessels is dictated by the transplanted tissue itself. When cell suspensions of the same tissue are introduced, host CNS tissue dominates as the local environment resulting in non-leaky vasculature within the graft.

Animals↗
PubMed1985

Differentiation of the mechanisms by which leukotrienes C4 and D4 elicit contraction of the guinea pig trachea.

The contractions elicited by leukotriene (LT) C4 and D4 in isolated guinea pig trachea were characterized under conditions in which LTC4 to LTD4 metabolism was blocked by the presence of 45 mM l-serine-borate complex (SB). The presence of SB caused a shift of the LTC4-concentration-response curve to the left by 7.5-fold, and blocked the bioconversion of LTC4 to LTD4 by the trachea as estimated by HPLC analysis of the LTs present in the tissue bath fluid. The potency of FPL 55712 as an antagonist of the LTC4-induced contractions in the presence of SB was 15-30-fold less than its potency as an antagonist of the LTD4-induced contractions. In contrast, another LT antagonist, SK&F 101132, equally antagonized the contractions elicited by LTC4 and LTD4 in either the presence or absence of SB. The differential antagonism of LTC4 and LTD4 implies the existence of multiple pharmacologic receptors for the LTs. The calcium channel entry blockers, nifedipine and verapamil, at concentrations as high as 10 microM, suppressed the maximal LTC4-induced contraction by no more than 20%, whereas the purported intracellular calcium antagonist, TMB-8, completely suppressed the LTC4 concentration-response curve in the presence of SB, a profile identical to that previously reported for LTD4. Thus, if multiple LT receptors exist, they appear to mobilize calcium in a qualitatively similar fashion following LT stimulation.

Airway Resistance↗
PubMed1986

Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells.

We have investigated the effects of extracellular and intracellular Ca deficits and of pharmacologic agents thought to inhibit Ca influx or intracellular Ca mobilization on vasopressin-evoked changes of cytosolic Ca2+ levels and PG synthesis in cultured rat mesenteric arterial vascular smooth muscle cells. Vasopressin rapidly increased cytosolic Ca2+ as well as PG synthesis. The increase of cytosolic Ca2+ and the rate of PG synthesis were both maximal within the first minute of incubation. An extracellular Ca deficit of short duration partially inhibited both vasopressin-evoked PG synthesis and the increase of cytosolic Ca2+ by 40 to 60%. Two procedures which deplete cells of some of their intracellular Ca, namely a 30 min incubation in EGTA-supplemented, Ca-lacking media, or a 1 min incubation with ionophore A23187 in Ca-deficient media, decreased PG synthesis by 65% to 100%. The addition of extracellular Ca to Ca-depleted cells restored the ability of vasopressin to stimulate PG synthesis. Two Ca channel antagonists, nifedipine or cinnarizine, had no effect on either vasopressin-evoked PG synthesis or increased cytosolic Ca2+, whereas TMB-8 (10 microM), a putative inhibitor of intracellular Ca mobilization, decreased PG synthesis by 75% by inhibiting acylhydrolase as well as cyclo-oxygenase activities, but had no effect on basal or vasopressin-evoked increase of cytosolic Ca2+, documenting that its inhibitory effect was not a consequence of decreased cytosolic Ca2+. These results demonstrate that decreased cellular Ca levels are associated with decreased cytosolic Ca2+ levels and PG synthesis, and support the hypothesis of a link between, on the one hand, cellular Ca and/or cytosolic Ca2+ and on the other hand, PG synthesis.

Animals↗
PubMed1992

PGE2 secretion from organ cultured gastric mucosa: correlation with cyclooxygenase activity and endogenous substrate release.

In gastrointestinal research the in vitro release of prostaglandins from incubated or cultured biopsies is a widely used method to estimate prostaglandin synthesis. We therefore investigated the rate limiting mechanisms of PGE2 release in organ cultured gastric mucosa of the rabbit, determining PGE2 secretion from organ cultured mucosal biopsies by radioimmunoassay and prostaglandin synthesizing capacity by in vitro incubation of mucosal homogenate or microsomes with [14C]-arachidonic acid. Freshly taken biopsies secreted PGE2 at an initial high rate, that decreased during the following 4 hrs of culture. This PGE2 release was dose dependently reduced by inhibitors of the prostaglandin cyclooxygenase. 5mM acetylsalicylic acid (ASA) maximally suppressed PGE2 secretion to 7% of controls, and the inhibition by ASA was quantitatively similar at every given culture period. PGE2 release was markedly increased by carbenoxolone but was only slightly activated by extracellular calcium and the Ca(++)-ionophore A23187. However, Ca++/A23187 were unable to maintain PGE2 secretion at the initial rate. PGE2 secretion was undisturbed in calcium-free medium but was reduced to 50-60% of controls by excess EDTA. The intracellular calcium chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N',-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) similarly inhibited PGE2 release to 72% of controls. In contrast, PGE2 release was unaffected by the intracellular calcium antagonist 3,4,5-trimethylene-bis(4-formylpyridinium bromide) dioxime (TMB-8), the calmodulin antagonists N-(6-aminohexyl)-1-5-chloro-1-naphthalenesulfonamide (W-7) and calmidazolium (compound R24571) or various direct inhibitors of endogenous arachidonic acid release like tetracaine, bromophenacyl bromide, neomycin or low dose quinacrine, indicating that the reduction of PGE2 release by EDTA or BAPTA may be mediated by mechanisms different from substrate release. In contrast, an inhibition of PGE2 secretion by quinacrine at high concentrations (greater than or equal to 0.8 mM) was attributed to a direct inhibition of the prostaglandin cyclooxygenase, similar to ASA. Finally, the reduction of the prostaglandin synthesizing capacity by ASA was strongly correlated with the inhibition of PGE2 secretion, also at low concentrations and minor degrees of inhibition. From these data we conclude, that the activity of the prostaglandin cyclooxygenase is rate limiting for PGE2 secretion from organ cultured mucosal biopsies rather than arachidonic acid release by a phospholipase A2. This should be considered for interpretation of studies based on prostaglandin release from cultured mucosa.

Animals↗
PubMed1993

Competence induction by PDGF requires sustained calcium influx by a mechanism distinct from storage-dependent calcium influx.

The significance and mechanism of extracellular calcium influx in the stimulation by PDGF of cell replication was investigated in density-arrested C3H 10T1/2 mouse fibroblasts. PDGF consistently stimulated a biphasic increase in the [Ca2+]i composed of a rapid transient release of calcium from intracellular storage sites followed by a sustained elevation, significantly greater than prestimulated levels, which was dependent upon the [Ca2+]e and persisted for at least 1 h. The percentage of cells incorporating [3H]-TdR into DNA after stimulation with PDGF+insulin was closely correlated with the magnitude of the sustained [Ca2+]i increase and to the [Ca2+]e. Selective inhibition of the sustained [Ca2+]i increase, by blocking calcium influx with La3+, completely inhibited progression to S phase without affecting the release of calcium from intracellular storage sites. Progression to S phase was inhibited by La3+ or the omission of added extracellular calcium only during PDGF exposure and not during treatment with insulin. PDGF-induced calcium influx was completely inhibited by La3+ whereas storage-dependent calcium influx (SDCI) induced by thapsigargin was unaffected. Pretreatment with TPA, forskolin, dibutyryl-cAMP, dibutyryl-cGMP, nifedipine, and TMB-8 had no effect on PDGF-induced calcium influx. These data suggest that the induction of replicative competence by PDGF is dependent upon the maintenance of a sustained increase in the intracellular calcium concentration due to the influx of extracellular calcium through a calcium influx pathway distinct from SDCI.

Animals↗
PubMed1993

Independent actions of asparagine and high levels of free Ca2+ in the induction of ornithine decarboxylase.

During growth stimulation of cells, Ca2+ and amino acids of the A, ASC and N transport systems are important for the induction of ornithine decarboxylase (ODC, L-ornithine carboxylase, EC 4.1.1.17). In order to clarify the relationship between Ca2+ and amino acids, we studied the induction of ODC by asparagine under three different Ca2+ states in H-35 rat hepatoma cells. First, in normal cells, extracellular Ca2+ above 0.1 mM and 10 mM asparagine separately stimulated ODC activity and their effects were approximately additive. In these normal cells, asparagine could act in the absence of medium Ca2+. TMB-8, a sequestered-Ca2+ release antagonist, had no effect on ODC induction whilst the asparagine action is sensitive to treatment with W7, a Ca-calmodulin antagonist, or lanthanum, a Ca2+ antagonist. Secondly, in cells treated with 0.5 mM EGTA in Ca(2+)-free medium, the asparagine action on ODC induction was blocked but the inhibition could be reversed by the addition of Ca2+ to the medium. Thirdly, ionomycin treatment in the absence of medium Ca2+ did not block the asparagine effect. Furthermore, in ionomycin-treated cells, the presence of high levels of medium Ca2+ increased ODC activity, but this increase was additive to, and could not replace, the action of asparagine. Our results indicate that the asparagine action does not depend on an increase of intracellular free-Ca2+.

Animals↗
PubMedDate not supplied

Histamine release from beta-escin-permeabilized rat peritoneal mast cells and its inhibition by intracellular Ca2+ blockers, calmodulin inhibitors and cAMP.

In order to study the intracellular events leading to histamine release, rat peritoneal mast cells were permeabilized using beta-escin, a triterpenoid. IP3 (0.5-10 microM) dose-dependently elicited significant histamine release from permeabilized mast cells in a Ca2+-free medium, as did GTP-gamma-S at concentrations higher than 5 microM, GTP-gamma-S induced IP3 production dose-dependently in association with histamine release. Histamine release induced by IP3 and GTP-gamma-S was inhibited by pretreatment with TMB-8, while neomycin pretreatment suppressed histamine release caused by GTP-gamma-S but not that caused by IP3.IP3 may cause Ca2+ release from the intracellular Ca2+ store as the key event leading to histamine release. At concentrations higher than 0.1 microM, Ca2+ dose-dependently induced histamine release. Both IP3-and Ca2+-induced histamine release from permeabilized mast cells was inhibited by pretreatment with calmodulin inhibitors (W-7 and calmidazolium), or with cytochalasin D or colchicine. Pretreatment with cAMP also inhibited the histamine release induced by either IP3 or Ca2+. From the present study, it can be assumed that (1) Ca2+ may act on a calmodulin-associated process(es) and cytoskeletal systems, in the process leading to histamine release, and (2) cAMP seems to inhibit histamine release, even in the stages following Ca2+ release from the intracellular Ca2+ store.

Animals↗
PubMedDate not supplied

Role of endoplasmic reticulum, an intracellular Ca2+ store, in histamine release from rat peritoneal mast cell.

By means of the Ca-antimonate precipitation technique, it was revealed that Ca2+ accumulates in the endoplasmic reticulum (ER) of rat peritoneal mast cells. The ER of rat mast cells was isolated using Percoll density gradient centrifugation, and its characteristics were studied. Although the uptake of 45Ca into the ER was enhanced by adenosine 5'-triphosphate (ATP) at concentrations lower than 2 mM, at higher concentrations ATP induced 45Ca release from the ER, suggesting that bidirectional translocation of Ca2+ takes place in the ER membrane. When apyrase was added to the reaction mixture to decompose all ATP molecules, the amount of 45Ca in the ER was decreased, indicating that ATP is necessary to retain Ca2+ in the ER. Not only inositol 1,4,5-trisphosphate (IP3) but also guanosine 5'-triphosphate (GTP) was effective in releasing Ca2+ from the ER at concentrations higher than 2 microM, while guanosine 5'-[gamma-thio]-triphosphate (GTP-gamma S), a non-hydrolysable analogue of GTP, was not effective. This may indicate that a hydrolysis of GTP is necessary for Ca2+ release from the ER. Intracellular Ca2+ blockers such as 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and dantrolene sodium were effective in inhibiting Ca2+ release from ER induced by IP3. The Ca2+ release due to IP3 was also inhibited by flunarizine, a Ca2+ channel blocker, and by oxatomide, an antiallergic drug. Since these two compounds are diphenylpiperazine derivatives, it is suggested that a diphenylpiperazine moiety may be effective in inhibiting Ca2+ release from the ER.

Adenosine Triphosphate↗
PubMedDate not supplied

Anaphylatoxin C3a induces rapid protein phosphorylation in guinea pig platelets.

We investigated C3a signal transduction using guinea pig platelets. Anaphylatoxin C3a induced cytosolic calcium influx and turnover of inositol phospholipids and caused protein phosphorylation via specific C3a receptors on guinea pig platelets. Phosphorylation of 40 kDa and 20 kDa proteins was detected within 30 s after C3a stimulation. From phosphoamino acid analysis of both proteins, it was found that about 80% was serine and 20% was threonine. Phosphorylation was decreased by pretreatment of platelets with calcium blockers, diltiazem and TMB-8. A protein kinase C inhibitor, staurosporine, effectively blocked phosphorylation of both proteins, but H-7 did not. The effects of these inhibitors on platelet aggregation were correlated with their effects on protein phosphorylation. These results suggest that protein-serine/threonine phosphorylation is important for C3a signal transduction in guinea pig platelets and that the isoenzyme of protein kinase C or another kinase probably participates in such protein phosphorylation. C3a also caused protein-tyrosine phosphorylation of 56 kDa and 33-36 kDa proteins within 30 s. Staurosporine effectively blocked phosphorylation of these bands. It is suggested that tyrosine kinase also may be involved in C3a signalling in guinea pig platelets.

Animals↗
PubMed1995

The raccoon spinocervical and spinothalamic tracts: a horseradish peroxidase study.

The locations of the cells of origin of the spinocervical tract (SCT) and spinothalamic tract (STT) were examined in relation to the somatotopic and laminar organization of the cervical enlargement of the raccoon dorsal horn (DH). In different animals, either the lateral cervical nucleus or the lateral thalamus was injected with a 2% solution of wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Following 24 h or 4 days, respectively, the animals were sacrificed and both injection and target sites (spinal cord segments C6-T2) were processed using the TMB method. All labelled cells were counted in every fifth 50 microns section. Following injection of WGA-HRP into the lateral cervical nucleus, all labelled SCT cells were located ipsilateral to the injection sites. Most (84%) were in laminae III and IV, laminae known from other studies to contain cells preferentially responsive to light tactile stimulation, with very few (3%) in lamina I. Nearly 50% of labelled cells were located in the medial 1/3 of the DH, the region of representation of the glabrous surfaces of the raccoon forepaw. The mean number of labelled SCT cells per section was 4.19. After tracer injections of the lateral thalamus, more than 75% of STT cells were located contralateral to the injection sites. Forty-three percent were located in lamina I and 24% were in lamina V, laminae whose cells have been shown to be responsive to more intense forms of stimulation, as well as to light touch. Only 22% were located in the medial 1/3 of the DH. The mean number of labelled STT cells per section was 0.83. The results suggest that the SCT may play a more critical role in relaying discriminative light tactile and nociceptive information from the glabrous surfaces of the forepaw, but that there may be a greater role for the STT in relaying nociceptive information from the forelimb as a whole.

Animals↗
PubMed1991

Light and electron microscopical visualization of anterogradely labelled corticospinal growth cones using a new combination of HRP staining techniques.

Up until now, the ultrastructural visualization of growth cones of developing long fibre tracts could only be achieved by horseradish peroxidase (HRP) application 'en route', resulting in axonal damage, which in turn may affect growth cone morphology. Besides, this technique results in labelling of passing fibres, thus hampering the identification of axon origin as well as the interpretation of growth cone configuration. In the present investigation a new combination of HRP staining and intensification techniques is presented which makes it possible to visualize anterogradely labelled corticospinal growth cones over long distances in developing rat spinal cord at the light as well as the electron microscopical level. HRP was applied to the originating cells of the corticospinal tract, located in the sensorimotor cortex, and after 24 h was visualized using a procedure which essentially consists of 3 subsequent steps: first a tetramethylbenzidine (TMB)/ammoniumheptamolybdate (AHM) reaction; second diaminobenzidine (DAB)/nickel (Ni) stabilization and finally glucose oxidase intensification. As was verified at the EM level, the staining procedure here described reveals a complete intense black staining of HRP-labelled growth cones of outgrowing corticospinal axons. Therefore, the method described here guarantees a correct analysis of growth cone morphology at the light microscopical and the ultrastructural level. The present procedure is especially valuable in studying the development of long central nervous fibre systems.

Animals↗
PubMed1995

Electron microscopic imaging of multiple markers in glutaraldehyde fixed CNS tissue of Macaca fascicularis: maximizing information from a single experimental animal.

Sensory and motor pathways in the central nervous system (CNS) of macaque monkeys were visualized by anterograde or retrograde axonal transport of wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) reacted with the chromagen tetramethylbenzidine (TMB), or by the use of anterograde degeneration after specific ablation lesions. To maximize information from each animal we combined the results of the anterograde and retrograde axonal transport with several pre- and post-embedding markers at both the light and electron microscopic levels while maintaining good preservation of tissue. Pre-embedding techniques included those for cytochrome oxidase activity and the calcium-binding proteins calbindin D-28k and parvalbumin. Post-embedding techniques included immunocytochemistry for gamma-aminobutyric acid (GABA) or other amino acid neurotransmitters. We believe that the methods described here provide superior tissue preservation, thus permitting a more detailed analysis of tissue prepared after experiments concerned with neural circuitry.

Animals↗
PubMed1995

A modification of biotinylated dextran amine histochemistry for labeling the developing mammalian brain.

Biotinylated dextran amine (BDA) has proven to be an excellent anterograde tracer in adult mammalian brains, having some advantages over other anterograde tracers such as Phaseolus vulgaris-leucoagglutinin (PHA-L) and biocytin. However, results are inferior when BDA is used in neonatal mammals. To improve the sensitivity and quality of BDA labeling in neonatal mammalian brains, the tetramethylbenzidine-sodium tungstate (TMB-ST) method for horseradish peroxidase (HRP) histochemistry was modified and used in BDA histochemistry. After BDA application to the visual cortex of neonatal rat and cat, contralateral and ipsilateral cortical and subcortical regions were examined for BDA-labeled exons and terminals. The modified BDA histochemistry produced corpus callosum (CC) axons in neonatal rat and cat that were heavily and continuously labeled. The distribution, trajectories, branching and termination of individual CC axons, and even possible axon-axon contracts, were clearly identified in exquisite detail, even at low magnification. The quality of BDA labeling in the ipsilateral lateral geniculate nucleus and superior colliculus was similar to that of the CC axonal labeling. These results indicate that the modified BDA histochemistry provides a very sensitive and reliable approach to revealing the detailed distribution and morphology of projecting axons and terminals in the developing mammalian nervous system.

Animals↗
PubMed1996

Anterograde axonal tracing with the subunit B of cholera toxin: a highly sensitive immunohistochemical protocol for revealing fine axonal morphology in adult and neonatal brains.

We report an improved immunohistochemical protocol for revealing anterograde axonal transport of the subunit B of cholera toxin (CTB) which stains axons and terminals in great detail, so that single axons can be followed over long distances and their arbors reconstructed in their entirety. Our modifications enhance the quality of staining mainly by increasing the penetration of the primary antibody in the tissue. The protocol can be modified to allow combination in alternate sections with tetramethylbenzidine (TMB) histochemical staining of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Using the protocol, we tested the performance of CTB as an anterograde tracer under two experimental paradigms which render other anterograde tracers less sensitive or unreliable: (1) labeling the entire retinofugal projection to the brain after injections into the vitreal chamber of the eye, and (2) labeling developing projections in the cortex and thalamus of early postnatal mammals. Qualitative comparisons were made with other tracers (Phaseolus vulgaris leucoagglutinin, dextran rhodamine, biotinylated dextran, free WGA, or WGA-HRP) that were used to label these same projections. From these observations it is clear that CTB, visualized with our protocol, provides more sensitive anterograde labeling of retinofugal projections as well as of axonal connections in the neonatal forebrain.

Animals↗
PubMed1983

Prenatal development of the optic projection in albino and hooded rats.

The development of retinofugal projections has been examined in albino and hooded rat embryos from embryonic day 16 to birth (E21.5). Horseradish peroxidase (HRP) was injected intraocularly through the uterine wall and its anterograde transport revealed with TMB and DAB. The retrograde transport of HRP or the fluorescent dyes Nuclear yellow, Fast blue and propidium iodide from optic tract, superior colliculus (SC) or lateral geniculate body (LG) injections was used to demonstrate the origin of the projections. Superficial projections to the contralateral SC were first identified at E16. A light projection to the entire medio-lateral extent of the ipsilateral SC could be detected a day later. The optic axons grow over the surface of the diencephalon at E16 and it was only at later stages that the fibers were observed to invade successively deeper parts of the LG. A superficial projection to the ipsilateral LG could first be detected at E17. Both the ipsilateral and contralateral projections grew through the entire dorso-ventral extent of the lateral geniculate body: some restriction of the axons to their normal adult termination zones could be detected by E21. No difference in the distribution of projections could be detected between the albino and pigmented rats although the projections were lighter, and possibly because of this were detected later, in the albino rats. At all the ages examined in this study labeled retinal ganglion cells were observed in the non-injected eyes after injection of label into the contralateral eye. The use of persistent fluorescent dyes showed that these retinal ganglion cells did not survive for more than 5 days postnatally. The projection to the uninjected eye came preferentially from ganglion cells in the lower nasal retina while the ipsilateral central projections came predominantly but not exclusively from the lower temporal retina of the injected eye. It appears, therefore, that the initial projections of optic axons in the rat are not limited to their normal termination zones and that the choice of pathway at the chiasm appears to be only loosely controlled.

Animals↗