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F G Wouterlood

Publications and source records attributed to F G Wouterlood.

At least 37 records · Page 2Linked to original sources
PubMed1994

Multiple anterograde tracing, combining Phaseolus vulgaris leucoagglutinin with rhodamine- and biotin-conjugated dextran amine.

The simultaneous use of different neuroanatomical anterograde tracers provides a potentially powerful method to study the convergence of afferent systems in a particular brain area. However, a simple routine procedure to apply multiple anterograde tracers in conjunction with their simultaneous visualization is still missing. We report an easy and straightforward application of three sensitive anterograde tracers: Phaseolus vulgaris leucoagglutinin (PHA-L), rhodamine-conjugated dextran amine (RDA) and biotin-conjugated dextran amine (BDA). These tracers can be visualized simultaneously and permanently through a triple-staining procedure with nickel-enhanced diaminobenzidine (DAB-Ni), DAB and 1-naphthol/Azur B as chromogens. Our test model comprised the projections from the nucleus reuniens thalami and entorhinal cortex. Both projection systems show a high degree of overlap in their terminal fields in the hippocampus. Two tracers were injected in the left and right entorhinal cortex, respectively; a third tracer was injected in the nucleus reuniens. This combination of injections provided a good opportunity to compare the three tracers in one and the same animal. PHA-L, RDA and BDA, injected in either of the injection sites, turned out to be equally sensitive and revealed the morphology of the involved projection systems in great detail. The triple-staining protocol yielded an excellent, simultaneous detectability of the three tracers with a remarkably low background level. Thus, the combination of the anterograde tracers PHA-L, RDA and BDA, in conjunction with the triple-staining procedure, offers a very attractive approach for neuroanatomical research.

Animals↗
PubMed1994

Combined anterograde tracing with biotinylated dextran-amine, retrograde tracing with fast blue and intracellular filling of neurons with lucifer yellow: an electron microscopic method.

In order to determine the presence of synaptic connectivity between fibres originating from a specific source and neurones with a known morphology and known fibre projection, we have introduced a method for electron microscopy that combines three techniques: retrograde fluorescent tracing, anterograde tracing using biotinylated dextran-amine and intracellular injection of Lucifer Yellow (LY) in lightly fixed brain slices. Neurones in the rat entorhinal cortex that project to the infralimbic cortex and that might be in synaptic contact with fibres originating in the dorsal subiculum served as a model. After surgical application of the tracers and a survival period enabling transport, the brain was fixed and vibratome slices 300 microns thick were prepared in which retrogradely labelled cells were intracellularly injected with LY. This substance and the transported biotinylated dextran-amine were converted into different electron-dense labels. First, LY immunocytochemistry was conducted, then followed by silver-gold enhancement of the immunoprecipitate. Subsequently, the tissue sections were treated with an avidin-biotin-horseradish peroxidase complex and subjected to a diaminobenzidine-peroxide reaction. This protocol resulted in labelling of biotinylated dextran-amine-positive fibres and terminals that could easily be differentiated from the LY-positive neuronal elements and also showed well preserved ultrastructural detail.

Amidines↗
PubMed1993

Identification of synaptic interactions of intracellularly injected neurons in fixed brain slices by means of dual-label electron microscopy.

The injection of the dye Lucifer Yellow (LY) into neurons in slices of fixed brain is used to associate cells displaying a particular dendritic geometry with a specific pattern of neuronal connectivity. In the present report we expand on this technique by combining it at the electron microscopic level with immunocytochemistry and/or degeneration for the study of synaptic relationships. As a model we use the projection neurons of nucleus accumbens. These neurons were retrogradely labeled in vivo with injections or a fluorescent tracer. Fast Blue, into the ventral mesencephalon. Using epifluorescent monitoring, these neurons were located in perfusion-fixed brain slices and intracellularly injected with LY. They were visualized in the light and electron microscope using a peroxidase-antiperoxidase immunocytochemical method. Certain afferent connections of these neurons were identified in the same tissue through the use of either dual-label immunocytochemistry or anterograde degeneration combined with a single-label immunoreaction. In the dual-label procedure, a silver-gold intensification of the diaminobenzidine (DAB) reaction product for the first antigen (LY) was contrasted with a nonintensified reaction product for the second antigen (tyrosine hydroxylase [TH]). Ultrastructurally, metallic gold particles appeared to be dispersed over the immunolabeled perikarya, dendrites, and, occasionally, axonal terminals of LY-injected neurons whereas the flocculent DAB reaction product was present in TH-containing axons and terminals. Following lesions of the ventral subiculum in the hippocampal formation, degenerating axon terminals were detected in nucleus accumbens along with immunoreacted, LY-injected neurons. The techniques outlined in this report should prove invaluable for the study of the synaptic interactions of identified neurons. They can be reliably reproduced with a high yield per experiment.

Animals↗
PubMed1993

The anterograde neuroanatomical tracer biotinylated dextran-amine: comparison with the tracer Phaseolus vulgaris-leucoagglutinin in preparations for electron microscopy.

We investigated the properties of biotinylated dextran-amine (BDA) as a neuroanatomical tracer at the electron microscopic level and we compared the results with those obtained previously with another tracer, the lectin Phaseolus vulgaris-leucoagglutinin (PHA-L). BDA was injected into various brain areas of rats. Following survival and fixation, vibratome sections were cut, subjected to a freeze-thaw treatment, and incubated overnight with avidin-biotin complex (ABC). Following reaction with diaminobenzidine (DAB)-hydrogen peroxide, the sections were processed for electron microscopy. In the electron microscope we observed that the reaction product occurred in the cytoplasm of cell bodies and in the matrices of dendrites, axons and axon terminals following ABC histochemistry of BDA-containing brain sections. The ultrastructural details of BDA-labelled neurones were generally better preserved than in PHA-L-labelled material, whereas at the same time penetration of the reagent into the sections was complete (incomplete in sections of PHA-L material). We conclude that the use of BDA as a neuroanatomical tracer in electron microscopy is a good substitute for PHA-L. The detection method for transported BDA is much faster and less complicated than that for PHA-L, while the results are better; that is, there is improved penetration coinciding with good preservation of ultrastructure. Keeping the limitations of BDA as a neuroanatomical tracer in mind, e.g., retrograde transport into local collaterals of axons that intermingle with anterogradely labelled axons, BDA seems well suited as a neuroanatomical tracer for electron microscopy.

3,3'-Diaminobenzidine↗
PubMed1992

Restricted origin and distribution of projections from the lateral to the medial septal complex in rat and guinea pig.

The origin and distribution of projections from the lateral to the medial septal complex were studied at the light- and electron-microscopical level in the rat and the guinea pig, with the use of sensitive anterograde tracing techniques. Injections in the lateral septal complex resulted in only weak to moderate labeling in the medial septal nucleus. In contrast, injections in all but the ventral subdivision of the lateral septal complex labeled restricted terminal arborizations in the angular portions of the vertical diagonal band. Analyses at the electron-microscopical level indicated that these fibers form both symmetrical and asymmetrical synapses with dendrites and somata, but not with axons. These findings are discussed in the light of a presumed functional circuit from the hippocampal formation, via the lateral septal complex, to cells in the medial septal complex that originate projections to the hippocampal formation.

Animals↗
PubMed1992

Techniques for converting Golgi precipitate in CNS neurons into stable electron microscopic markers.

Direct electron microscopy of nervous tissue stained with the Golgi impregnation method is unsatisfactory because the cytoplasm of the cell bodies and processes of the impregnated neurons are completely filled with a compact precipitate of electron dense silver chromate. This precipitate entirely obscures the cytological details of the impregnated neurons. Because of its solidity and instability in aqueous solutions, the silver chromate is also a source of inconvenience during the preparation of the ultrathin sections. This review summarizes methods that have been developed with the aim of replacing the Golgi precipitate in CNS neurons with a more convenient electron dense material--for example, heavy metal salts or metallic particles. Conversion of the precipitate into a stable electron dense marker is done before the material is embedded for electron microscopy. The methods include lead, gold, and bromide substitution, treatment with ammonia, direct chemical reduction into metallic silver, and photoreduction of the silver chromate into silver through irradiation with ultraviolet light.

Aminophenols↗
PubMed1992

Simultaneous characterization of efferent and afferent connectivity, neuroactive substances, and morphology of neurons.

We present a method for establishing in a single experiment four characteristics of individual neurons: the efferent and afferent connectivity, the morphology, and the content of a particular neuroactive substance. The connectivity of the neurons is determined by retrograde fluorescent tracing with Fast Blue and anterograde tracing with the lectin Phaseolus vulgaris leucoagglutinin (PHA-L). After fixation, the brain is cut into 300-micron thick slices. Neurons containing retrogradely transported Fast Blue are intracellularly injected with the fluorescent dye Lucifer Yellow to fill their dendritic trees. The slices are then resectioned at 20-40 microns. One section through the soma of a Lucifer Yellow-filled neuron is selected for the detection of a neuroactive substance contained by this cell [immunofluorescence, secondary antiserum conjugated to tetramethylrhodamine (TRITC)]. Using appropriate filtering, it can be determined in the fluorescence microscope whether a Lucifer Yellow-containing cell body has also been labeled with TRITC, i.e., whether it is immunoreactive for this neuroactive substance. The adjacent sections are subjected to dual peroxidase immunocytochemistry with different chromogens to visualize the PHA-L-labeled afferent fibers (nickel-enhanced diaminobenzidine, blue-black reaction product) and to stabilize the Lucifer Yellow (diaminobenzidine, brown reaction product) in the dendrites of the intracellular injected cells. The other sections are used for electron microscopic visualization of the transported PHA-L. The relationships between the PHA-L-labeled afferent fibers (blue color) and the dendrites of the intracellularly Lucifer Yellow-injected, retrogradely Fast Blue-labeled cells (brown color) are studied by light microscopy. The electron microscope supplies ultrastructural data on the PHA-L-labeled axon terminals.

Afferent Pathways↗
PubMed1992

Subicular efferents to histaminergic neurons in the posterior hypothalamic region of the rat studied with PHA-L tracing combined with histidine decarboxylase immunocytochemistry.

The projections from the subiculum to histaminergic cells in the posterior hypothalamic region of the rat were studied by means of anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin (PHA-L) combined with histidine decarboxylase (HDC)-immunohistochemistry. PHA-L was injected at various loci along the dorsoventral and proximodistal axes of the subiculum. This resulted in labeling of the fornix and of terminal plexuses at various locations in the diencephalon and the mammillary body. Following deposition of PHA-L in the proximal part of the dorsal subiculum, labeled fibers in the posterior hypothalamus were confined to the mammillary nuclei, whereas after injections of PHA-L in the distal part of the dorsal subiculum and the entire ventral subiculum, labeled fibers were also present in clusters of histaminergic cells located around the mammillary nuclei. The density of the PHA-L labeled fibers within these clusters increased from low to moderate in association with a shift of the injection sites from dorsal to ventral and from proximal to distal parts of the subiculum, i.e., the highest fiber labeling was seen after injections of PHA-L in the distal part of the ventral subiculum. In the latter experiments, PHA-L labeled fibers reached HDC-immunoreactive neurons in the tuberal magnocellular nucleus, the deepest layer of the caudal magnocellular nucleus, the two bridges of histaminergic cells in the posterior hypothalamus, and the histaminergic neurons scattered in the supramammillary region. A few labeled fibers invaded the postmammillary caudal magnocellular nucleus. The presence of varicosities on the PHA-L labeled fibers in close proximity to the cell bodies and dendrites of the histaminergic neurons suggest the existence of synaptic contacts.

Animals↗
PubMed1991

Ultrastructure of aberrant serotonin-immunoreactive fibers in the caudate putamen complex of the aged rat.

Degeneration of neurons in the central nervous system is associated with morphological changes. Previous observations made at the light microscopical level indicated degeneration of serotonin-immunoreactive (IR) fibers in the aged rat brain. In this study, a comparison at the ultrastructural level was made between serotonin-IR normal thin and aberrant swollen varicose fibers in the caudate-putamen complex of the aged rat. Ultrastructural features such as the size and content of the thin varicose fibers resembled those in the caudate-putamen complex of the young rat as reported by others. The aberrant profiles were swollen, reaching a size of 6 microns. Their vesicles varied in size and were no longer uniformly round. Moreover, distorted mitochondria and membrane-filled vacuolelike structures were a common feature of the aberrant profiles. These changes are indicative of a degenerative process and give further evidence that, whereas many serotonergic fibers are preserved at high age, other serotonergic fibers are degenerating in the caudate-putamen complex of the aged rat.

Aging↗
PubMed1991

Ultrastructure of giant and small thalamic terminals of cortical origin: a study of the projections from the barrel cortex in mice using Phaseolus vulgaris leuco-agglutinin (PHA-L).

By means of tracing with the lectin Phaseolus-vulgaris leucoagglutinin (PHA-L), we examined in the thalamus of the mouse, the axon terminals of fibers originating in the barrel cortex. Vibratome sections of the brain were subjected to PHA-L immunocytochemistry and processed for light and electron microscopy. We observed small (0.5-0.8 microns in diameter) varicosities of labeled fibers in the nucleus ventrobasalis (VB) and the nucleus posterior (PO) as well as labeled giant terminals (3-5 microns in diameter) in PO. The analysis involved examination of serial sections and computer-aided reconstruction of several terminals. The small varicosities in VB appear to be small axon terminals forming distinct asymmetric synapses with small dendritic profiles. Some labeled terminals are apposed to, but not synaptically related with, the cell bodies of neurons in VB that are retrogradely labeled with PHA-L. The small varicosities seen with the light microscope in PO are terminals forming asymmetric synapses with dendritic shafts. The giant terminals in PO appear as large, vesicle-filled profiles forming part of synaptic glomeruli, i.e. complexes of one corticothalamic terminal engulfing several excrescences of a single dendrite. A giant terminal forms several asymmetric synapses (about 8) with these excrescences, as well as numerous (up to 15) puncta adhaerentia. The glomeruli are enveloped in glial lamellae, and they are often found at the bifurcations of primary dendritic segments. We suggest that the small terminals in VB are in the service of feedback signalling from the barrel cortex to its principal thalamic relay nucleus; the functional importance of this projection may reside in increased spatio-temporal discrimination. We interpret the giant terminals in PO as elements serving feed-forward processing, allowing the barrel cortex to influence, via PO, parts of the motor pathway modulating the animal's ongoing behavior.

Animals↗
PubMed1990

Projection from the nucleus reuniens thalami to the hippocampal region: light and electron microscopic tracing study in the rat with the anterograde tracer Phaseolus vulgaris-leucoagglutinin.

In order to study the morphological substrate of possible thalamic influence on the cells of origin and area of termination of the projection from the entorhinal cortex to the hippocampal formation, we examined the pathways, terminal distribution, and ultrastructure of the innervation of the hippocampal formation and parahippocampal region by the nucleus reuniens of the thalamus (NRT). We employed anterograde tracing with Phaseolus vulgaris-leucoagglutinin (PHA-L). Injections of PHA-L in the NRT produce fiber and terminal labeling in the stratum lacunosum-moleculare of field CA1 of the hippocampus, the molecular layer of the subiculum, layers I and III/IV of the dorsal subdivision of the lateral entorhinal area (DLEA), and layers I and III-VI of the ventral lateral (VLEA) and medial (MEA) divisions of the entorhinal cortex. Terminal labeling is most dense in the stratum lacunosum-moleculare of field CA1, the molecular layer of the ventral part of the subiculum, MEA, and layer I of the perirhinal cortex. In layer I of the caudal part of DLEA and in MEA, terminal labeling is present in clusters. Injections in the rostral half of the NRT produce the same distribution in the hippocampal region as those in the caudal half of the NRT, although the projections from the rostral half of the NRT are much stronger. A topographical organization is present in the projections from the head of the NRT, so that the dorsal part projects predominantly to dorsal parts of field CA1 and the subiculum and to lateral parts of the entorhinal cortex, whereas the ventral part projects in greatest volume to ventral parts of field CA1 and the subiculum and to medial parts of the entorhinal cortex. The distribution of the reuniens fibers coursing in the cingulate bundle was determined by comparing cases with and without transections of this bundle. The fibers carried by the cingulate bundle exclusively innervate field CA1 of the hippocampus, the dorsal part of the subiculum, and the presubiculum and parasubiculum. They participate in the innervation of the ventral part of the subiculum and MEA. Electron microscopy was used to visualize the axon terminals of PHA-L-labeled reuniens fibers. These terminals possess spherical synaptic vesicles and form asymmetric synaptic contacts with dendritic spines or with thin shafts of spinous dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Hippocampal and midline thalamic fibers and terminals in relation to the choline acetyltransferase-immunoreactive neurons in nucleus accumbens of the rat: a light and electron microscopic study.

The synaptic interactions between terminals of allocorticostriatal and thalamostriatal fibers and the cholinergic neurons in the nucleus accumbens were investigated using degeneration and dual labelling immunocytochemistry in Wistar rats. The presumptive cholinergic neurons were labelled with antibodies directed against choline acetyltransferase and the afferent fibers were labelled anterogradely with Phaseolus vulgaris-leucoagglutinin. Fibers from the subiculum of the hippocampal formation and from the midline and intralaminar thalamus project densely into the medial nucleus accumbens where they overlap a relatively dense population of choline acetyltransferase-immunoreactive neurons. Varicosities containing Phaseolus vulgaris-leucoagglutinin juxtapose the immunoreactive neurons. To study the possibility that the cholinergic neurons could be the synaptic targets of these incoming fibers, the subiculum, the fornix, and the midline/intralaminar thalamus were lesioned in separate animals and brain sections were immunoprocessed for choline acetyltransferase and studied with the electron microscope. In addition, dual-labelling electron microscopic immunocytochemistry was employed. In total, 164 synaptic terminals from the subiculum/hippocampus and 130 from the midline/intralaminar thalamus were examined; all formed asymmetrical synaptic specializations. No hippocampal endings were seen to contact the somata or primary dendrites of the choline acetyltransferase-immunoreactive neurons; however, three were found in synaptic contact with distal, immunolabelled dendritic shafts. Most hippocampal terminals established contacts with unlabelled spines. Fifteen percent of the thalamic endings were found to synapse on the somata and the primary and distal dendrites of the choline acetyltransferase-immunoreactive neurons. The remaining thalamic terminals established synaptic junctions with small unlabelled dendrites or spines. These findings have important implications not only for our understanding of the synaptic organization of the hippocampal and thalamic projections to the nucleus accumbens, but also for the contribution of the cholinergic neurons to the circuitry of this nucleus.

Animals↗
PubMed1990

Hippocampal fibers make synaptic contacts with glutamate decarboxylase-immunoreactive neurons in the rat nucleus accumbens.

Axons arising in the hippocampal formation form asymmetric synapses with glutamate decarboxylase-immunoreactive neurons in nucleus accumbens. The postsynaptic targets included many spines, and less frequently dendritic shafts and perikarya. Some immunolabelled neurons resemble medium-sized, spiny projection neurons which suggests that the input from the hippocampal formation exerts a strong and direct influence on output pathways of nucleus accumbens.

Animals↗
PubMed1990

Efferent projections from the lateral septal nucleus to the anterior hypothalamus in the rat: a study combining Phaseolus vulgaris-leucoagglutinin tracing with vasopressin immunocytochemistry.

The tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into the lateral septum of the rat at different rostrocaudal locations to study the efferent septal projections to the anterior hypothalamus. For spatial correlation of these septofugal elements with the vasopressinergic system a dual immunocytochemical technique was used (i) to demonstrate nerve fibers and their corresponding bouton-like structures labeled with the tracer, and (ii) to identify vasopressin in the same section. The hypothalamic paraventricular and supraoptic nuclei, the accessory hypothalamic magnocellular system, and the suprachiasmatic nucleus are recipients of PHA-L-labeled fibers from all parts of the lateral septum. Close appositions between (i) these axons and their varicosities, and (ii) vasopressin-immunoreactive perikarya and their processes, putatively indicating functional interrelationships, were observed in all these nuclear areas, especially in their neuropil formations.

Animals↗
PubMed1990

Combination of anterograde tracing with Phaseolus vulgaris-leucoagglutinin, retrograde fluorescent tracing and fixed-slice intracellular injection of lucifer yellow.

A method is described for visualizing, in one histological section, three successive components of a putative neuronal network in the central nervous system: (i) fibres from neurones in an area A that innervate (ii) dendrites of morphologically identified neurones in an area B projecting (iii) to a specific target area C. The tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) is injected in area A in order to anterogradely label fibres and terminal varicosities in area B. In the same surgical session, the fluorescent tracer Fast blue is injected in area C to retrogradely label neuronal perikarya in area B. After the fixation of the brain and the preparation of 200-microns thick slices, cell bodies in area B containing retrogradely transported fluorescent label are intracellularly injected with the fluorescent dye Lucifer yellow. This reveals the full morphology of these cells. Hereafter, the slices are resectioned at 30 microns and subjected to dual immunocytochemistry with different chromogens in order to visualize the transported PHA-L (chromogen nickel-enhanced diaminobenzidine, blue-black reaction product) and to stabilize the Lucifer yellow (chromogen diaminobenzidine, brown reaction product). As a result, the PHA-L labelled fibres are stained blue-black, whereas the perikarya, dendritic trees and part of the axonal configurations of the retrogradely labelled, Lucifer yellow injected cells are stained brown. The relationships between the PHA-L labelled afferent fibres and the dendrites of the intracellularly Lucifer yellow-injected, retrogradely Fast blue-labelled cells can now be studied in detail.

Amidines↗
PubMed1990

Immunocytochemistry on free-floating sections of rat brain using microwave irradiation during the incubation in the primary antiserum: light and electron microscopy.

We studied the effects of microwave irradiation during the incubation of free-floating brain sections with primary antibodies against gamma-aminobutyric acid (GABA), enkephalin and vasopressin. Vibratome sections of perfusion-fixed rat brain were incubated: (a) overnight at room temperature (20-22 degrees C), (b) during various periods of time under microwave irradiation, such that the induced temperatures did not exceed 10 degrees C, (c) same as (b) but with induced temperatures not exceeding 40 degrees C, (d) without microwave irradiation, at 4-10 degrees C (temperature control for (b)), (e) same as (d) but at 40 degrees C (temperature control for (c)). During the incubation-irradiation we continuously monitored the temperature and controlled it by cooling and by manipulating the energy output of the magnetron. The peroxidase immunocytochemical procedure was completed using for all sections the same incubation parameters. Selected GABA-immunoreacted sections were examined in the electron microscope. Incubation at 10 degrees C in the primary antiserum as short as 30 min, with or without microwave irradiation, already results in (weak) binding of the antibodies to immunoreactive structures. One or 2 h of incubation in the primary antiserum in the microwave oven at 40 degrees C or at the same temperature outside the microwave oven results in excellent staining of GABA-immunoreactive structures and of good staining of enkephalin- or vasopressin-immunoreactive structures. The ultrastructural details were much better preserved in incubated-irradiated sections than in sections incubated overnight and only slightly less preserved than in the other control sections. There is no improved penetration of the antibodies into the sections. We conclude that by using microwave technology or by raising the temperature of the incubation medium, the time of incubation, at least in these antisera, can be shortened drastically, whereas the ultrastructural details remain well preserved.

Animals↗