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PubMed1981

Anatomical evidence for the influence of degenerating pathways on regenerating optic fibers following surgical manipulations in the visual system of the goldfish.

We have used [3H]proline radioautography to trace regenerating optic fibers in the goldfish following: (1) the removal of the right tectal lobe and the right eye, and (2) the removal of both tectal lobes. Our results indicate that following the removal of the right tectal lobe and the right eye, both the denervated tectal efferent pathways, and the denervated visual pathways and terminal zones of the enucleated eye were penetrated by the regenerating optic fibers. In addition, following bilateral lobectomy, the denervated tectal efferent pathways were bilaterally penetrated by the regenerating fibers. Since, in both types of operations, these denervated pathways and terminal zones should undergo degeneration, our results support the suggestion that the presence of degenerating axonal debris and proliferating glia may play an important role in guiding regenerating optic fibers in the visual system of the goldfish.

Animals↗

PubMed1982

Peripheral nerve autografts to the rat spinal cord: studies with axonal tracing methods.

In young adult female rats, autologous sciatic nerve segments were transplanted to the thoracic region of the spinal cord. The grafts became well innervated but led to no obvious functional improvement. The origin and termination of axons in the grafts was studied by retrograde neuronal labeling with horseradish peroxidase (HRP) and radioautographic axonal tracing. Studies with HRP indicated that some axons in the grafts originated from intrinsic CNS neurons with their cell bodies in nearby segments of the spinal cord and that others arose from dorsal root ganglia at the level of the grafts and at least 7 segments distal to them. After tritiated amino acids were injected into lumbar dorsal root ganglia, labeled axons could be followed into the grafts but not into the rostral spinal cord stumps. Together with other experimental observations, these results demonstrate a correlation between success or failure of elongation of dorsal root fibers and peripheral or central ensheathment at the axonal tip. The corticospinal tract was studied both with radioautography and retrograde axonal transport of HRP but no extension of its axons into peripheral nerve grafts was detected under these experimental conditions. The findings implicate both neuroglial and axonal factors in the feeble regenerative response usually seen after injury to the spinal cord.

Afferent Pathways↗

PubMed1982

The retinofugal pathways in a primitive actinopterygian, the chondrostean Acipenser güldenstädti. An experimental study using degeneration, radioautographic and HRP methods.

Experimental study of the retinofugal pathways in Acipenser güldenstädti was carried out on 109 specimens using 3 experimental tracing methods: Fink-Heimer (after retinal ablation), radioautography (after intraocular injection of tritiated markers) and HRP (after intraocular injection of HRP or iontophoretic deposit of HRP on the optic nerve). The optic fiber was found to partially decussate at the chiasm and to project to 5 contralateral regions: (1) hypothalamus (area optica hypothalami); (2) thalamus (area optica dorsalis thalami, area optica mediale thalami, nucleus thalamicus tractus optici marginalis, nucleus laminaris ventralis); (3) pretectum (nucleus pretectalis ventralis, nucleus commissurae posterioris, nucleus intercalaris lateralis); (4) optic tectum (superficial layers); and (5) mesencephalic tegmentum (area optica accessoria). The ipsilateral component was well developed and innervated the same regions mentioned above. A few contralateral optic fibers crossed again in the posterior commissure and terminated within ipsilateral visual pretectal structures. Although the architecture of the visual centers was less elaborated in this paleopterygian than in neopterygians (holosteans, teleosteans), we observed that the general organization of the retinal contralateral projections in this fish was comparable to that of more advanced actinopterygians. Our results indicate that this pattern was probably set at a very early date, perhaps as soon as the emergence of the chondrostean grade at the beginning of the Devonian.

Animals↗

PubMed1983

Evidence for the presence of serotonergic perikarya in the fetal rat pancreas as demonstrated by the high affinity uptake of [3H] 5-HT.

Evidence has been obtained for the presence of serotonergic fibers in the adult rat pancreas; however, the location of the serotonergic cell bodies remains unknown. Fetal rat pancreata (18, 20 and 22 days) were demonstrated to possess a high-affinity uptake of serotonin. Radioautography revealed the uptake sites to be similar to those seen in the adult, indicating the presence of serotonergic fibers in early development. In addition, cell bodies, possibly representing primitive neurons, were demonstrated to be heavily labelled. This suggests a possible intra-pancreatic location for the cell bodies of the pancreatic nerves. The preservation of the specific serotonin uptake in 18-day fetal tissue after 4 days in organ culture substantiates this view.

Animals↗

PubMed1983

Synaptic interaction of vagal afferents and catecholaminergic neurons in the rat nucleus tractus solitarius.

Combined radioautography and immunocytochemistry were used to define the ultrastructure and synaptic relations between vagal sensory afferents and catecholaminergic (CA) neurons of the A2 group located within the nucleus tractus solitarius (NTS) of rat brain. The vagal afferents were radioautographically labeled by tritiated amino acids anterogradely transported from the nodose ganglion. Immunocytochemical labeling for tyrosine hydroxylase (TH) served for the identification of catecholaminergic neurons. The radiographically labeled axons seen by light microscopy were widely distributed throughout the more caudal NTS. The reduced silver grains were more densely distributed within the NTS located homolateral to the injected nodose ganglion. The radioautographically labeled processes were localized in regions containing catecholaminergic neurons as indicated by immunoreactivity for TH. Electron microscopic analysis of the medial NTS at the level of the obex demonstrated that the reduced silver grains were localized within axon terminals. The radioautographically labeled terminals were 2-3 microns in diameter, contained numerous small, clear and a few large, dense vesicles, and formed predominately axodendritic synapses. Many of the recipient dendrites contained immunoreactivity for TH. In rare instances, vagal afferents formed synaptic appositions with both TH-labeled and unlabeled axon terminals and neuronal soma. This study provides the first ultrastructural evidence that the catecholaminergic neurons within the NTS receive direct synapses from sensory neurons in the nodose ganglion.

Animals↗

PubMed1984

Quantitative determination of angiotensin II binding sites in rat brain and pituitary gland by autoradiography.

Rat brain and pituitary angiotensin II (AII) binding sites were quantitated by incubation of tissue sections with 125I-[Sar1] AII, Ultrofilm radioautography, computerized densitometry, and comparison with 125I-standards at appropriate film exposure times. The highest number of AII binding sites was found in anterior pituitary and the circumventricular organs, organon subfornicalis and organon vasculosum laminae terminalis.

Angiotensin II↗

PubMed1984

Enteric receptors for 5-hydroxytryptamine.

[3H]5-hydroxytryptamine [( 3H]5-HT) was used as a radioligand to study enteric 5-HT receptors. Membranes were derived from preparations of longitudinal muscle with adherent myenteric plexus and of mucosa-submucosa dissected from guinea pig and rabbit small intestines. Filtration and radioautographic analyses were used. Specific [3H]5-HT binding was found in both preparations. Binding was saturable and dissociable with equilibrium dissociation constants (Kd) of 2.7 and 1.4 nM, respectively. A kinetic estimate of Kd (1.5 nM) was similar to that determined by saturation analysis and the Hill coefficient approximated unity. Ring-hydroxylation of indoles was found to be a requirement for antagonism of [3H]5-HT binding. On the other hand, substitutions could be made in the aliphatic side chain of tryptamines without destroying the affinity of analogues for the binding sites. The inability of antagonists to displace [3H]5-HT indicated that the binding sites were not muscarinic or nicotinic receptors, alpha- or beta-adrenoceptors, H1 or H2 histamine receptors, dopamine receptors or either the S1 or S2 types of 5-HT receptor that have been found in the brain. Frozen section dry-mount radioautography revealed the [3H]5-HT binding sites to be located in ganglia of the myenteric plexus and at the boundary between the mucosa and submucosa. The similarity between the structure-activity requirements for affinity at the [3H]5-HT binding sites and activation of neural or M receptors for 5-HT in the gut, as well as the characteristics and location of the binding sites suggests that they are enteric neural receptors for 5-HT.

Animals↗

PubMed1984

Astroglial proliferation and phenotype are modulated by neuronal plasma membrane.

The rate of proliferation of rat astroglia cultured in a serum-free medium, estimated by tritiated thymidine radioautography, was diminished by more than 50% by addition of rat central nervous system axolemmal fragments to the culture medium. Addition of the axolemmal fragments also induced a phenotypic alteration of the cultured astroglia, from cells of irregular shape containing a fine meshwork of intracytoplasmic glial fibrils to star-shaped cells with thicker, cable-like glial fibrils.

Animals↗

PubMed1985

The organization of the hypothalamic pathways mediating affective defense behavior in the cat.

The purpose of this study was to describe the hypothalamic pathways which mediate affective defense in the cat utilizing the methods of [14C]2-deoxyglucose (2-DG) and [3H]leucine radioautography in concert with the technique of electrical brain stimulation. The feline affective defense response, characterized by pupillary dilatation, piloerection, ear retraction, hissing, growling and striking with the forepaws, was elicited consistently by stimulation of sites within the ventromedial hypothalamus and anterior aspect of the medial hypothalamus. In one series of experiments, 2-DG autoradiography was employed to describe the brain regions activated following stimulation of sites in the region of the ventromedial hypothalamus from which affective defense had been elicited. Ventromedial hypothalamic stimulation produced activation primarily in forebrain regions situated rostral to the level of the stimulating electrode. These structures included principally the anteromedial hypothalamus and medial preoptic area, as well as the bed nuclei of the stria terminalis and anterior commissure, diagonal band and lateral septal area. The caudal extent of activation included only the dorsal and perifornical hypothalamus at the level of the stimulation site. In a second series of experiments, affective defense sites in the anteromedial hypothalamus were stimulated and the regional distribution of 2-DG label was identified. In contrast to the results obtained from ventromedial hypothalamic stimulation, these experiments revealed a marked descending distribution of label within the posterior hypothalamus, midbrain central gray and ventral tegmental area. Results obtained from studies in which tritiated amino acids were injected into affective defense sites in both the ventromedial nucleus and anteromedial hypothalamus confirmed the general findings observed with 2-DG autoradiography. From these observations, we have concluded that the organization of the pathway mediating affective defense behavior from the ventromedial hypothalamus to the midbrain involves an initial synapse within the region of the anteromedial hypothalamus and a second synapse in the midbrain central gray substance. The significance of the anteromedial hypothalamus for the expression of affective defense behavior was considered in the Discussion.

Aggression↗

PubMed1985

Characterization of specific binding sites labeled by [3H]LSD in coronal sections of paraformaldehyde-fixed rat brain.

Specific, high-affinity binding of [3H]lysergic acid diethylamide (LSD) in coronal sections of paraformaldehyde-fixed rat brain is described. Intracardiac perfusion of paraformaldehyde selectively altered 40% of the total binding sites normally labeled by [3H]LSD in unfixed sections. Competition by unlabeled LSD and serotonin (5-HT) was not altered by the fixation procedure. Competition by spiperone, however, revealed that the fixation procedure preferentially altered sites for which spiperone has high affinity. This technique should facilitate the combination of neuroanatomical techniques such as radioautography and immunocytochemistry.

Animals↗

PubMed1985

Localization of [3H]serotonin in neuroblastoma x glioma hybrid cells.

Synthesis, localization and release of serotonin (5-HT) were studied in cholinergic neuroblastoma X glioma NG108-15 cells. The content of 5-HT and tryptophan hydroxylase activity rose substantially when hybrid cells were differentiated by prostaglandin E1 plus theophylline, or dibutyryl cAMP. Localization of [3H]5-HT taken up into differentiated NG108-15 cells was examined by electron microscopic radioautography. Silver grains were observed mostly in neurites, indicating that neurites of differentiated NG108-15 cells are the preferential uptake site of [3H]5-HT. Statistical analysis of the results of electron microscopic radioautographs revealed that silver grains had a high affinity for dense core vesicles of 60-170 nm diameter, though grains were also located over endoplasmic reticulum, mitochondria and cytosol. Dense core vesicles were abundant in neurites, and less numerous in cell bodies of the hybrid cells. [3H]5-HT taken up into NG108-15 cells was released by potassium stimulation in the presence of Ca2+. The results indicate that NG108-15 hybrid cells manifest many properties comparable to those of serotonergic neurons.

Animals↗

PubMed1986

GABA and serotonin (5-HT) pattern in the supraependymal fibers of the rat epithalamus: combined radioautographic and immunocytochemical studies. Effect of 5-HT content on [3H]GABA accumulation.

Numerous studies have suggested the serotoninergic nature of the supraependymal plexuses; moreover, several supraependymal fibers are also able to take up [3H]GABA and could be GABA-containing fibers. In this approach, by combined immunocytochemistry and radioautography, we analyzed and compared the distribution of endogenous and exogenous GABA and 5-HT in the supraependymal layer, after inhibition of their respective catabolisms. The majority of the supraependymal fibers are reactive to GABA and 5-HT antisera which indicates that they could contain both GABA and 5-HT. Furthermore it is possible to show that endogenous 5-HT containing fibers are able to accumulate [3H]GABA and conversely. These data point to a functional role for both neurotransmitters in these nerve elements. On the other hand, GABA and 5-HT contents may be connected since p-chlorophenylalanine treatment which inhibits 5-HT synthesis increased [3H]GABA labelling of these plexuses. Finally, several supraependymal fibers are also able to take up [3H]glutamate (but not [3H]glutamine); this compound might be accumulated as GABA precursor and/or as neurotransmitter.

Animals↗

PubMed1987

Serotonin innervation in adult rat neostriatum. I. Quantified regional distribution.

The distributional features of the serotonin (5-HT) innervation in adult rat neostriatum were examined and quantified using two complementary chemoanatomical methods: 5-HT-immunohistochemistry on serial histological sections and radioautography after [3H]5-HT uptake in whole cerebral hemisphere slices. As visualized and measured after peroxidase-antiperoxidase immunostaining, the neostriatal 5-HT fiber network pervading the entire neostriatum was 2-3 times denser in its ventral than dorsal parts, and showed a slight rostrocaudal increase in density. Its axonal length ranged from 1.06 to 4.18 m per mm3 of striatal tissue. Radioautographic counts of the [3H]5-HT-labeled axon varicosities within comparable sectors of the neostriatum showed good correlation with this distribution pattern. As extrapolated after appropriate corrections for incomplete detection at the chosen exposure time and from the thickness of sections examined, the number of neostriatal 5-HT varicosities (innervation density) ranged from 1.5 to 4.8 millions and averaged 2.6 millions per mm3 of tissue. These quantitative results provided new insights into the topographical organization of the dorsal raphe-neostriatal 5-HT projection system. They already allow for meaningful correlations with currently available microchemical data on intrastriatal 5-HT levels and should also be of considerable significance when as precise information becomes available on the number and localization of different 5-HT receptors and uptake carriers within rat neostriatum.

Animals↗

PubMed1987

Opioid receptors in rat neostriatum: radioautographic distribution at the electron microscopic level.

The distribution of mu-opioid receptors, selectively labeled in vitro with a monoiodinated Met-enkephalin analog [( 125I]FK 33-824), was analyzed by light and electron microscopic radioautography in sections from the neostriatum of the rat. In the light microscope, patches of high receptor densities were detected amidst a moderately labeled matrix. The number of silver grains, as counted in 1-micron thick plastic-embedded sections, was 3 times greater inside the patches than in the intervening matrix. In both compartments, the proportion of labeled binding sites associated with the neuropil was significantly higher (greater than 70%) than that associated with nerve cell bodies or myelinated fascicles. Quantitative analyses of electron microscopic radioautographs revealed that the majority of silver grains corresponding to specifically bound [125I]FK molecules originated from radioactive sources associated with apposed neuronal membranes. Of the total number of specific binding sites, 53% was associated with axodendritic, 18% with axoaxonic and 3% with axosomatic interfaces. The occurrence of multiple labeled foci along the plasma membrane of certain perikarya and dendrites suggested that some of the binding sites might be associated with somato/dendritic elements. The high incidence of labeling along axoaxonic interfaces indicated that others were linked to the membrane of axons and/or axon terminals. A major finding of the present study was that only a small proportion of specific FK binding sites (7% of total) was associated with synaptic junctions. Labeled synapses were primarily of the asymmetric type and were found predominantly on dendritic branches and spines. A few were observed on nerve cell bodies. Labeled symmetric synapses were rare and encountered exclusively on dendritic branches. The high frequency with which specifically labeled binding sites were found to be associated with neuronal interfaces involving axonal processes strongly suggests that even if non-junctional these binding sites correspond to functional receptors. Whether these receptors are activated by endogenous ligand molecules released by the labeled terminals themselves or from terminals located at a distance from the labeled interfaces remains to be determined.

Animals↗

PubMed1987

Serotonin axon terminals in the ventral tegmental area of the rat: fine structure and synaptic input to dopaminergic neurons.

The serotoninergic (5-hydroxytryptamine, 5-HT) innervation of the rat ventral tegmental area (VTA) was examined by light and electron microscopic radioautography following intraventricular infusion of [3H]5-HT. The [3H]5-HT labeled processes were characterized with respect to their regional distribution, ultrastructure and relationships with all neurons, including dopaminergic neurons, identified in the same sections using immunocytochemistry for the localization of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH). By light microscopy, [3H]5-HT labeled axons and axonal varicosities were detected throughout the interfascicular nucleus and ventral portion of the VTA. By electron microscopy, [3H]5-HT-labeled axons were found to be mainly small and unmyelinated, although a few showed several lamellae of myelin. The labeled varicosities measured 0.6 micron in mean diameter and contained many small, round or flattened agranular vesicles and a few large granular vesicles. More than 18% showed synaptic specializations in single thin sections. Most of these synapses were asymmetric and established on dendritic shafts. Based on the probability of seeing such synaptic specializations in single thin sections, it was estimated that as many as 50% of the labeled 5-HT terminals formed synaptic contacts in the VTA. In dually labeled light microscopic sections, [3H]5-HT-accumulating processes often appeared adjacent to TH-immunoreactive perikarya and proximal dendrites. Electron microscopy demonstrated that terminals with radioautographic labeling for 5-HT formed conventional synapses both with TH-labeled and unlabeled dendrites in the VTA. Many additional 5-HT terminals lacking recognizable synaptic densities were directly apposed to TH-labeled dendrites and were isolated from the rest of the neuropil by thin glial leaflets. These results suggest that 5-HT neurons innervate both dopaminergic and non-dopaminergic neurons in the VTA and may influence mesocortical and mesolimbic efferent systems through synaptic as well as non-synaptic mechanisms.

Animals↗

PubMed1988

Effects of colchicine on the intraneuronal transport of secretory material prior to the axon: a morphofunctional study in hypothalamic neurosecretory neurons of the rat.

The effects of colchicine on neurosecretory neurons of the rat hypothalamus were studied by immunocytochemistry, high-resolution radioautography, and conventional electron microscopy. In control rats, intraneuronal immunocytochemical labeling of vasopressin, oxytocin and somatostatin occurred essentially in the Golgi apparatus, the neurosecretory granules and to a lesser extent, the endoplasmic reticulum. These immunostaining patterns were dramatically modified 24 h after the administration of colchicine: immunoreactive peptides were located in granular or tubular structures accumulated at the periphery of the perikarya, but the Golgi stacks were not immunostained. Two h after the administration of tritiated leucine, quantitative analysis of radioautographic labeling of supraoptic perikarya revealed large amounts of radioactive protein in the Golgi saccules of neurosecretory neurons in control rats, but in the neurons of colchicine-treated rats, radioautographic labeling was mainly located in granular structures accumulated at the periphery of the perikarya, with no significant labeling on the Golgi stacks. Lastly, 3 noteworthy effects of colchicine on the ultrastructural morphological features of these neurosecretory neurons consisted in: (1) a dramatic disorganization of the Golgi complexes, (2) an accumulation of electron-dense proteic material within the lumen of cisternae of both the rough and smooth endoplasmic reticulum and, (3) a marked depolymerization of perikaryal microtubules, specifically those associated with the Golgi stacks. Taken together, these data do not fit the prevailing concept that the colchicine-induced accumulation of secretory material within the perikarya of neurosecretory neurons essentially results from the blockade of axoplasmic transport mechanisms. Instead, they support the idea that the effects of colchicine are related to the inhibition of the intraneuronal transport of newly synthesized secretory material from the endoplasmic reticulum to the Golgi apparatus, suggesting that the microtubules associated with the Golgi stacks are possible sites of colchicine action.

Animals↗

PubMed1989

Serotonin innervation in adult rat neostriatum. II. Ultrastructural features: a radioautographic and immunocytochemical study.

High-resolution radioautography after cerebroventricular administration of tritiated serotonin (5-HT) and PAP immunocytochemistry with an antiserum against 5-HT-glutaraldehyde conjugate (kindly donated by M. Geffard) were used in parallel to investigate the intrinsic and relational fine structural features of 5-HT axon varicosities (terminals) in the neostriatum of the adult rat. The uptake-labeled varicosities were examined in single thin sections from a paraventricular sector of neostriatum, whereas their immunostained counterparts were viewed in serial thin sections from the same paraventricular sector plus a dorsal neostriatal sector. The two approaches yielded complementary results in terms of varicosity dimensions, synaptic features and appositional relationships. Serotonin axon terminals were generally small and, as measured in immunostained material, even smaller in the dorsal than in the paraventricular neostriatum. Their internal features, best viewed in radioautographs, included small pleomorphic synaptic vesicles with occasional large granular vesicles and mitochondria. Junctional 5-HT terminals from both the paraventricular and the dorsal neostriatal sectors synapsed exclusively, and with equal frequency, on dendritic spines or shafts, almost always with asymmetrical membrane differentiations. The proportion of junctional varicosities, however, was very low in serial (immunocytochemical) as well as single (radioautographic) thin sections. Only 10-13% of 5-HT varicosities from either the paraventricular or the dorsal neostriatum exhibited a synaptic junction, in contrast with a junctional incidence of at least 70% for randomly selected axonal varicosities similarly sampled in the surrounding neuropil. Serotonin axon terminals, whether or not synaptic, were closely apposed to a variety of structures comprising mostly other axon terminals, dendritic spines and branches, but rarely neuronal somata. The synaptic and appositional features of immunostained 5-HT varicosities were similar for both the dorsal and the paraventricular neostriatum. In this context, it is likely that the effects of 5-HT in the neostriatum are exerted upon a multiplicity of cellular target sites in addition to the restricted number of dendritic spines and shafts synaptically contacted by this type of monoamine terminal.

Animals↗

PubMed1989

Is the supply of axoplasmic proteins a burden for the cell body? Morphometry of sensory neurons and amino acid incorporation into their cell bodies.

Since the perikaryon is considered to be the source of all axoplasmic proteins, we estimated the amount of protein synthesized in cell bodies and axoplasmic volumes of sensory neurons of two anuran species (Xenopus and Caudiverbera) to detect a correlation between these variables. The range of cell body volumes was 1:28 and 1:38 in Xenopus and Caudiverbera, respectively, while that of axoplasmic volumes was 1:5000-6000. The protein synthesis in glial and neuronal cell bodies was assessed with pulses of labeled amino acids followed by radioautography. No obvious correlation was found between axoplasmic volume and either rate or amount of amino acid incorporated into cell bodies. The rate of amino acid incorporation into glial and neuronal cell bodies was of the same order of magnitude. Results suggest that the maintenance of the axoplasm does not seem to be a burden for the perikaryon.

Amino Acids↗