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A Rustioni

Publications and source records attributed to A Rustioni.

At least 37 records · Page 2Linked to original sources
PubMed1995

Central release of tracer after noxious stimulation of the skin suggests non-synaptic signaling by unmyelinated fibers.

Injury to a peripheral nerve causes central changes of various nature and complexity reflecting activation of multiple signaling mechanisms. In a previous study we reported that nerve lesion triggers central release of a tracer, wheatgerm-agglutinin conjugated to horse-radish peroxidase, by unmyelinated fibers in the spinal cord. The released tracer occupies the space between nerve terminals and dendrites without extending into the synaptic cleft. We interpreted this to suggest release of unidentified endogenous factor(s) at nonsynaptic sites, which may contribute to the signaling of peripheral injury to the central nervous system. For such signaling to occur, a message must first be communicated along the axon. This message may depend on axonal transport and/or altered electrical activity. In pilot experiments we observed that application of tetrodotoxin (to block impulse conduction) to the intact nerve did not result in tracer release. We hypothesized that the message might be the sustained discharge of C fibers that occurs after injury. We show here that selective activation of C fibers (by applying mustard oil to the hindlimb of anesthetized rats) causes central release of tracer previously transported from the sciatic nerve to superficial laminae of the dorsal horn.

Animals↗
PubMed1995

An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry.

Immunocytochemistry for amino acids with post-embedding gold is compatible with glutaraldehyde fixation, osmication, and embedding in epoxy-based plastics, but immunogold detection of larger molecules in the central nervous system commonly requires special procedures, e.g. minimizing exposure to glutaraldehyde, eliminating osmium, cryosectioning, and/or embedding in acrylic plastics. These make samples more difficult to prepare and view and may compromise structural preservation. We report a new technique, fixing with high levels of glutaraldehyde, replacing osmium with tannic acid followed by other heavy metals and p-phenylenediamine, and embedding in Epon. This method optimizes antigenicity while retaining the structural preservation and convenient handling of standard embedding techniques. Compared to standard Epon embedment, labeling for neuropeptides in brain and spinal cord is improved. Moreover, the present method yields excellent labeling of glutamate receptors (difficult to identify with traditional post-embedding techniques) and enables simultaneous visualization of associated neurotransmitters.

Animals↗
PubMed1995

Synaptic interactions between primary afferent terminals and GABA and nitric oxide-synthesizing neurons in superficial laminae of the rat spinal cord.

The superficial laminae (I and II) of the spinal dorsal horn receive small caliber primary afferent fibers responsive to noxious stimulation, and contain local circuit neurons that modulate afferent input. Many of these neurons are GABAergic; about a third of these also synthesize nitric oxide. We identified three main morphological types of primary afferent terminals in superficial laminae after injections of a tracer selective for small caliber afferents into the sciatic nerve of rats. The relative densities of the three types varied through the dorsoventral extent of laminae I and II. Synaptic contacts of each type with GABA- and nitric oxide synthase (NOS)-containing dendrites and axon terminals were determined by preembedding and postembedding immunocytochemistry. Nonglomerular primary afferent terminals, likely to originate from peptidergic unmyelinated fibers, were not seen in synaptic contact with either GABA- or NOS-containing neurons. Primary afferent terminals at the center of type 1 glomeruli (C1) and at the center of type 2 glomeruli (C2) are likely to originate from unmyelinated and small myelinated fibers, respectively. GABAergic terminals contacted more C2 than C1 terminals, suggesting more effective presynaptic inhibition of C2 terminals. Many GABAergic terminals were also positive for NOS, but all GABAergic terminals presynaptic to primary afferent terminals were negative for NOS. Only C2 terminals established frequent synapses with NOS-positive dendrites. (ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1994

Amino acid immunocytochemistry of primary afferent terminals in the rat dorsal horn.

We combined transganglionic tracing methods with postembedding electron microscopic immunocytochemistry to determine whether identified primary afferent fibers terminating in spinal laminae I-IV may use glutamate and aspartate as neurotransmitters. Sciatic injections of wheat-germ agglutinin conjugated to horseradish peroxidase labeled fine afferent fibers with terminals in laminae I-II of the lumbar spinal cord, whereas injections of the B subunit of cholera toxin conjugated to horseradish peroxidase labeled primary afferent terminals in deeper laminae. Many labeled primary afferent terminals in superficial laminae were involved in glomerular synaptic arrangements; others established nonglomerular contacts. Most glomerular arrangements were clearly immunopositive for glutamate, compared with dendrites, astrocytes, or terminals immunopositive for gamma-aminobutyric acid (GABA). The degree of enrichment varied in labeled terminals of different morphological types. Aspartate was enriched, though to a lesser degree than glutamate, in labeled central terminals of glomeruli in superficial laminae. Labeled primary afferent terminals in laminae III-IV were immunopositive for glutamate, though at lower levels than glomerular terminals in superficial laminae. Aspartate was not enriched in these terminals compared with dendrites, glia, and GABA-positive terminals. These results support a neurotransmitter role for glutamate in primary afferents to the dorsal horn. Quantitative differences in the content of glutamate in identified primary afferent terminals may be related to functional differences. Enrichment of aspartate in terminals in superficial but not deep laminae is compatible with a role for this amino acid in sustained, NMDA-mediated phenomena characteristic of activity in fine caliber afferents.

Afferent Pathways↗
PubMed1994

Tyrosine phosphorylation in rat spinal cord after sciatic nerve transection.

Immunocytochemistry using antibodies against phosphotyrosine was employed to identify changes in tyrosine phosphorylation in the rat spinal cord consequent to sciatic nerve injury. Increased immunostaining in the spinal gray matter, dorsal columns and gracile nucleus on the side of the lesion became evident after 3 days and was more pronounced with longer survival times up to 3 weeks (the longest survival tested). This increase was most prominent in the fourth lumbar segment (the focus of termination of sciatic nerve afferents). Immunostaining was ain astroglial cells and their processes in the dorsal horn; stained microglia were also seen. Immunopositivity also increased in glial cells surrounding motoneurons at the same levels. These changes suggest that a diffusible growth factor released centrally by injured nerve fibers activates tyrosine phosphorylation in glial cells via receptor tyrosine kinases.

Animals↗
PubMed1994

Enrichment of glutamate immunoreactivity in lemniscal terminals in the ventropostero lateral thalamic nucleus of the rat: an immunogold and WGA-HRP study.

BACKGROUND: The ventropostero lateral nucleus (VPL) is a thalamic somatosensory center receiving inputs from limbs and trunk; some of this input is via terminals of the dorsal column medial lemniscal pathway. These fibers convey non-noxious somesthesic information. METHODS: In this study the neurochemical content of lemniscal afferents in VPL of rats was investigated at the electron microscopic level by combining anterograde transport of horseradish peroxidase conjugated to wheat germ agglutinin, injected in the dorsal dorsal column nuclei, with postembedding immunogold labeling for glutamate (Glu). RESULTS: Anterograde labeling in VPL was detected only in myelinated axons and in large terminals containing round synaptic vesicles, interpreted as lemniscal afferents. Quantitative evaluation of gold particle density showed enrichment of Glu immunolabeling in the identified lemniscal terminals with respect to other neuronal profiles. Observation of serial sections immunoreacted for Glu demonstrated consistency of labeling, whereas in alternate sections immunoreacted for Glu and for the inhibitory amino acid GABA these two antigens were always present in distinct types of terminals. CONCLUSIONS: These findings are in agreement with several lines of evidence, obtained with different experimental approaches, supporting the hypothesis that Glu plays a major role in conveying sensory stimuli to the thalamus from second order neurons in the dorsal column nuclei.

Animals↗
PubMed1993

Neurons in rat cerebral cortex that synthesize nitric oxide: NADPH diaphorase histochemistry, NOS immunocytochemistry, and colocalization with GABA.

Neurons that stain for NADPH diaphorase, which colocalizes with nitric oxide synthase (NOS), are scattered uniformly across neocortex, and denser in entorhinal cortex. In the primary sensorimotor cortex, 0.5-2% of neurons contain NOS. These are most numerous in layers II-III, whereas NOS-positive fibers are concentrated in layers IV and VI. Most stained neurons are aspiny bipolar cells. Some in deep layers are multipolar; very few are pyramidal-shaped. In layer IV, NOS-positive neurons and their dendrites are confined to the septa between barrels. Retrograde tracing experiments demonstrate that NOS-positive cells are local circuit neurons. Double staining demonstrates that NOS-positive neurons also contain GABA.

Amino Acid Oxidoreductases↗
PubMed1993

Amino acid immunoreactivity in corticospinal terminals.

Anterogradely labeled corticospinal axons and their terminals were identified after injections of wheat germ-agglutinin conjugated to horseradish peroxidase in the sensorimotor cortex of rats. Thin myelinated axons were labeled in the corticospinal tract. Terminal labeling was densest in the internal basilar nucleus and laminae III-IV of the dorsal horn throughout the spinal cord; electron microscopical observations were mainly from the cervical enlargement. Labeled terminals were most often small and dome-shaped with densely packed, clear round vesicles and sparse mitochondria. These terminals established asymmetric synapses with small dendrites or spines and were never involved in axoaxonic contacts. Postembedding immunocytochemistry was used to study the subcellular distribution of glutamate, aspartate, and gamma-aminobutyric acid (GABA). Corticospinal terminals appeared enriched in glutamate, but not GABA. Some corticospinal terminals appeared enriched in aspartate, though the labeling was less selective than in the case of glutamate. GABA immunolabeling was very dense in about 20% of terminals. These were most often small, rich in mitochondria, and made symmetric synapses; they were not anterogradely labeled from the cortex. Quantitative analysis on double immunolabeled material allowed a direct comparison of particle density for different antigens in the same section. Terminals with a high density of particles coding for glutamate were not enriched with GABA, and terminals immunolabeled for GABA were not enriched with glutamate. There was no significant correlation between glutamate and aspartate immunolabeling in corticospinal terminals; a subpopulation of these terminals may be enriched in aspartate. Aspartate immunolabeling was consistently higher in dendrites postsynaptic (in the plane of section) to corticospinal terminals than in other dendrites.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1993

Glutamate immunocytochemistry in the dorsal horn after injury or stimulation of the sciatic nerve of rats.

Glutamate is a major neurotransmitter of fine afferent fibers to the spinal cord. Neuropeptides are also released by the same fibers. We explored, by quantitative immunocytochemistry, the effects of two experimental manipulations of peripheral nerves on the levels of these two classes of mediators. Glutamate levels in the superficial dorsal horn of rats increased after chronic loose ligature of the sciatic nerve, a model for hyperpathic peripheral neuropathy. A similar increase was observed acutely, after stimulation of C fibers, but not A fibers, in the sciatic nerve. In contrast, immunostaining for substance P and calcitonin gene-related peptide decreased in the same region with both manipulations. The decrease in immunocytochemical levels of peptides is in agreement with previous observations and can result from activity-related depletion. We propose that the increase in glutamate levels reflects differences in the regulation and kinetics of amino acid versus peptide neuromediators.

Animals↗
PubMed1992

Nitric oxide synthase and GABA colocalize in lamina II of rat spinal cord.

A dense plexus of fibers in the substantia gelatinosa contains nitric oxide synthase (NOS). Using electron microscopic double-labeling immunocytochemistry for NOS combined with GABA or glutamate, we find that all NOS-positive terminals in this region also contain GABA but are not enriched in glutamate. In an attempt to verify that NOS-positive terminals do not originate from primary afferents, we combined NOS immunocytochemistry with anterograde tracing from the sciatic nerve. An intrinsic spinal origin for the NOS-positive plexus is suggested. The results are discussed in the light of the possible involvement of nitric oxide in hyperalgesia.

Afferent Pathways↗
PubMed1992

NADPH diaphorase in the spinal cord of rats.

To identify spinal neurons that may synthesize nitric oxide, cells and fibers histochemically stained for NADPH diaphorase (a nitric oxide synthase) were studied in the spinal cord of rats. The histochemical reaction gave an image similar to the best Golgi impregnations, staining cells down to their finest processes. Transverse, horizontal, and parasagittal 50 and 100 microns sections were used to follow dendritic and axonal arborizations of stained neurons. Major cell groups were identified in the superficial dorsal horn and around the central canal (at all spinal levels), and in the intermediolateral cell column (at thoracic and sacral levels). Scattered positive cells were also found in deeper dorsal horn, ventral horn, and white matter. In some cases, axons of cells in the dorsal horn could be traced into the white matter; many of these cells resembled neurons projecting to various supraspinal targets. Stained cells in the intermediolateral column, which sent their axons into the ventral root, were presumed to be preganglionic autonomic neurons. Dense plexes of fibers were stained in laminae I and II and in the intermediolateral column. A large number of NADPH diaphorase-positive neurons in the spinal cord appear to be involved in visceral regulation. Fibers of the intermediolateral system had a special relationship with vasculature, suggesting that nitric oxide may help to couple neural activity with regional blood flow in the spinal cord. The abundance of NADPH diaphorase-positive neurons and fibers in the superficial dorsal horn suggests that nitric oxide may also be involved in spinal sensory processing.

Amino Acid Oxidoreductases↗
PubMed1992

Dorsal root ganglion neurons projecting to the dorsal column nuclei of rats.

Dorsal root ganglion (DRG) neurons may give origin to ascending branches that terminate in the dorsal column nuclei (DCN); uncertainties still exist with regard to the proportion of these neurons in different DRGs and to the type of these neurons. The percentage and size of neurons that project to the DCN were determined in a large number of DRGs by means of the retrograde transport of colloidal gold-labeled wheat germ agglutinin conjugated to enzymatically inactive horseradish peroxidase (WGAapoHRP-AU). A total of 16,239 neurons was tallied in 80 DRGs from nine rats; 3,240 (20%) of these were retrogradely labeled by the tracer injected in the DCN. Percentages of DCN projecting neurons vary considerably at different segmental levels: they are higher in cervical (up to 63%) than in thoracic (up to 31% for T1, up to 12% for thoracic DRGs below T1) or lumbar DRGs (up to 15%). At cervical levels highest percentages were encountered in C6, C7, and C8 and lowest percentages in C2-C4. At lumbar levels highest percentages were encountered in L4 and lowest in L1 and L6. When considering the soma size of DRG neurons it appears that: 1) there are more large cells, labeled and unlabeled, at cervical (38%) than at lumbar levels (30%) and more at lumbar than at thoracic levels (23%); 2) at every level, most labeled, i.e., projecting, neurons are large; and 3) DRGs with the highest proportions of large vs. small cells contain the highest percentages of DCN projecting neurons. These results represent the first attempt at establishing the percentages and soma size of DCN projecting neurons from a large number of DRGs and at comparing the contribution to these nuclei from cervical, thoracic, and lumbar DRGs. Some of the differences in the ratio of projecting neurons at different levels may be explained on the basis of well-known anatomical features, e.g., the projections to the Clarke's column of many DRG neurons in lumbar ganglia. The contribution of virtually exclusively large DRG neurons to the DCN, suggested by indirect or incomplete evidence, is demonstrated by the present retrograde labeling and soma size measurements. The results relate to the functional component of peripheral receptors that relay their input via the dorsal columns and do not seem to support a recent suggestion that a sizeable fraction of unmyelinated primary afferents ascend in the dorsal columns to terminate in the DCN.

Animals↗
PubMed1992

Substance P innervation of the rat and cat thalamus. I. Distribution and relation to ascending spinal pathways.

An antiserum for substance P (SP) with minimal cross-reactivity for other tachykinins was employed to map the distribution of SP-positive nerve fibers and terminals in the thalamus of cats and rats with special emphasis on the innervation by these fibers of nuclei related to the somatosensory system. In both species SP innervation is predominantly along the midline, in medial and posterior thalamic regions, and sparser in sensory relays for specific modalities. Among the most densely innervated nuclei are the parafascicular, paraventricular, rhomboid, central medial and parts of mediodorsal, lateral posterior, and ventral lateral geniculate. SP innervation of somatosensory-related nuclei is also evident in central lateral nucleus, posterior complex (PO), and in ventroposterolateral (VPL) nucleus of both cats and rats. In VPL of cats SP fibers and terminals are present along its ventral and lateral border, a paralaminar area in which spinothalamic fibers have been shown to terminate and where neurons responsive to noxious stimuli have been reported. Also in rats the SP innervation of VPL is similar to that of spinothalamic tract fibers. The SP innervation of somatosensory thalamic nuclei may be supplied, at least in part, by spinothalamic afferent as suggested by the depletion of SP after anterolateral chordotomy but not after ablation of the dorsal column nuclei. The presence of SP-positive spinothalamic neurons in the spinal cord is reported in the following paper.

Amino Acid Sequence↗
PubMed1992

Substance P innervation of the rat and cat thalamus. II. Cells of origin in the spinal cord.

Evidence in the preceding paper suggests that fibers and terminals immunopositive for substance P (SP) in somatosensory thalamic nuclei are part of the spinothalamic tract (STT). In this paper, more direct evidence on this point is provided by immunocytochemistry for SP on the cervical spinal cord, alone or combined with the retrograde transport of colloidal gold-labeled wheat germ agglutinin conjugated to enzymatically inactive horseradish peroxidase (WGAapoHRP-Au). In cats and rats pretreated with colchicine and/or anterolateral chordotomy (to increase SP content in cell bodies), many small to large cell bodies are SP-immunopositive especially in laminae I and V, but also in more ventral laminae of the upper cervical cord. SP neurons are also present in the dorsolateral funiculus (in the lateral spinal nucleus, LSN, in rats) but not in the lateral cervical nucleus or in the internal basilar nucleus. In both species there is a considerable degree of overlap in the distribution of SP-positive neurons and that of STT neurons. SP immunocytochemistry in rats after WGAapoHRP-Au injection in the somatosensory thalamus reveals SP-positive STT neurons in LSN, in lamina I and in lamina V, and, to a lesser extent, in more ventral laminae. These results demonstrate that SP is a marker and/or neuromediator for some STT neurons. Together with the evidence discussed in the preceding paper, the results also suggest that SP-positive neurons may be involved in the transmission of nociceptive input.

Animals↗
PubMed1992

Peripheral injury and anterograde transport of wheat germ agglutinin-horse radish peroxidase to the spinal cord.

Previous observations have revealed labeling in the extracellular space surrounding boutons and unmyelinated fibers in superficial laminae of the spinal cord after injection of the tracer wheat germ agglutinin conjugated to horseradish peroxidase in dorsal root ganglia. The degree of extracellular labeling appeared related to the extent of the damage to the ganglia at the time of the injection. To determine whether injury might produce extracellular labeling, we investigated the effects of unilateral nerve crush or transection on spinal labeling after bilateral injections of the tracer into sciatic nerves. Confirming previous reports, labeling was confined to small dorsal root ganglion cells and to spinal laminae I and II, suggesting a selective affinity of this tracer for unmyelinated fibers. Labeling of both ganglion neurons and superficial spinal laminae was increased on the injured side, probably as a result of increased efficiency of receptor-mediated endocytosis. Electron microscopical observations revealed that the tracer was largely confined to unmyelinated dorsal root fibers bilaterally; a higher percentage of these fibers were labeled on the injured side. In the dorsal horn, the tracer was predominantly within unmyelinated axons and their terminals on the control side, whereas most of the labeling was extracellular and transneuronal on the injured side. The extracellular labeling surrounded unmyelinated fibers and their terminals in the spinal cord, but was excluded from the synaptic cleft. The demonstration that injury is accompanied by significantly increased release of this tracer from the terminals of unmyelinated fibers into the extracellular space suggests that endogenous substances may be released after peripheral lesions as a central signal of injury.

Animals↗
PubMed1992

Techniques to optimize post-embedding single and double staining for amino acid neurotransmitters.

We report a number of technical refinements for single and double staining with post-embedding electron microscopy for glutamate, aspartate, and gamma-aminobutyric acid. Best results were obtained with 2.5% glutaraldehyde in the fixative and by minimizing the duration of plastic polymerization and the interval between cutting and reacting. Quantitative documentation of the ability of exogenous glutamate, aspartate, and gamma-aminobutyric acid to block their immune staining is provided. Increased intensity of staining with the glutamate and aspartate antisera resulted from preincubation of glutamate antiserum with aspartate and aspartate antiserum with glutamate. To perform double staining with antisera raised in the same species, it was necessary to block antigenicity of the first antiserum; best results were obtained with hot paraformaldehyde fumes. By using a detergent instead of etching, these methods permitted the simultaneous visualization of tracers to identify neuroanatomic pathways.

Afferent Pathways↗
PubMed1991

Aspartate-like immunoreactivity in primary afferent neurons.

There is now good evidence that amino acids act as neurotransmitters in primary afferent neurons of dorsal root ganglia. Glutamate is the primary candidate for such a role, and there are reasons to believe that release of glutamate may be accompanied by the release of other neuroactive substances. Using immunocytochemical techniques, we have tested the hypothesis that some dorsal root ganglion neurons contain elevated levels of aspartate as well as glutamate. Antisera raised against conjugates of aspartate or glutamate were used for this purpose. Blocking experiments confirmed that these antibodies were specific to their antigens in cryostat sections of dorsal root ganglia. Aspartate immunoreactivity was found in approximately 30% of neurons in cervical dorsal root ganglia. The relation between cell size and staining intensity for aspartate was examined using quantitative video microscopy; the great majority of cells immunopositive for aspartate were small (15-30 microns in diameter); about 85% of these cells stained for aspartate, although staining intensities varied over a wide range. By reacting consecutive sections with anti-aspartate and anti-glutamate it was shown that elevated levels of aspartate were found in the same cells which contained elevated levels of glutamate. By measuring the staining intensity of individual cells for both aspartate and glutamate, it was also shown that there was a positive correlation between staining intensity and, presumably, concentration of the two amino acids. The presence of high levels of aspartate in terminals located in the superficial laminae of the dorsal horn was verified by pre- and post-embedding immunocytochemistry with the electron microscope. Aspartate was demonstrated in scalloped terminals, including dark scalloped terminals believed to be associated with unmyelinated fibers of nociceptors. This evidence supports the hypothesis that aspartate as well as glutamate is present in the cell bodies and terminals of nociceptive primary afferents, and may be released by the terminals of these afferents to activate neurons in the superficial laminae of the dorsal horn.

Afferent Pathways↗