Biomedical subjects

V A Pieribone

Publications and source records attributed to V A Pieribone.

32 records · Page 2Linked to original sources

PubMed1994

The reticulospinal glutamate synapse in lamprey: plasticity and presynaptic variability.

1. The glutamatergic synapses formed between the unbranched giant reticulospinal axons onto spinal neurons in lamprey offer a central vertebrate synapse in which the presynaptic element can be impaled with one or several microelectrodes, which may be used for recording as well as microinjection of different substances. To provide a basis for the use of this synapse in studies of release mechanisms, we have examined the use-dependent modulation of the synaptic response under conditions of conventional cell body stimulation, and during direct stimulation of the presynaptic axon. 2. To examine the stability of the mixed electrotonic and chemical reticulospinal excitatory postsynaptic potential (EPSP) over time, action potentials were evoked at a rate of 1 Hz for 800-1000 trials. In three out of seven synapses the chemical component remained at a similar amplitude, while in four cases a progressive decrease (up to 35%) occurred. The electrotonic component remained at a similar amplitude in all cases. 3. During paired pulse stimulation of the reticulospinal cell body (pulse interval 65 ms) the chemical EPSP component showed a net facilitation in all cases tested [from 0.64 +/- 0.35 to 0.89 +/- 0.48 (SD) mV, n = 13], while the peak amplitude of the electrotonic component was unchanged (1.37 +/- 0.68 and 1.36 +/- 0.66 mV, respectively). Recording of the axonal action potential during paired pulse stimulation showed that the width of the first and second action potential did not differ [1/2 width (2.48 +/- 0.39 ms and 2.48 +/- 0.42 ms, respectively; n = 8)]. 4. The degree of facilitation varied markedly between different synapses, ranging from an increase of a few percent to a two-fold increase (24 +/- 16% mean change of total EPSP amplitude, corresponding to 44 +/- 26% mean change of chemical EPSP amplitude). This type of variability was also observed in synapses made from the same unbranched reticulospinal axon onto different postsynaptic cells. 5. When paired pulse stimulation was applied to the reticulospinal axon in the very vicinity of the synaptic area (0.1-1 mm) a net depression of the chemical component occurred in 11 out of 19 cases, and in the remaining cases the level of net facilitation was lower as compared with cell body stimulation (range between +17 and -23% change of total EPSP amplitude; mean -5%; n = 19). 6. To test if the change of the EPSP plasticity during local stimulation correlated with an increased transmitter release, two microelectrodes were placed in the same reticulospinal axon at different distances from the synaptic area.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Distribution of alpha 1 adrenoceptors in rat brain revealed by in situ hybridization experiments utilizing subtype-specific probes.

The distribution of neurons in the rat CNS that synthesize mRNA for the alpha 1A/D and alpha 1B adrenoceptors was revealed by the in situ hybridization method. Forty-eight-mer DNA probes were synthesized to two different and unique regions of both the alpha 1A/D and alpha 1B mRNAs. Tissue sections from all levels of the CNS and some peripheral ganglia were incubated in a hybridization cocktail containing one of these four probes. The two mRNAs were expressed in a discrete and often complementary manner to each other, and identical hybridization patterns were seen for the probes directed against the same mRNA. The alpha 1A/D probes hybridized heavily with neurons in the internal granular and internal plexiform layers of the olfactory bulb, in layers II-V of most areas of the cerebral cortex, and in the lateral aspect of the lateral amygdaloid nucleus, with pyramidal neurons of CA1-CA4 regions, hilar and granular neurons of the dentate gyrus, and neurons in the reticular thalamic nucleus, cranial and spinal motor nuclei, and the inferior olivary nucleus. Light labeling was seen in a variety of other regions in the brain and spinal cord. The alpha 1B probes hybridized heavily with neurons in the mid layers of cerebral cortex and with virtually all neurons in the thalamus, except the reticular and habenular nuclei. In addition, labeling was seen in the lateral and central amygdaloid nuclei, in brainstem and spinal motor nuclei, over most neurons of the dorsal and medullary raphe nuclei and neurons of the intermediolateral cell column in the spinal cord. Light labeling was seen in the septal nucleus, the horizontal limb of the diagonal band, the paraventricular and lateral hypothalamic nuclei, the pontine and medullary reticular formation, and in most laminae in the spinal cord. The patterns of labeling obtained with the alpha 1B probes resemble the labeling seen in previous autoradiographic ligand binding studies utilizing "general" alpha 1 ligands, while the labeling patterns seen with the alpha 1A/D probes do not correspond to any published alpha 1 receptor distribution pattern, indicating that this mRNA likely encodes for a novel adrenoceptor. The present findings further expand the heterogeneity of adrenoceptor mRNAs presented in two accompanying studies (Nicholas et al., 1993a,b). This differential distribution of adrenoceptors subtypes provides a framework for the functional diversity to the apparently widespread, diffuse, and rather homogeneous noradrenergic innervation of the CNS.

Animals↗

PubMed1993

Evolutionary conservation of synaptosome-associated protein 25 kDa (SNAP-25) shown by Drosophila and Torpedo cDNA clones.

The neuron-specific proteins SNAP-25 (synaptosome-associated protein 25 kDa), synaptobrevin and syntaxin, are localized to presynaptic terminals in mammals and have been found to associate with proteins involved in vesicle docking and membrane fusion. We describe here SNAP-25 cDNA clones from the fruit fly Drosophila melanogaster and the ray Torpedo marmorata. In situ hybridization showed that SNAP-25 mRNA is exclusively found in brain and ganglia in Drosophila with a pattern suggesting expression in most neurons. The Drosophila and Torpedo proteins show 61 and 81% amino acid identity to mouse SNAP-25, a degree of conservation similar to that previously reported for synaptobrevin. None of the SNAP-25 sequences has a membrane-spanning region, but all contain a cluster of cysteine residues that can be palmitoylated for membrane attachment. SNAP-25 displays sequence similarity to syntaxin A and B. These data show that SNAP-25 and synaptobrevin, which are both implicated in vesicle docking and/or membrane fusion, have both been highly conserved during evolution. This supports the existence of a basic molecular machinery for synaptic vesicle docking in vertebrate and invertebrate synapses.

Amino Acid Sequence↗

PubMed1993

CGRP-like immunoreactivity in A11 dopamine neurons projecting to the spinal cord and a note on CGRP-CCK cross-reactivity.

Using the indirect immunofluorescence technique and double labelling procedures combined with retrograde tracing it could be demonstrated that the A11 dopamine cell group, located at the border between the diencephalon and mesencephalon of the rat brain and some of which project to the spinal cord, contains calcitonin gene-related peptide (CGRP)-like immunoreactivity. Thus, another catecholamine group in the rat brain has been shown to have a coexisting peptide. One of the CGRP antisera used in the present study also stained cholecystokinin (CCK) containing neurons in various brain areas. Absorption and displacement experiments using immunohistochemistry and radioimmunoassay showed that this cross-reactivity was confined to the C-terminal portion of the peptide molecule. Therefore, the present results suggest that CGRP antisera used for immunohistochemistry and radioimmunoassay should be tested for possible cross-reactivity with CCK.

Animals↗

PubMed1993

Cellular localization of messenger RNA for beta-1 and beta-2 adrenergic receptors in rat brain: an in situ hybridization study.

Selective, 35S-labeled, oligonucleotide probes were designed from sequences of the rat beta-1 and beta-2 adrenoceptor messenger RNAs for use in situ hybridization experiments on sections of unfixed rat brain and spinal cord. After hybridized sections were exposed to film or dipped in autoradiographic emulsion, specific and selective labeling patterns characteristic for each receptor messenger RNA and region of the central nervous system were observed. For example, labeling for beta-1 messenger RNA was found in the anterior olfactory nucleus, cerebral cortex, lateral intermediate septal nucleus, reticular thalamic nucleus, oculomotor complex, vestibular nuclei, deep cerebellar nuclei, trapezoid nucleus, abducens nucleus, ventrolateral pontine and medullary reticular formations, the intermediate gray matter of the spinal cord and in the pineal gland, while beta-2 messenger RNA labeling was strongest in the olfactory bulb, piriform cortex, hippocampal formation, thalamic intralaminar nuclei and cerebellar cortex. In some of these regions the beta-1 labeling seemed mainly confined to the cell nucleus. Whether or not this apparently nuclear labeling is specific, i.e. indicates synthesis of beta-1 receptor, remains to be established. However, all labeling patterns described disappeared when excess unlabeled probes were added to their respective radiolabeled probes or when sense probes were employed. Since the in situ method labels only cell bodies that produce the messenger RNA for these two beta receptor subtypes, a comparison between these maps and those of past autoradiographic studies mapping the location of central beta receptors using drugs as radioligands may produce further insights regarding the pre- and postsynaptic localization of these receptors in the various parts of the central nervous system circuitry.

Adrenergic Fibers↗

PubMed1992

Serotonin-, substance P- and glutamate/aspartate-like immunoreactivities in medullo-spinal pathways of rat and primate.

Serotonergic neurons of the medulla oblongata have been proposed to play a role in the control of sensory, motor and autonomic cells in the spinal cord. Many of these raphe neurons have been shown to contain the undecapeptide substance P as well as the tripeptide thyrotropin-releasing hormone, but evidence for the presence of an excitatory amino acid in these pathways has not yet been documented. In colchicine-treated rats, we have used a combination of retrograde tracing and tri-color immunohistofluorescence techniques to study co-localization of serotonin- and substance P- with glutamate- or aspartate-like immunoreactivities in medullary neurons and the possible spinal projections of these cells. In addition, the distributions of serotonin-, substance P- and glutamate-immunoreactive terminal fields in the dorsal, ventral and lateral horns of the spinal cord were examined with tri-color immunofluorescence in the rat and the primate Macaca fasciculata. In colchicine-treated rats, glutamate- and aspartate-like immunoreactivity was found in practically all serotonin- and substance P-immunoreactive neurons of the B1, B2 and B3 cell groups. Some of these neurons also contained wheat-germ agglutinin conjugated to inactivated horseradish peroxidase and colloidal gold particles retrogradely transported from the spinal cord. In the spinal cords of non-colchicine-treated monkeys and rats, striking co-localization of serotonin, substance P- and glutamate-like immunoreactivities was seen in large boutons, surrounding the dendrites and cell bodies of large alpha motor neurons in the ventral horn. These observations suggest the existence of spinally projecting serotonin/substance P neurons containing excitatory amino acids such as glutamate or aspartate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Differential expression of mRNAs for neuropeptide Y-related peptides in rat nervous tissues: possible evolutionary conservation.

Neuropeptide Y (NPY) is the only member of its peptide family that has been isolated from the mammalian CNS. We have recently found that two different NPY-related molecules are present in the CNS of a cyclostome, the river lamprey (Lampetra fluviatilis) (Söderberg et al., 1991). Here we show that this is also true for the rat CNS, by demonstrating expression of peptide YY (PYY) mRNA in brainstem neurons distinct from those neurons that express NPY mRNA. Dissimilar oligonucleotide DNA probes complementary to 3' untranslated regions of the rat PYY, NPY, and pancreatic polypeptide (PP) mRNA were used in in situ hybridization experiments on sections of rat brain and spinal cord, visceral organs, and peripheral nerve ganglia. The PYY probe hybridized with two populations of neurons in the brainstem: one dispersed along the midline in the rostral medulla and another in the lateral caudal medulla (A1 region). No additional labeling was detected in the remainder of the neuraxis. In the periphery, PYY hybridization was seen only in endocrine cells of the colon, and not in sympathetic ganglia or the adrenal gland, suggesting that previous observations of PYY immunoreactivity in these latter structures were due to antibody cross-reactivity with NPY. The NPY probe did not hybridize with cells on the midline region that contains PYY neurons, but it did label large numbers of neurons throughout the neuraxis. No expression of PP mRNA was detected in the CNS. Northern blot analysis failed to detect PYY mRNA in the CNS, further supporting the observation that PYY is only expressed by a discrete collection of CNS neurons. The anatomy of PYY- and NPY-expressing cells in the CNS and gut shows a striking similarity between rat and lamprey (Brodin et al., 1989), vertebrates that diverged evolutionarily about 450 million years ago, suggesting that both peptide systems have been conserved throughout vertebrate evolution.

Animals↗

PubMed1991

Subregions of the periaqueductal gray topographically innervate the rostral ventral medulla in the rat.

Previous anatomical and physiological studies have revealed a substantial projection from the periaqueductal gray (PAG) to the nucleus paragigantocellularis (PGi). In addition, physiological studies have indicated that the PAG is composed of functionally distinct subregions. However, projections from PAG subregions to PGi have not been comprehensively examined. In the present study, we sought to examine possible topographic specificity for projections from subregions of the PAG to PGi. Pressure or iontophoretic injections of wheat germ agglutinin-conjugated horseradish peroxidase, or of Fluoro-Gold, placed into the PGi of the rat retrogradely labeled a substantial number of neurons in the PAG from the level of the Edinger-Westphal nucleus to the caudal midbrain. Retrogradely labeled neurons were preferentially aggregated in distinct subregions of the PAG. Rostrally, at the level of the oculomotor nucleus, labeled neurons were i) compactly aggregated in the ventromedial portion of the PAG corresponding closely to the supraoculomotor nucleus of the central gray, ii) in the lateral and ventrolateral PAG, and iii) in medial dorsal PAG. More caudally, retrogradely labeled neurons became less numerous in the dorsomedial PAG but were more widely scattered throughout the lateral and ventrolateral parts of the PAG. Only few retrogradely labeled neurons were found in the ventromedial part of the PAG at caudal levels. Injections of retrograde tracers restricted to subregions of the PGi suggested topography for afferents from the PAG. Injections into the lateral portion of the PGi yielded the greatest number of labeled neurons within the rostral ventromedial PAG. Medially placed injections yielded numerous retrogradely labeled neurons in the lateral and ventrolateral PAG. Injections placed in the rostral pole of the PGi (medial to the facial nucleus) produced the greatest number of retrogradely labeled neurons in the dorsal PAG. To examine the pathways taken by fibers projecting from PAG neurons to the medulla, and to further specify the topography for the terminations of these afferents in the PGi, the anterograde tracer Phaseolus vulgaris-leucoagglutinin was iontophoretically deposited into subregions of the PAG that contained retrogradely labeled neurons in the above experiments. These results revealed distinct fiber pathways to the rostral medulla that arise from the dorsal, lateral/ventrolateral, and ventromedial parts of the PAG. These injections also showed that there are differential but overlapping innervation patterns within the PGi. Consistent with the retrograde tracing results, injections into the rostral ventromedial PAG near the supraoculomotor nucleus yielded anterograde labeling immediately ventral to the nucleus ambiguus in the ventrolateral medulla, within the retrofacial portion of the PGi.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗

PubMed1991

Adrenergic innervation of the rat nucleus locus coeruleus arises predominantly from the C1 adrenergic cell group in the rostral medulla.

Focal iontophoretic injections of the retrograde tracer Fluoro-Gold into the locus coeruleus were combined with immunocytochemistry for phenylethanolamine N-methyltransferase, the final enzyme in the synthesis of epinephrine. Retrograde labeling confirmed recent findings that the major afferents to the locus coeruleus are present in the ventrolateral (nucleus paragigantocellularis) and dorsomedial medulla (nucleus prepositus hypoglossi), areas containing the C1 and C3 adrenergic cell groups, respectively. The Fluoro-Gold label revealed morphologic details of locus coeruleus afferent cells. Labeled neurons in the prepositus hypoglossi region were typically round (10 microns diameter) or ellipsoidal and compressed against the ventricle wall (10 x 20 microns), while those in the paragigantocellularis were most often multipolar and ellipsoidal or triangular in shape (10 x 20-20 x 30 microns). Double labeling in the same tissue sections revealed that locus coeruleus afferent neurons are intercalated among phenylethanolamine N-methyltransferase-positive C1 and C3 neurons. Twenty-one per cent of locus coeruleus afferent neurons in paragigantocellularis stained for phenylethanolamine N-methyltransferase while only 4% of locus coeruleus afferents in the prepositus hypoglossi area exhibited phenylethanolamine N-methyltransferase immunoreactivity. In paragigantocellularis, doubly labeled neurons were usually the smaller locus coeruleus afferents, while in the prepositus hypoglossi phenylethanolamine N-methyltransferase labeling was found in all cell types that project to the locus coeruleus. Phenylethanolamine N-methyltransferase-positive fibers from the C1 and C3 cell groups form an adrenergic fiber bundle in the dorsomedial medulla; in the pons, these fibers appear to exit this bundle and innervate the locus coeruleus. Fibers from the neurons of the C3 cell group also appear to ascend on the dorsal surface of the medulla to innervate the locus coeruleus. The phenylethanolamine N-methyltransferase fiber innervation in the locus coeruleus was dense and highly varicose. Phenylethanolamine N-methyltransferase innervation in the dorsal pons was not restricted to the locus coeruleus but was also prominent in neighboring areas such as Barrington's nucleus and the lateral dorsal tegmental nucleus of Gudden.

Afferent Pathways↗

PubMed1990

Anatomical evidence for multiple pathways leading from the rostral ventrolateral medulla (nucleus paragigantocellularis) to the locus coeruleus in rat.

Using retrograde transport of Fluoro-Gold (FG) combined with immunofluorescence for phenylethanolamine-N-methyltransferase (PNMT), we have examined afferents to the nucleus locus coeruleus (LC) from the rostral ventrolateral medulla (nucleus paragigantocellularis; PGi) in rats sustaining lesions of the medullary adrenergic bundle (MB). In lesioned rats, very few adrenergic LC-projecting neurons persist in the PGi ipsilateral to the lesion, representing a 90% decrease in comparison to non-lesioned animals. These results indicate that almost all adrenergic input to the LC from C1 neurons in PGi is conveyed by the MB. In contrast, the number of non-adrenergic LC afferent neurons in the PGi ipsilateral to the lesion only decreased by 48% after such lesions. Thus, this pathway also provides non-adrenergic projections to LC from PGi, but many of these are conveyed by other route(s) as well.

Animals↗

PubMed1989

Diverse afferents converge on the nucleus paragigantocellularis in the rat ventrolateral medulla: retrograde and anterograde tracing studies.

The nucleus paragigantocellularis in the ventrolateral medulla has been implicated in cardiovascular, pain, and analgesic functions; and it has also been found to be a major afferent to the pontine nucleus locus coeruleus. In the present study, afferents to the nucleus paragigantocellularis were identified in the rat by means of the retrograde tracers wheat germ agglutinin-conjugated horseradish peroxidase or Fluoro-Gold. Projections to the nucleus paragigantocellularis arise from a wide variety of nuclei with autonomic, visceral, and sensory-related functions. Major afferents with consistent and robust retrograde labeling include most laminae of the spinal cord, the caudal lateral medulla, the contralateral paragigantocellularis, the nucleus of the solitary tract, the A1 area, the lateral parabrachialis, the Kölliker-Fuse nucleus, the periaqueductal gray, and a preoculomotor nucleus in the ventral central gray, the supraoculomotor nucleus. Other notable afferents, seen only after large caudal injections into the nucleus paragigantocellularis, include the lateral hypothalamus, the paraventricular nucleus of the hypothalamus, and the medial prefrontal cortex. Minor afferents include the gigantocellular nucleus, the area postrema, the caudal raphe groups, the inferior colliculus, the A5 area, and the locus coeruleus. The projection from the supraoculomotor nucleus, not previously reported as an afferent to the ventrolateral medulla, was confirmed with anterograde tracing by means of Phaseolus vulgaris-leucoagglutinin. Iontophoretic deposits of Phaseolus vulgaris-leucoagglutinin into the nucleus of the solitary tract (commissuralis level) or into the periaqueductal gray also yielded terminal fiber labeling in the nucleus paragigantocellularis. Fibers from the supraoculomotor nucleus and the nucleus of the solitary tract were densest in the lateral aspect of the nucleus paragigantocellularis (corresponding to the rostroventrolateral reticular nucleus), while fibers from the periaqueductal gray were more medially located. Previous studies have defined inputs to the rostral ventrolateral medulla from the cochlear nucleus as well as from the colliculi. In the present study, deposits of wheat germ agglutinin-conjugated horseradish peroxidase or Phaseolus vulgaris-leucoagglutinin into the cochlear nucleus or the superior colliculus yielded only sparse anterograde labeling in the nucleus paragigantocellularis, but heavily labeled adjacent areas. The inferior collicular injections yielded strong but restricted anterograde labeling in the rostromedial paragigantocellularis, medial to the facial nucleus. These results indicate that the paragigantocellularis area receives inputs from diverse brain structures. Neurons in the nucleus paragigantocellularis afferent to the locus coeruleus, being distributed throughout this region, may provide a channel where several types of information are integrated and transmitted to the extensive locus coeruleus noradrenergic efferent network...

Afferent Pathways↗

PubMed1988

The iontophoretic application of Fluoro-Gold for the study of afferents to deep brain nuclei.

A method is described for identifying the afferents to confined areas within the central nervous system using iontophoretic application of the fluorescent tracer, Fluoro-Gold (FG). Unlike other fluorescent tracers, it is possible to make focal iontophoretic injections through small-tipped micropipettes, and electrophysiological recordings from the injection pipette can be used to define structures prior to injections. Retrograde labeling with FG appears to be as sensitive as wheatgerm agglutinin-conjugated horseradish peroxidase visualized with tetramethylbenzidine. Furthermore, iontophoretically applied FG does not appear to be taken up and transported retrogradely by fibers of passage. Finally, retrograde transport of FG can be combined with immunofluorescence without appreciable loss of sensitivity in either label.

Animals↗

PubMed1988

Adrenergic and non-adrenergic neurons in the C1 and C3 areas project to locus coeruleus: a fluorescent double labeling study.

Following iontophoretic injections of the retrograde tracer Fluoro-gold into the rat locus coeruleus (LC), retrogradely labeled neurons were seen predominantly in the area of C1 adrenergic neurons in the ventrolateral medulla (nucleus paragigantocellularis; PGi) and in the area of C3 adrenergic neurons in the dorsomedial medulla (nucleus prepositus hypoglossi; PrH). Subsequent immunofluorescence for phenylethanolamine-N-methyltransferase indicated that adrenergic and non-adrenergic LC projecting neurons in both areas are interdigitated, and that 21% of LC afferent neurons in the PGi are adrenergic while only 4% of LC afferent neurons in the area of PrH are adrenergic.

Adrenergic Fibers↗

PubMed1986

The brain nucleus locus coeruleus: restricted afferent control of a broad efferent network.

Dense, focal injections of wheat germ agglutinin conjugated-horseradish peroxidase in the locus coeruleus of rats labeled afferent neurons in unexpectedly few brain regions. Major inputs emanate from only two nuclei--the paragigantocellularis and the prepositus hypoglossi, both in the rostral medulla. The dorsal cap of the paraventricular hypothalamic nucleus and the spinal intermediate gray are possible minor afferents to locus coeruleus. Other areas reported to project to locus coeruleus (for example, amygdala, nucleus tractus solitarius, and spinal dorsal horn) did not exhibit consistent retrograde labeling. Anterograde tracing and electrophysiologic experiments confirmed the absence of input to locus coeruleus from these areas, which instead terminate in targets adjacent to locus coeruleus. These findings redefine the anatomic organization of the locus coeruleus, and have implications for hypotheses concerning the functions of this noradrenergic brain nucleus.

Afferent Pathways↗