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V M Pickel

Publications and source records attributed to V M Pickel.

At least 181 records · Page 10Linked to original sources
PubMed1986

Autoradiographic detection of [125I]-secondary antiserum: a sensitive light and electron microscopic labeling method compatible with peroxidase immunocytochemistry for dual localization of neuronal antigens.

We examined whether autoradiographic localization of [125I]-antirabbit immunoglobulin (IgG) was suitable for light and electron microscopic detection of a rabbit antiserum to the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), and whether autoradiographic and peroxidase labeling could be combined for simultaneous immunocytochemical identification of TH and neuropeptides in brain. Adult rat brains were fixed by aortic arch perfusion with acrolein and paraformaldehyde. Vibratome sections of the fixed tissues were incubated with various dilutions of TH antiserum followed by [125I]-secondary IgG. These sections were then directly processed for autoradiography or were incubated with rabbit antiserum to substance P (SP) or methionine [Met5]-enkephalin (ME). These latter sections were then processed by the peroxidase-antiperoxidase (PAP) or conjugated peroxidase methods followed by autoradiography. Exposure periods of 12-20 days for light microscopy or 90 days for electron microscopy yielded substantial accumulations of silver grains even at the highest (1:30,000) dilution of TH antiserum. At this dilution, immunoreactivity for TH was virtually nondetectable by PAP and conjugated peroxidase methods. The differential sensitivities of the autoradiographic versus peroxidase methods provided a means for separable identification of rabbit antiserum to TH and to SP or ME. Ultrastructural analysis of the catecholaminergic neurons in the medial nuclei of the solitary tract (NTS) showed selective cytoplasmic localization of silver grains for [125I]-labeling of TH in perikarya, dendrites, and terminals. Within single thin sections prepared for dual labeling, the peroxidase marker for SP and for ME was differentially localized with respect to autoradiographic labeling of TH.

Animals↗
PubMed1986

Phenylethanolamine N-methyltransferase-containing neurons in rat retina: immunohistochemistry, immunochemistry, and molecular biology.

We sought to characterize in detail neurons in rat retina that contain phenylethanolamine N-methyltransferase (PNMT), the epinephrine biosynthetic enzyme. Cell bodies and processes of PNMT-containing neurons in retina were identified by immunohistochemistry. The coexistence of other catecholamine biosynthetic enzymes in the same cells was also investigated. Biochemical, molecular biological and immunochemical methods were applied to determine whether retinal PNMT is similar to the adrenal enzyme, since regulation of PNMT in retina and adrenal appears to be different. The results show that there are two types of PNMT-containing cells: those containing PNMT exclusively and those containing PNMT with two other catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC), but not dopamine beta-hydroxylase (DBH). PNMT-only cell bodies are localized in the inner nuclear layer (INL) and the ganglion cell layer (GCL). Their processes are observed in outer and inner strata of the inner plexiform layer (IPL). Only a small fraction of PNMT neurons in INL also contain TH and AADC. These cells send their processes to the adjacent stratum of the IPL. Antibodies to bovine adrenal DBH, however, fail to localize DBH in any rat retinal cells. Immunochemical titration shows that PNMT from both retina and adrenal gland has the same immunoreactivity. Furthermore, a PNMT-cDNA probe hybridizes equally with PNMT-mRNA isolated from both the retina and the adrenal gland. These results indicate that PNMT is identical in these tissues.

Adrenal Glands↗
PubMed1986

Tyrosine hydroxylase in the rat parabrachial region: ultrastructural localization and extrinsic sources of immunoreactivity.

We sought to determine the ultrastructural localization and the extrinsic sources of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), in the lateral parabrachial region (PBR) of adult male rats. In the first portion of the study, a rabbit antiserum to TH was immunocytochemically localized in coronal sections through the lateral PBR from acrolein-fixed brains using the peroxidase-antiperoxidase method. Electron-microscopic analysis revealed that perikarya and dendrites with peroxidase immunoreactivity for TH constituted only 17% of the total labeled profiles. Afferents to the TH-labeled perikarya and dendrites usually failed to exhibit immunoreactivity and were thus considered noncatecholaminergic. Somatic synapses were most commonly detected on small immunoreactive perikarya in the central lateral nucleus of the PBR. Other labeled perikarya located in the dorsal lateral or ventral lateral nuclei received few somatic synapses and were morphologically distinct in terms of their larger size, infolded nuclear membrane, and abundance of cytoplasmic organelles. Axons and axon terminals with peroxidase immunoreactivity constituted the remaining labeled profiles in the lateral PBR. These terminals primarily formed symmetric synapses with unlabeled and a few labeled dendrites. The labeled axon terminals were categorized into 2 types: Type I was small (0.3-0.6 micron), contained many small clear vesicles, and exhibited few well-defined synaptic densities. The second type was large (0.8-1.4 micron), contained both small clear and large dense core vesicles, and exhibited well-defined synaptic densities. The 2 types of terminals were morphologically similar to dopaminergic terminals. The location of catecholaminergic neurons contributing to the TH-labeled terminals was determined by combining peroxidase-antiperoxidase immunocytochemistry for TH with retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). The tracer was unilaterally injected into the PBR of anesthetized adult rats. Immunocytochemical labeling for TH was seen as a brown reaction product within neurons in known catecholaminergic cell groups. A black granular reaction product formed by a cobalt-intensified and diaminobenzidine-stabilized tetramethyl benzidine reaction for WGA-HRP was evident within many TH-labeled and unlabeled neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Dual peroxidase and colloidal gold-labeling study of angiotensin converting enzyme and angiotensin-like immunoreactivity in the rat subfornical organ.

The cellular relationships between angiotensin converting enzyme (ACE) (EC 3.4.14.1) and angiotensin-like immunoreactivity (AGLI) were examined in the subfornical organ (SFO). Brains from adult rats were fixed by vascular perfusion with 3.75% acrolein and 2% paraformaldehyde. The region containing the SFO was then sectioned on a vibrating microtome. Partially permeabilized sections were immunocytochemically labeled using the peroxidase-antiperoxidase (PAP) or combined PAP and immunogold methods. Goat antiserum to ACE was localized to both non-neuronal and neuronal cells within the SFO. Intense peroxidase immunoreactivity for ACE was associated with the ventricular and basal surface of ependymal cells, the luminal surface of the vascular endothelium, portions of glial membranes exposed to extracellular spaces, and membranous organelles within neuronal processes. Two antisera raised in rabbits against angiotensin II showed peroxidase immunoreactivity within the extracellular spaces and throughout the cytoplasm of numerous axon terminals and a few perikarya and dendrites in the SFO. Axon terminals and dendrites also showed aggregates of AGLI in smooth membranes and vesicles near the plasmalemma. Gold labeling for AGLI was evident in only 6% of the axon terminals and in a smaller number of dendrites containing peroxidase immunoreactivity for ACE. The low incidence of terminals containing both markers appeared to at least partially reflect limited penetration of the 10 nm gold particles. These results provide the first ultrastructural evidence that ACE is associated with the plasmalemma and membranous organelles strategically located for interaction with precursors of angiotensin II or other peptides within the cerebrospinal fluid, extracellular spaces and neurons of the SFO.

Angiotensin I↗
PubMed1985

The anatomy of neuropeptide-Y-containing neurons in rat brain.

The distribution of neuropeptide Y in the central nervous system of adult male rats was investigated using indirect immunofluorescence, the peroxidase-antiperoxidase technique and by radioimmunoassay of microdissected brain regions. The different methods were in good agreement and showed that neuropeptide Y had a widespread distribution and was present in extremely high concentrations. The highest concentrations of neuropeptide Y were found in the paraventricular hypothalamic nucleus and hypothalamic arcuate nucleus, which also contained the highest density of immunoreactive fibers and numbers of perikarya, respectively. The suprachiasmatic nucleus, median eminence, dorsomedial hypothalamic nucleus and paraventricular thalamic nucleus showed high concentrations as well as high densities of fibers. Moderate concentrations were found in the bed nucleus of the stria terminalis, although a high density of fibers was found. Areas with moderate concentrations and densities of fibers were the medial preoptic area, anterior hypothalamic area, periventricular nucleus, posterior hypothalamus and the medial amygdaloid nucleus. The nucleus of the solitary tract contained a low concentration of neuropeptide Y although a high number of immunoreactive perikarya was found in colchicine-treated rats. Low concentrations were also measured in the cerebral cortex, yet relatively high numbers of cell bodies and fibers were found dispersed through the cortex. The extremely high concentrations and widespread distribution of neuropeptide Y in the central nervous system suggests a number of important physiological roles for this neurotransmitter candidate.

Animals↗
PubMed1984

Enkephalin-like immunoreactivity in rat area postrema: ultrastructural localization and coexistence with serotonin.

The ultrastructure of enkephalin-containing neurons and their capacity to take-up [3H]serotonin were examined in the area postrema. Untreated adult rats and rats with intraventricular infusions of 10(-4) M tritiated serotonin, 5-hydroxytryptamine [( 3H]5-HT) were perfused with 4% paraformaldehyde and 0.2-0.5% glutaraldehyde. Coronal Vibratome sections through the area postrema from both groups of animals were immunocytochemically labeled with an antiserum to leucine Leu5-enkephalin. The sections from the animals infused with the isotope subsequently were processed for autoradiography. Enkephalin-like immunoreactivity (ELI) was detected in perikarya, dendrites, axons and axon terminals most frequently located along the ventricular and ventrolateral portions of the area postrema. The labeled perikarya were few in number and were characterized by a thin rim of cytoplasm containing peroxidase immunoreactivity. Dendrites and terminals containing ELI formed synapses primarily with unlabeled axon terminals and dendrites, respectively. However, terminals containing ELI also formed synaptic junctions with other unlabeled axon terminals. Appositions between enkephalin-containing processes and modified glia were occasionally seen near the ventricular surface. In sections processed for both immunocytochemistry and autoradiography, approximately 5% of the terminals containing ELI showed uptake of [3H]5-HT. We conclude that neurons containing ELI are primarily, but not exclusively, associated with other intrinsic neurons or afferents in the rat area postrema and that some of the enkephalin-labeled terminals have the capacity to take-up serotonin. Specificity of uptake of [3H]5-HT in neurons containing endogenous serotonin and factors which may contribute to the low probability of detecting both peroxidase and autoradiographic markers in single sections are discussed.

Animals↗
PubMed1984

Ultrastructural localization of tyrosine hydroxylase in rat nucleus accumbens.

Immunocytochemical localization of tyrosine hydroxylase (TH) was used to determine the ultrastructural morphology and synaptic associations of catecholaminergic terminals in the nucleus accumbens of the rat. The brains were fixed by vascular perfusion with 4% paraformaldehyde and 0.2% glutaraldehyde. Coronal sections cut with a vibrating microtome were incubated with rabbit antiserum to TH then immunocytochemically labeled by the peroxidase-antiperoxidase method. Immunoreactivity for the enzyme was found within unmyelinated axons and axon terminals. These terminals contained either all small clear or combined small clear and large dense core vesicles. Approximately 40% of the labeled terminals formed symmetric synapses with unlabeled proximal or distal dendritic shafts. The dendrites showed a spare distribution of spines. Axosomatic synapses and axonal associations of the TH-containing terminals also were detected. The recipient perikarya were usually 10-20 micrometers in diameter and contained an indented nucleus and abundant cytoplasm. The content of large dense vesicles and synaptic associations with somata and proximal dendrites suggest that a certain proportion of the TH-containing terminals within the nucleus accumbens are morphologically distinct from catecholaminergic terminals within the dorsal striatum. These differences are discussed in relation to neuropeptides and functions of the dopaminergic mesolimbic and nigrostriatal pathways.

Animals↗
PubMed1984

Substance P, neurotensin, enkephalin, and catecholamine-synthesizing enzymes: light microscopic localizations compared with autoradiographic label in solitary efferents to the rat parabrachial region.

The immunocytochemical localizations of substance P, neurotensin, enkephalin and the catecholamine-synthesizing enzymes tyrosine hydroxylase and dopamine-beta-hydroxylase were examined in the rat parabrachial region. The immunoreactivity for each marker was compared with the distribution of superimposed autoradiographic labeling of parabrachial afferents after unilateral injection of 3H-amino acids into the caudal portion of the medial nucleus of the solitary tract (m-NTS). Substance-P- and neurotensinlike immunoreactivity (SPLI and NTLI, respectively) were localized primarily in varicose processes in the ventrolateral quadrant of the parabrachial region. The SPLI and NTLI were differentially localized with respect to each other; however, both peptides were detected in regions of the parabrachial containing dense autoradiographic label. In contrast, enkephalinlike immunoreactivity (ELI), tyrosine hydroxylase, and dopamine-beta-hydroxylase were detected in processes and a few perikarya located outside the ventrolateral parabrachial region. The ELI was primarily in the dorsolateral, and the catecholamine-synthesizing enzymes were primarily in the medial parabrachial regions which contained sparse autoradiographic labeling of transported amino acids. We conclude that in the rat parabrachial region, SPLI and NTLI are contained within two distinct populations of afferents which may originate from perikarya in the caudal m-NTS, whereas ELI and the catecholamines are more likely to be found in other afferents or possibly in intrinsic neurons.

Animals↗
PubMed1984

Chemical and structural analysis of the relation between cortical inputs and tyrosine hydroxylase-containing terminals in rat neostriatum.

Levels of tyrosine hydroxylase (TH) and the ultrastructural relation between axons from cerebral cortex and TH containing, predominantly dopaminergic terminals were examined in the adult rat neostriatum at 2 and 12 days following unilateral decortication. The caudate nuclei from the unlesioned and lesioned hemispheres were biochemically assayed for TH processed for light or electron microscopic localization of the enzyme. At both time intervals examined, there was no statistically significant alteration in TH activity or apparent change in the intensity of reactive labeling visualized by light microscopy. However, electron microscopic examination of the caudate nucleus homolateral to the decortication at two days following surgery revealed the presence of numerous small, osmiophilic boutons which were much less frequently seen on the contralateral side. Further ultrastructural examination showed that the osmiophilic boutons formed predominantly asymmetric, axodendritic synapses. In sections containing both degenerating and TH labeled terminals, two patterns of connectivity could be discovered. First and most commonly, the degenerating and TH-labeled terminals formed synapses with the same dendrite or dendritic spine. Less frequently, the two types of terminals were in direct contact with each other. In this axo-axonic relation, the outer membranes between the terminals were in apposition but usually failed to exhibit pre- or postsynaptic specializations. These findings indicate that the cortical and dopaminergic nigral efferents have actions on common recipient neurons in the rat caudate nucleus and provide support for a possible direct axonal interrelationship between these two primary inputs.

Animals↗
PubMed1984

Serotoninergic terminals: ultrastructure and synaptic interaction with catecholamine-containing neurons in the medial nuclei of the solitary tracts.

The ultrastructural morphology of serotoninergic terminals and their synaptic relation with catecholaminergic neurons were examined in the medial nuclei of the solitary tracts (m-NTS) using combined autoradiographic and immunocytochemical methods. Adult rats were pretreated with a monoamine oxidase inhibitor and subjected to a 2-hour intraventricular infusion of 50 nM tritiated 5-hydroxytryptamine (3H-5HT). At the termination of the infusion, the brains were fixed by aortic arch perfusion with a mixture of 4% paraformaldehyde and 0.5% glutaraldehyde. Coronal Vibratome sections through the NTS and more rostral raphe nuclei were immunocytochemically labeled with specific antiserum to serotonin or tyrosine hydroxylase and then processed for autoradiography. By light microscopy, concentrations of reduced silver grains indicating uptake of 3H-5HT usually paralleled the localization of peroxidase immunoreactivity for serotonin in neuronal perikarya of the rostral raphe nuclei and in varicosities in the brainstem. The 3H-5HT-containing varicosities were found throughout the medial and commissural portions of the NTS, where they were frequently associated with processes showing immunoreactivity for the catecholamine-synthesizing enzyme tyrosine hydroxylase. Ultrastructural examination of the m-NTS revealed that the silver grains for 3H-5HT were accumulated over axon terminals. The 5HT-labeled terminals contained a heterogeneous population of vesicles and formed both symmetric and asymmetric synapses with dendrites. The recipient dendrites were either, unlabeled or showed immunoreactivity for tyrosine hydroxylase. These findings support a direct serotoninergic modulation of catecholaminergic neurons within the rat m-NTS.

Animals↗
PubMed1984

Ultrastructural relation between cortical efferents and terminals containing enkephalin-like immunoreactivity in rat neostriatum.

The interrelationships between cortical efferents and terminals containing enkephalin-like immunoreactivity (ELI) were examined by combining anterograde degeneration with electron microscopic immunocytochemistry in the adult rat neostriatum. Two days following unilateral removal of the cerebral cortex, the brains were fixed by aortic arch perfusion, then sectioned and processed for the immunocytochemical localization of an antiserum directed against methionine (Met5)-enkephalin. The observed relationships between the degenerating cortical efferents and immunocytochemically labeled terminals were of two types. In the first, the degenerating and ELI containing terminals converged on the same unlabeled dendrite or dendritic spine. In the second, terminal and preterminal axons of the ELI containing neurons had one surface directly apposed to the plasma membrane of a degenerating axon terminal. These findings support the concept that neurons containing opioid peptides and cortical efferents modulate the output of common recipient neurons and may also directly interact with each other through presynaptic axonal mechanisms in the rat neostriatum.

Animals↗
PubMed1984

Ultrastructural localization of monoamines and peptides in rat area postrema.

An overall schema for the synaptic interactions of monoaminergic and peptidergic neurons and their relation to the ventricle and to blood vessels within the rat area postrema is presented. The specific markers include: 1) the immunocytochemical localization of the catecholamine-synthesizing enzyme tyrosine hydroxylase, and the neuropeptides enkephalin and substance P; and 2) the radioautographic localization of [3H]serotonin (5-hydroxytryptamine) and 3H-labeled amino acids anterogradely transported from the nodose ganglion.

Amines↗
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↗
PubMed1983

Substance P and enkephalin in transected axons of medulla and spinal cord.

The accumulation of transported materials in cut axons is demonstrated by the light and electron microscopic immunocytochemical localization of substance P and enkephalin in the caudal medulla and cervical spinal cord of adult rat. Two days following unilateral knife-cuts in the caudal medulla or spinal (C2-C3) levels, substance P and enkephalin-like immunoreactivity (SPLI and ELI) are detected in lesioned axons located rostral and caudal to the transection. Rostrally, SPLI and ELI are detected in the lateral reticular region and ventrolateral fasciculus corresponding to the location of previously identified bulbospinal pathways. Caudally, previously unidentified, propriospinal pathways showing SPLI are detected in the dorsal columns and in the dorsolateral fasciculus. In contrast, ELI is found caudal to the transection only in the reticular region of the medulla. For both peptides, immunoreactivity is present throughout axons containing numerous large, dense core, and small clear vesicles. These results support the concept of both particulate and soluble modes of transport for substance P and enkephalin within axons of the central nervous system.

Animals↗
PubMed1983

Immunohistochemical localization of choline acetyltransferase using a monoclonal antibody: a radioautographic method.

Monoclonal antibodies to rat striatal choline acetyltransferase were produced by fusion of sensitized mouse lymphocytes with murine plasmacytoma (NS1) cells. Two stable anti-choline acetyltransferase lines were established by limiting dilution cloning. Specificity of antibody was established by the following criteria: (1) on an enzyme linked immunosorbant assay, antibodies reacted against choline acetyltransferase which was highly purified; (2) by immunoprecipitation, monoclonal antibody bound to its antigen and precipitated choline acetyltransferase activity from solution, when used in conjunction with rabbit antimouse IgG; and (3) monoclonal antibody was shown to specifically localize cholinergic neurons. The monoclonal antibody to choline acetyltransferase was radiolabeled in culture by incubating hybridomas in medium containing 3H-labeled amino acids. This 3H-labeled antibody was used for radioautography on cryostat sections of rat peripheral and central nervous systems. In a sampling of areas, highly specific labeling of cholinergic structures was afforded at both light and electron microscopic levels. Double labeling of tyrosine hydroxylase, a catecholaminergic marker, and choline acetyltransferase was carried out by reacting sections first with the 3H-labeled antibody to choline acetyltransferase and then with rabbit antibody to tyrosine hydroxylase. The choline acetyltransferase label was radioautographically processed and tyrosine hydroxylase was visualized by the peroxidase-antiperoxidase method. The combined techniques of peroxidase and radioautographic histochemistry provide permanent electron dense labels which can be examined simultaneously within a single histologic section.

Animals↗
PubMed1983

Antibodies to the beta-adrenergic receptor: attenuation of catecholamine-sensitive adenylate cyclase and demonstration of postsynaptic receptor localization in brain.

Antibodies to the beta 2-adrenergic receptor of frog erythrocytes have been raised in rabbits by immunization with purified receptor preparations. Binding of the antibodies to the receptors was demonstrated by immunoprecipitation and by the altered mobility of the antibody-bound receptors on steric-exclusion HPLC columns. As assessed by a radioimmunoassay developed with the antibody, beta 2-adrenergic receptors from several sources showed various degrees of immunological crossreactivity whereas several beta 1-adrenergic receptors did not crossreact. The antibody appeared to not bind at the ligand binding site of the receptor and did not perturb antagonist radioligand binding to the receptor. Nonetheless, the antibodies selectively attenuated catecholamine-stimulated adenylate cyclase. This suggests that the antibodies recognize and bind to domains of the receptor other than the binding site and that may be involved in coupling to other components of the adenylate cyclase system. Immunocytochemical techniques were used with the antibodies to delineate a postsynaptic localization of beta-adrenergic receptors in rat and frog brain. Thus, these anti-beta-adrenergic receptor antibodies provide a useful reagent for probing beta-adrenergic receptor structure, function, and localization.

Adenylyl Cyclases↗
PubMed1982

Antibodies to rat choline acetyltransferase for immunochemistry and immunocytochemistry.

Choline acetyltransferase was purified from rat striata by several sequential column chromatographic separations on Sepharose 4B, hydroxylapatite, Sephadex G-150 and CM-Sephadex, and finally by elution of enzymatically active protein from gel slices. Conventional rabbit and monoclonal antibodies to the enzyme were produced. Monoclonal antibodies were radiolabeled in vivo and used for specific immunoradioautographic localization of choline acetyltransferase in cholinergic neurons by light and electron microscopy.

Animals↗