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V M Pickel

Publications and source records attributed to V M Pickel.

At least 145 records · Page 8Linked to original sources
PubMed1991

Vesicular and cytoplasmic localization of neurotensin-like immunoreactivity (NTLI) in neurons postsynaptic to terminals containing NTLI and/or tyrosine hydroxylase in the rat central nucleus of the amygdala.

Neurotensin and catecholamines in the central nucleus of the amygdala (CNA) have both been implicated in the integration of autonomic responses to stress. We examined whether there might be a cellular substrate for interactions involving these putative neurotransmitters in the CNA. Sections of acrolein-fixed rat brain were processed either (1) for the ultrastructural localization of a rat antiserum against neurotensin using the peroxidase-antiperoxidase (PAP) method, or (2) for the dual localization of rat neurotensin antiserum and rabbit antiserum against the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), using the PAP method and immunoautoradiography. The rat polyclonal antiserum against neurotensin was shown in immunoblots to recognize neuromedin N and Lys-Arg-neurotensin (LANT-6) in addition to neurotensin. In single and dual labeling studies, the neurotensin-like immunoreactivity (NTLI) was detected in perikarya and processes. The NTLI was localized predominantly to dense core vesicles in one group of perikarya and dendrites, while a second group had labeling both in dense core vesicles and more diffusely throughout the cytoplasm. Terminals also showed NTLI, particularly in association with dense core vesicles. The labeled terminals formed primarily symmetric junctions with both cell bodies and dendrites. In the dual labeling study, perikarya contained only NTLI while terminals contained TH and/or NTLI. Terminals containing TH or NTLI separately innervated cell bodies and dendrites displaying NTLI, and formed separate or convergent inputs onto unlabeled neuronal targets. Terminals colocalizing both TH and NTLI formed junctions only on unlabeled dendrites. These findings show that in the rat CNA two populations of neurons differ with respect to their distribution of NTLI, and that the output from neurons containing NTLI is modulated by direct synaptic input from terminals containing neurotensin and/or catecholamines. Release of neurotensin and catecholamines, most likely dopamine, from the same or separate terminals on common targets in the CNA may account for certain similarities in their stress-related functions.

Amygdala↗
PubMed1991

Plasmalemmal appositions between cholinergic and non-cholinergic neurons in rat caudate-putamen nuclei.

We have observed that in rat caudate-putamen nuclei, neurons immunolabeled for choline acetyltransferase were sometimes in direct apposition to unlabeled perikarya and dendrites [Pickel V. M. and Chan J. (1990) J. Neurosci. Res. 25, 263-280]. Similar juxtapositions between plasmalemmas of nerve cells each receiving input from one common terminal have been associated with activation of certain central neurons [Theodosis D. T. and Poulain D. A. (1989) Brain Res. 484, 361-366]. Thus, we sought to determine the relative abundance and ultrastructure of the appositions and the frequencies of shared synapses between choline acetyltransferase-labeled and unlabeled neurons in the rat striatum. A monoclonal antibody raised against choline acetyltransferase was localized in semi-adjacent ultrathin sections through 24 neurons in the dorsolateral caudate-putamen nuclei. Five of these choline acetyltransferase-labeled perikarya showed direct somatic appositions with unlabeled neurons. The remaining 19 of the choline acetyltransferase-labeled perikarya did not show somatic appositions with unlabeled perikarya; however, when traced through multiple (20-100) semi-adjacent sections their dendrites always showed extensive plasmalemmal juxtapositions with one or more unlabeled perikarya. The apposed perikarya had round nuclei and other characteristics of medium, spiny neurons. The majority of the apposed cholinergic and non-cholinergic neurons were postsynaptic to at least one common unlabeled terminal. These terminals usually formed symmetric junctions. At sites of appositions, the plasmalemmas of choline acetyltransferase-immunoreactive soma or dendrites and unlabeled neurons were closely spaced without intervening astrocytic processes. The appositions lacked the ultrastructural features typical of gap-junctions, but did occasionally show parallel arrays of thin (1-2 nm) electron-dense bands. In both labeled and unlabeled perikarya, the nuclei were separated from the appositional zones by narrow (0.7-3.3 microns) rims of cytoplasm. This cytoplasmic rim contained subsurface cisternae and other less specialized smooth and rough endoplasmic reticulum, and vesicular structures. The findings suggest that in the caudate-putamen nuclei (1) the tonically active cholinergic neurons [Wilson C. J. et al. (1990) J. Neurosci. 10, 508-519] may modulate or be modulated by non-cholinergic spiny neurons through non-synaptic somatic or dendritic appositions, and (2) that both neurons may be simultaneously inhibited by shared afferent input. Activation of this system could facilitate coordinated movements through synchronization of cholinergic interneurons and spiny projection neurons containing GABA or other transmitters.

Animals↗
PubMed1990

In the rat medial nucleus accumbens, hippocampal and catecholaminergic terminals converge on spiny neurons and are in apposition to each other.

The nucleus accumbens septi (Acb) represents an interface between limbic and motor systems and a site for modulation of these integrative functions by ascending catecholaminergic, principally dopaminergic, axons. This modulatory regulation is most likely attributed to pre- or postsynaptic associations between limbic telencephalic and brainstem afferents. In the present investigation, we examined the ultrastructure and synaptic associations of hippocampal afferents, as well as their relation to catecholaminergic terminals, in the medial Acb of adult rats. Hippocampal afferents were identified by anterograde transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) injected in the ventral subiculum, and by anterograde degeneration seen 2-3 days following lesion of the fimbria. Specific comparisons between these methods were made (1) to determine whether similar populations of terminals were labeled and (2) to assess the feasibility of combining degeneration with immunoperoxidase labeling for the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH). Hippocampal afferents labeled with HRP were finely myelinated or unmyelinated and gave rise to small terminals (mean diameter 0.58 micron) containing mostly clear, round vesicles. Of the HRP-labeled terminals which made recognizable junctions, 85% (104/122) formed asymmetric synapses with the heads of dendritic spines. The remainder either formed asymmetric axodendritic synapses or symmetric junctions. Degenerating terminals were significantly smaller (mean diameter 0.35 micron) than terminals labeled with HRP. However, these also formed principally asymmetric axospinous synapses (89/102, 87%). Whether identified by HRP transport or anterograde degeneration, the hippocampal afferents comprised approximately 10% of all terminals and 30% of all asymmetric axospinous synapses in the medial Acb. In contrast to hippocampal afferents, TH-labeled terminals formed primarily symmetric contacts with dendritic shafts and the heads and necks of spines. Quantitative analysis of sections containing both anterograde degeneration and TH-immunoreactivity showed that 25% (26/104) of associations formed by degenerating hippocampal terminals involved convergent inputs with TH-labeled terminals on the same postsynaptic structure. These included dual input either to the same spine head or to different parts of the same dendrite. In addition, the plasma membranes of hippocampal and TH-labeled terminals were often directly apposed to each other (10/58, 17% of axo-axonal associations formed by degenerating terminals), without recognizable synaptic specializations.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗
PubMed1990

Spiny neurons lacking choline acetyltransferase immunoreactivity are major targets of cholinergic and catecholaminergic terminals in rat striatum.

The ultrastructural substrate for functional interactions between intrinsic cholinergic neurons and catecholaminergic afferents to the caudate-putamen nucleus and nucleus accumbens septi (NAS) was investigated immunocytochemically. Single sections of glutaraldehyde-fixed rat brain were processed 1) for the immunoperoxidase labeling of a rat monoclonal antibody against the acetylcholine-synthesizing enzyme choline acetyltransferase (CAT) and 2) for the immunoautoradiographic localization of a rabbit polyclonal antiserum against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). The ultrastructural morphology and cellular associations did not significantly differ in the caudate-putamen versus NAS. Immunoperoxidase reaction for CAT versus NAS. Immunoperoxidase reaction for CAT was seen in perikarya, dendrites, and terminals, whereas immunoautoradiography for TH was in terminals. The perikarya and dendrites immunolabeled for CAT were large, sparsely spiny, and postsynaptic mainly to unlabeled axon terminals. Only 2-3% of the CAT-labeled terminals (n = 136) and less than 1% of the TH-labeled terminals (n = 86) were apposed to, or formed synapses with, perikarya or dendrites immunoreactive for CAT. Most unlabeled and all labeled terminals formed symmetric synapses. In the same sample, 18% of the CAT and 16% of the TH-labeled terminals were directly apposed to each other. Unlabeled dendritic shafts received the major (40% for CAT versus 23% for TH) synaptic input from cholinergic terminals, while unlabeled spines received the major (47% for TH versus 23% for CAT) synaptic input from catecholaminergic terminals. Neither the unlabeled dendrites or spines received detectable convergent input from CAT and TH-labeled terminals. Thirteen percent of the CAT-labeled and 14% of TH-labeled terminals were in apposition to unlabeled terminals forming asymmetric, presumably excitatory, synapses with unlabeled dendritic spines. We conclude that in both the caudate-putamen and NAS cholinergic and catecholaminergic terminals 1) form symmetric, most likely inhibitory, synapses primarily with non-cholinergic neurons, 2) differentially synapse on shafts or spines of separate dendrites, and 3) have axonal appositions suggesting the possibility of presynaptic physiological interactions. These results support the hypothesis that the cholinergic-dopaminergic balance in striatal function may be mediated through inhibition of separate sets of spiny projection neurons with opposing excitatory and inhibitory functions.

Adrenergic Fibers↗
PubMed1990

Optimization of differential immunogold-silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding.

The limited success of immunogold labeling for pre-embedding immunocytochemistry of neuronal antigens is largely attributed to poor penetration of large (5-20 nm) colloidal gold particles. We examined the applicability of using silver intensification of 1 nm colloidal gold particles non-covalently bound to goat anti-rabbit immunoglobulin (1) for single labeling of a rabbit antiserum against the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), and (2) for immunogold localization of rabbit anti-TH simultaneously with immunoperoxidase labeling of a mouse monoclonal antibody against the opiate peptide, leucine-enkephalin (LE). Vibratome sections were collected from acrolein fixed brains of adult rats. These sections were immunolabeled without use of freeze-thawing or other methods that enhance penetration, but damage ultrastructure. By light microscopy, incubations in the silver intensifier (Intense M, Janssen) for less than 10 min at room temperature resulted in a brownish-red reaction product for TH. This product was virtually indistinguishable from that seen using diaminobenzidine reaction for detection of peroxidase immunoreactivity. Longer incubations produced intense black silver deposits that were more clearly distinguishable from the brown immunoperoxidase labeling. However, by light microscopy, the gold particles seen by electron microscopy were most readily distinguished from peroxidase reaction product with shorter silver intensification periods. The smaller size of gold particles with shorter periods of silver intensification also facilitated evaluation of labeling with respect to subcellular organelles. Detection of the silver product did not appear to be appreciably changed by duration of post-fixation in osmium tetroxide. In dual-labeled sections, perikarya and terminals exhibiting immunogold-silver labeling for TH were distinct from those containing immunoperoxidase labeling for LE. These results (1) define the conditions needed for optimal immunogold-silver labeling of antigens while maintaining the ultrastructural morphology in brain, and (2) establish the necessity for controlled silver intensification for light or electron microscopic differentiation of immunogold-silver and peroxidase reaction products and for optimal subcellular resolution.

Animals↗
PubMed1990

Neuropeptide Y in cortex and striatum. Ultrastructural distribution and coexistence with classical neurotransmitters and neuropeptides.

NPY-neurons in the striatum and cortex have many morphological and chemical features in common. They are intrinsic, medium sized, aspiny and exhibit ultrastructural characteristics typical of neurons undergoing active synthesis and release of peptides. Most of the NPY-neurons in the two regions coexist with somatostatin, exhibit high levels of NADPH-diaphorase and are resistant to degeneration associated with Huntington's disease. Ultrastructural analysis suggests that the ensheathment by glia and sparsity of asymmetric (putatively excitatory) inputs may render NPY neurons resistant to excitotoxicity. Although NPY-neurons receive few inputs, they make numerous contacts with dendrites within a small region of the neuropil. Among their targets are GABAergic neurons. These NPY-receptive GABA neurons differ from other GABAergic neurons in the vicinity in that they receive few other inputs along their somata and proximal dendrites. This suggests that NPY may exert more influence on a specific class of GABAergic neurons. Many more of the NPY-terminals are found at sites that would be strategic for the simultaneous modulation of the release of transmitters and postsynaptic responses. The differences among NPY-neurons in the striatum versus cerebral cortex are mainly chemical. Most notably, the NPY-neurons are GABAergic in the cortex and not GABAergic in the striatum. In addition, some of the NPY-axons in the ventral portions of striatum and cerebral cortex may be catecholaminergic, and thus originate in brainstem areas recognized to contain NPY and epinephrine or norepinephrine. NPY- and catecholaminergic fibers converge onto same dendrites. Thus, the two transmitters may interact through intercellular biochemical pathways postsynaptically. Finally, the sites where the two fibers directly contact each other may be where NPY stimulates the turnover of dopamine.

Animals↗
PubMed1990

Ultrastructural localization of tyrosine hydroxylase in the rat ventral tegmental area: relationship between immunolabeling density and neuronal associations.

Dopaminergic neurons of the A 10 cell group in the rat ventral tegmental area (VTA) exhibit electrical and dye coupling. Also, the activity of these neurons at least partially reflects their content of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. We examined the ultrastructural localization of TH to determine the morphological features of dopaminergic neurons in the VTA and the relationships between their TH immunoreactivity content and afferent input. Antiserum against the trypsin-treated form of TH was localized using peroxidase-antiperoxidase (PAP) and immunoautoradiographic methods. Immunoreactivity was detected in perikarya, dendrites, and terminals. The perikarya contained the usual organelles, as well as cilia, lamellar bodies, and subsurface cisterns. Qualitative evaluation of peroxidase reaction product and quantitative analysis of the number of silver grains/unit area revealed varying amounts of TH immunoreactivity in nuclei and cytoplasm. Lightly or intensely labeled nuclei were not necessarily associated with corresponding cytoplasmic labeling density. However, cytoplasmic labeling directly corresponded to the relative frequencies of neuronal appositions and synaptic input. Those neurons with less dense cytoplasmic PAP product received fewer synaptic contacts and were less frequently in apposition to other TH-labeled soma and dendrites than neurons displaying relatively more dense cytoplasmic PAP product. Analysis of single sections revealed that 67% (n = 71) of all TH-labeled somata and 15% (n = 2431) of all TH-labeled dendrites were in apposition to other TH-labeled soma or dendrites. TH-labeled terminals were rarely detected and contained relatively low levels of immunoreactivity. The majority of labeled terminals (n = 29/46) formed synapses with labeled soma and dendrites. Unlabeled terminals (n = 2424) in contact with TH-labeled dendrites appeared to form predominantly symmetric synapses. Ten percent (n = 248) of the unlabeled terminals dually synapsed onto adjacent immunoreactive dendrites, perikarya, or dendrite and perikaryon. We conclude that in the rat VTA, (1) detected TH immunoreactivity in cytoplasm, but not nucleus, corresponds to the level of feedback principally from nondopaminergic afferents; (2) dendrodendritic as well as axodendritic synapses between TH-immunoreactive neurons may mediate dopaminergic autoinhibition; and (3) gap junction-like appositions between neurons and convergent inputs from unlabeled terminals onto TH-immunoreactive profiles provide an anatomical substrate whereby cellular activities might be coordinated under certain conditions.

Animals↗
PubMed1989

Ultrastructural localization of choline acetyltransferase in the rat rostral ventrolateral medulla: evidence for major synaptic relations with non-catecholaminergic neurons.

Pharmacological and biochemical studies suggest that interactions between cholinergic and catecholaminergic and catecholaminergic neurons, particularly those of the C1 adrenergic cell group, in the rostral ventrolateral medulla (RVL) may be important in cardiovascular control. Ultrastructural localization of choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine, and its relation to neurons exhibiting immunoreactivity for catecholamine- (tyrosine hydroxylase; TH) or adrenaline (phenylethanolamine-N-methyltransferase; PNMT) -synthesizing enzymes were examined in the RVL using dual immunoautoradiographic and peroxidase anti-peroxidase (PAP) labeling methods. By light microscopy, the ChAT-immunoreactive neurons were located both dorsally (i.e. the nucleus ambiguus) and ventromedially to those labeled with TH or PNMT (TH/PNMT). A few ChAT-labeled processes were dispersed among TH/PNMT-containing neurons with the majority of overlap immediately ventral to the nucleus ambiguus. By electron microscopy, ChAT-immunoreactivity (ChAT-I) was detected in neuronal perikarya, dendrites, axons and axon terminals and in the vascular endothelial cells of certain blood vessels. The ChAT-labeled perikarya in the ventromedial RVL were medium-sized (15-20 microns), elongated, contained abundant cytoplasm and had had slightly indented nuclei. Synaptic junctions on ChAT-immunoreactive perikarya and dendrites were primarily symmetric with 64% (45 out of 70) of the presynaptic terminals unlabeled. The remaining terminals were immunoreactive for ChAT (30%) or TH/PNMT (6%). Terminals with ChAT-I were large (0.8-2.0 microns) and contained numerous small clear vesicles and 1-2 dense core vesicles. Seventy-seven percent (112 out of 145) of the ChAT-labeled terminals formed symmetric synapses with unlabeled perikarya and dendrites, whereas only 8% were with TH/PNMT-labeled perikarya and dendrites, and 15% were with ChAT-immunoreactive perikarya and dendrites. We conclude (1) that cholinergic neurons in the RVL principally terminate on and receive input from non-catecholaminergic neurons, and (2) that the reported sympathetic activation following application of cholinergic agents to the RVL may be mediated by cholinergic inhibition of local inhibitory interneurons. The observed synapses between ChAT and TH/PNMT-containing neurons suggests that cholinergic and adrenergic neurons additionally may exert a minor reciprocal control on each other and thus may modulate their response to the more abundant input from afferents containing other transmitters.

Animals↗
PubMed1989

Cellular substrates for interactions between neurons containing phenylethanolamine N-methyltransferase and GABA in the nuclei of the solitary tracts.

Adrenaline and gamma-aminobutyric acid (GABA) have been implicated in autonomic functions involving the intermediate and caudal portions of the medial nuclei of the solitary tracts (m-NTS). We sought to determine whether there was a cellular basis for direct intracellular or synaptic interactions between these transmitters in neurons in the m-NTS of rat brain by using dual-labeling immunocytochemical methods. Light microscopy revealed immunoautoradiographic labeling for the adrenaline-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) in perikarya and processes in close proximity to cells demonstrating peroxidase reaction product for GABA. Electron microscopy of the intermediate m-NTS at the level of the area postrema further established the localization of immunoautoradiographic and peroxidase labels for PNMT and GABA in common as well as separate perikarya and dendrites. All axon terminals were labeled separately for PNMT and GABA. The PNMT-labeled terminals formed both symmetric and asymmetric synapses, whereas the GABA-labeled terminals formed exclusively symmetric synapses. Twenty-four percent (n = 42) of the PNMT- and 39% (n = 128) of the GABA-labeled terminals formed synaptic junctions on unlabeled soma and dendrites. Occasionally both types of terminals converged on a common unlabeled dendrite and on GABA-labeled dendrites. Only 3% of the PNMT- and 12% of the GABA-containing terminals formed synapses on PNMT-labeled soma and dendrites, whereas 7% of each type formed synapses with GABA-labeled profiles. The remaining labeled terminals lacked synaptic relations within the sections examined. The autoradiographic results were confirmed and extended by means of immunogold labeling for PNMT in combination with peroxidase-antiperoxidase localization of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). GAD-labeled terminals formed symmetric synapses with dendrites that were either unlabeled or contained low levels of PNMT (gold particles) or PNMT and GAD. We conclude that in caudal, more cardiovascular portions of the NTS, adrenaline and GABA may coexist, but they are more commonly detected in separate populations of neurons having receptive sites for both transmitters and innervating certain common target neurons.

Animals↗
PubMed1989

Cytoplasmic loop of beta-adrenergic receptors: synaptic and intracellular localization and relation to catecholaminergic neurons in the nuclei of the solitary tracts.

Pharmacological studies suggest that beta-adrenergic receptors (beta AR) in the medial nuclei of the solitary tracts (m-NTS) facilitate presynaptic release of catecholamines and also function at postsynaptic sites. We have localized the antigenic sites for a monoclonal antibody against a peptide corresponding to amino acids 226-239 of beta AR in the m-NTS of rat brain. By light microscopy, immunoperoxidase labeling for this antibody was detected in somata and proximal processes of many small cells that were distributed throughout the rostrocaudal extent of the m-NTS. Electron microscopy confirmed the cytoplasmic localization of beta AR in perikarya and proximal dendrites of neurons. Immunoreactivity occurred as discrete patches associated with cytoplasmic surfaces of plasma membrane and with irregularly-shaped saccules with clear lumen in the immediate vicinity. Select regions of nuclear envelopes, mitochondrial membranes, and rough endoplasmic reticulum were also immunoreactive along their cytoplasmic surfaces. In contrast, the Golgi apparatus was labeled, but infrequently. Immunoreactivity was also detected at numerous post- and occasional presynaptic membrane specializations of select axodendritic junctions. Dual labeling for the beta AR-antibody by the immunoperoxidase method and for a rabbit antiserum against the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), by the immunoautoradiographic method within the same sections, further established the precise cellular relations between beta AR and catecholaminergic neurons. Immunoreactivity for beta AR was detected in numerous perikarya and proximal dendrites that did not show detectable levels of TH. However, a few cells were dually labeled for both antigens, as seen by both light and electron microscopy. The TH-labeled terminals formed synapses at junctions both with and without beta AR-like immunoreactivity. These results from the single and dual labeling studies: (1) confirm biochemical predictions that amino acids 226-239 of beta AR protein reside intracellularly; (2) provide the first ultrastructural evidence for beta AR localization within both pre- and postsynaptic membrane specializations of a subset of catecholaminergic synapses; and (3) suggest select intracellular sites that may be involved with synthesis and/or internalization and degradation of the receptor protein.

Animals↗
PubMed1989

Ultrastructural localization of phenylethanolamine N-methyltransferase-like immunoreactivity in the rat locus coeruleus.

Adrenergic afferents from the rostral ventrolateral medulla are known to modulate the activity of noradrenergic neurons of the locus coeruleus (LC). The light and electron microscopic localization of a polyclonal antiserum directed against the adrenaline synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT) was used to determine the identity and targets of the adrenergic afferents to the LC of the rat brain. By light microscopy, varicose processes showing intense PNMT-like immunoreactivity (LI) were seen throughout the neuropil surrounding neuronal perikarya which in adjacent sections were shown to contain immunoreactivity for the noradrenaline synthesizing enzyme, dopamine-beta-hydroxylase. Electron microscopy confirmed that these labeled varicose processes were primarily axon terminals. Terminals containing PNMT-LI constituted 30% (141 out of 464) of all identifiable terminals within the LC. These terminals were 0.5-1.8 micron in diameter and contained many small, clear and from 2 to 10 larger dense-core vesicles. The targets of the terminals with PNMT-LI were principally unlabeled (i.e. non-PNMT-containing) perikarya and dendrites. The synaptic junctions on perikarya were rare and exclusively symmetric; whereas, those on proximal (large) dendrites were somewhat more numerous and included symmetric as well as asymmetric membrane specializations. However, the vast majority (85% from a total of 141) of the terminals with PNMT-LI formed asymmetric synaptic junctions on unlabeled distal (small) dendrites and dendritic spines. In rare instances, the PNMT-immunoreactive terminals also formed synaptic junctions with other similarly labeled terminals. These findings provide the first ultrastructural evidence that adrenergic terminals in the LC (1) are one of the more prevalent synaptic inputs to the principally noradrenergic neurons; (2) have both symmetric and asymmetric synaptic specializations conventionally associated with inhibition and excitation, respectively; and (3) may modulate other adrenergic terminals through presynaptic mechanisms. In addition to the varicose processes, light microscopy revealed diffuse PNMT-LI throughout the LC. The ultrastructural correlate of this labeling was seen as patches of peroxidase product within the cytoplasm of a few perikarya and dendrites and throughout the cytoplasm of astrocytes identified by their discrete bundles of microfilaments. The detection of PNMT-LI in cells that are not known to synthesize adrenaline is surprising and suggests either a functional diversity for PNMT or amino acid sequence homologies with related enzymes which are enriched in the LC.

Animals↗
PubMed1989

Neuropeptide Y-like immunoreactivity in neurons of the solitary tract nuclei: vesicular localization and synaptic input from GABAergic terminals.

The ultrastructural localization of neuropeptide Y-like immunoreactivity (NPY-LI) was examined in the medial nuclei of the solitary tracts (mNTS) of adult rat brain. Peroxidase-antiperoxidase (PAP) reaction product was localized extensively to the central lumen of large (100-150 nm), dense-core vesicles. The labeled vesicles were seen in axon terminals of untreated, control animals and in perikarya and dendrites of rats receiving intraventricular injections of colchicine 24 h prior to sacrifice. The labeled terminals were of two types. The first type contained numerous small, clear vesicles that were rimmed with peroxidase product and 1-6 large, dense-core vesicles that were labeled throughout their central lumen. The second type contained a more homogeneous population of labeled large, dense-core vesicles. Axon terminals showing NPY-LI formed either asymmetric synapses with unlabeled dendrites or were without recognized junctions. Within labeled terminals, as well as within perikarya and dendrites, the majority of the dense-core vesicles were located near non-synaptic portions of the plasmalemma that were heavily ensheathed with glial processes. Only a few unlabeled terminals penetrated the glial investments to form synaptic contacts on soma or dendrites containing NPY-LI. These synaptic contacts were of both symmetric and asymmetric types. Combined immunoperoxidase labeling for glutamic acid decarboxylase and immunogold labeling for NPY further established that at least some of the terminals forming symmetric junctions on the NPY-immunoreactive dendrites were GABAergic. These results provide ultrastructural evidence that in the mNTS, NPY-LI is localized principally to large dense-vesicles within neurons whose output is partially regulated by GABA. The preferential distribution of the labeled vesicles along non-synaptic, glial-invested portions of the plasmalemma suggests that neuronal NPY may modulate the activity of nearby astrocytes. Additionally, the localization of NPY-LI in terminals containing a mixed population of synaptic vesicles and forming asymmetric axodendritic junctions suggests that NPY and/or coexisting transmitter may also exert certain known hypotensive effects by excitation of local intrinsic or projection neurons in this brain region.

Animals↗
PubMed1989

Glucocorticoid-induced PNMT-immunoreactive sympathetic cells in the superior cervical ganglion of the rat.

Light and electron microscopic immunocytochemical techniques were used to study the effect of glucocorticoids on the development of phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive cells in the superior cervical ganglion (SCG) of early postnatal rats. Rats were injected daily with hydrocortisone acetate on postnatal days 2-6. The first PNMT-immunoreactive cells were detected 6 hours after the first glucocorticoid injection and their number increased after subsequent injections. No PNMT-immunoreactive cells were detected in uninjected controls. PNMT-immunoreactive fibres were seen in the ganglion 6 hours after the first glucocorticoid injection. The PNMT-immunoreactive cells consistently showed processes 2 days after beginning the glucocorticoid treatment, and long processes and fibre networks were seen in ganglia of 7-day-old rats. However, no PNMT-immunoreactive fibres were seen in the iris, which is innervated by the SCG. Ultrastructurally, most of the PNMT-immunoreactive cells had the look of small granule-containing (SGC) cells, including heterochromatin clumps along the nuclear envelope and in the center of the nucleoplasm as well as dense core vesicles. SGC cells, nonimmunoreactive to PNMT antiserum, also were seen. However, some PNMT-immunoreactive cells showed ultrastructural characteristics of nerve cells. In contrast to the SGC cells, these cells were characterized by a voluminous cytoplasm, dispersed nuclear heterochromatin, and a lack of granular vesicles. These results demonstrate that glucocorticoids induce PNMT immunoreactivity both in SGC cells and also in cells with characteristics of principal neurons.

Animals↗
PubMed1989

Adrenergic neurons in the rostral ventrolateral medulla: ultrastructure and synaptic relations with other transmitter-identified neurons.

The first part of this chapter demonstrates that the C1 adrenergic neurons have high mitochondrial content and a close proximity to capillaries and glia suggestive of a high metabolic activity and a possible chemosensory function. Adrenergic terminals arising primarily from these neurons (1) can influence sympathetic nerve discharge through direct contacts on sympathetic preganglionic neurons in the IML of the spinal cord; and (2) are one of the more prevalent synaptic inputs to the principally noradrenergic neurons in the locus coeruleus. In both the IML and locus coeruleus, adrenergic terminals may be either excitatory (asymmetric synapses) or inhibitory (symmetric synapses) depending on their distribution on the post-synaptic target. The second part of this chapter shows that C1 adrenergic neurons in the RVL are modulated by synaptic associations with a variety of transmitter systems (see schematic Fig. 8). Specifically, C1 adrenergic neurons receive (1) major inhibitory input (symmetric synapses) from GABA-ergic and opioid terminals as well as from unidentified (unlabelled) transmitter-containing terminals; (2) major excitatory input (asymmetric synapses) from terminals containing substance P as well as other unidentified terminals and (3) minor inputs from cholinergic, adrenergic and noradrenergic pathways. Moreover, cholinergic terminals in the RVL form symmetric synapses mainly on unidentified transmitter-containing neurons rather than the C1 neurons suggesting that the reported cardiovascular effects of cholinergic agents in the RVL are most likely mediated via inhibitory interneurons. Within the RVL, adrenergic and noradrenergic terminals innervate cholinergic and opioid neurons. Thus, these results not only provide direct evidence that a number of transmitters modulate the activity of C1 adrenergic neurons, but also suggest new directions for studies of functional interactions involving catecholaminergic regulation of other transmitter-containing neurons within the RVL.

Animals↗
PubMed1989

Ultrastructural basis for interactions between central opioids and catecholamines. I. Rostral ventrolateral medulla.

Opioids and some alpha 2-adrenergic agonists are both known for their potent hypotensive actions following local application to the rostral ventrolateral medulla (RVL), in particular the region containing the C1 adrenergic neurons. We sought to determine whether coexistence and/or synaptic interactions might account for the commonality of cardiovascular responses to opioids and catecholamines in the RVL. Dual light and electron microscopic (EM) immunoperoxidase labeling of a rat monoclonal antibody against the opioid peptide Leucine5 (Leu5)-enkephalin and immunoautoradiographic localization of a rabbit antiserum against the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) were examined in single sections through the RVL of adult colchicine-pretreated rats. Cross-reactivity of the enkephalin antibody was most intense with Leu5-enkephalin. Methionine5-enkephalin as well as dynorphin A, but not beta-endorphin, were also recognized by the antisera. By light microscopy, the Leu5-enkephalin-like immunoreactivity (LE-LI) was identified by peroxidase reaction product in perikarya and processes. Most of the perikarya containing LE-LI were located dorsolaterally or ventromedially to those showing immunoautoradiographic labeling for TH. However, a few perikarya appeared to contain both LE-LI and TH-immunoreactivity (TH-I) which were difficult to differentiate by light microscopy. By EM, perikarya and dendrites immunoreactive for LE, TH, and both LE and TH were readily distinguishable. Perikarya and dendrites immunoautoradiographically labeled for TH alone were more numerous than those containing LE-LI or TH-I and LE-LI. Axon terminals also were immunolabeled either for one or both reaction products. However, the TH-labeled neurons constituted one of the primary (42% from a total of 118) targets of terminals containing LE-LI. Additionally, some of these terminals containing LE-LI shared a common target with TH-labeled terminals. These common target neurons contained either TH-I or TH-I and LE-LI. In most cases, the identified junctions were symmetric and the terminals with LE-LI (0.4-1.2 microns in diameter) contained either (1) a few small clear vesicles (scv's) and numerous intensely immunoreactive large (100-150 nm) dense-core vesicles (dcv's); or (2) many scv's and from 0-6 dcv's of a somewhat smaller (80-120 nm) diameter. The latter type of terminal was more consistently dually labeled for TH. The remaining terminals containing LE-LI formed synaptic junctions with unlabeled perikarya or dendrites (32%), were in apposition to other unlabeled as well as TH or LE- and TH-containing terminals (4%) or were without recognizable specializations within the plane of section (22%).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Ultrastructural basis for interactions between central opioids and catecholamines. II. Nuclei of the solitary tracts.

Interactions between central opioids and catecholamines are thought to underlie the ability of adrenergic agonists both to lower blood pressure and alleviate certain symptoms of opiate withdrawal. We examined the cellular substrate for interactions between neurons containing enkephalin-like opioid peptides and catecholamines in cardiovascular portions of the medial nuclei of the solitary tracts (m-NTS) of adult rats. Single sections were dually labeled using a double-bridged peroxidase method for the localization of a monoclonal leucine (Leu5)-enkephalin-antibody and immunoautoradiography for the localization of polyclonal antibodies against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Light microscopy revealed a few perikarya and numerous varicosities containing Leu5-enkephalin-like immunoreactivity (LE-LI). These were distributed among TH-labeled perikarya and processes throughout the rostrocaudal NTS. Electron microscopy of the m-NTS at the level of the area postrema further established the single as well as dual localization of TH and LE-LI in individual perikarya, dendrites, and axon terminals. Silver grains indicative of TH-labeling were usually distributed throughout the cytoplasm, whereas the peroxidase reaction product for LE-LI was localized principally to large (80-150 nm), dense-core vesicles. Immunoautoradiographic labeling for TH was detected in 118 terminals within a series of sections containing 183 terminals with LE-LI. Of these, 26% of the TH-labeled terminals and 32% of the enkephalin-containing terminals formed symmetric synapses with unlabeled dendrites, while only 7% of each type formed symmetric synapses with TH-labeled dendrites. In favorable planes of sections, the unlabeled as well as TH-labeled dendrites received convergent input from both types of terminals. A few of the remaining terminals that contained either TH or LE-LI formed asymmetric junctions with unlabeled distal dendrites; the others were without recognizable synaptic specializations within the plane of section. Approximately 20% of the TH-labeled terminals and 6% of the terminals containing LE-LI were dually labeled for both antibodies. These were invested with astrocytic processes characterized by bundles of intermediate filaments. We conclude that within cardiovascular portions of the m-NTS, opioid peptides and catecholamines contained within the same or separate terminals modulate the activity of target neurons through direct symmetric, probably inhibitory, synaptic junctions and may additionally modulate the activity of neighboring astrocytes through exocytotic release from large dense-core vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗