Numerous Large Dense-core Vesicles Research Articles

Transcriptional Orchestration of the Regulated Secretory Pathway in Neurons by the bHLH protein DIMM

The Drosophila basic helix-loop-helix (bHLH) gene dimmed (dimm) promotes a neurosecretory/neuroendocrine phenotype in cells but is not associated with specific neuropeptides or neurohormones. Rather, it is expressed by those peptidergic neurons that project long axons and appear to produce large amounts of secretory peptides. Here, we genetically transform nonpeptidergic neurons in Drosophila to study DIMM's action mechanisms. Nonpeptidergic neurons normally fail to accumulate ectopic neuropeptides. We now show that they will do so when they are also forced to express ectopic DIMM. Furthermore, mass spectrometry shows that photoreceptors, which are normally nonpeptidergic, fail to process an ectopic neuropeptide precursor to make bioactive peptides but will do so efficiently when DIMM is co-misexpressed. Likewise, photoreceptors, which normally package the fast neurotransmitter histamine within small clear synaptic vesicles, produce numerous large dense-core vesicles (LDCVs) when they misexpress DIMM. These novel LDCVs accumulate ectopic neuropeptide when photoreceptors co-misexpress a neuropeptide transgene. DIMM-expressing photoreceptors no longer accumulate histamine and lose synaptic organelles critical to their normal physiology. These findings indicate that DIMM suppresses conventional fast neurotransmission and promotes peptidergic neurosecretory properties. We conclude that DIMM normally provides a comprehensive transcriptional control to direct the differentiation of dedicated neuroendocrine neurons.

Pro‐opiomelanocortin colocalizes with corticotropin‐ releasing factor in axon terminals of the noradrenergic nucleus locus coeruleus

We previously demonstrated that the opioid peptide enkephalin and corticotropin-releasing factor (CRF) are occasionally colocalized in individual axon terminals but more frequently converge on common dendrites in the locus coeruleus (LC). To further examine potential opioid cotransmitters in CRF afferents we investigated the distribution of pro-opiomelanocortin (POMC), the precursor that yields the potent bioactive peptide beta-endorphin, with respect to CRF immunoreactivity using immunofluorescence and immunoelectron microscopic analyses of the LC. Coronal sections were collected through the dorsal pontine tegmentum of rat brain and processed for immunocytochemical detection of POMC and CRF or tyrosine hydroxylase (TH). POMC-immunoreactive processes exhibited a distinct distribution within the LC as compared to the enkephalin family of opioid peptides. Specifically, POMC fibers were enriched in the ventromedial aspect of the LC with fewer fibers present dorsolaterally. Immunofluorescence microscopy showed frequent coexistence of POMC and CRF in varicose processes that overlapped TH-containing somatodendritic processes in the LC. Ultrastructural analysis showed POMC immunoreactivity in unmyelinated axons and axon terminals. Axon terminals containing POMC were filled with numerous large dense-core vesicles. In sections processed for POMC and TH, approximately 29% of POMC-containing axon terminals (n = 405) targeted dendrites that exhibited immunogold-silver labeling for TH. In contrast, sections processed for POMC and CRF showed that 27% of POMC-labeled axon terminals (n = 657) also exhibited CRF immunoreactivity. Taken together, these data indicate that a subset of CRF afferents targeting the LC contain POMC and may be positioned to dually impact LC activity.

Expression of vanilloid receptor TRPV1 in the rat trigeminal sensory nuclei.

Little is known about the central projection patterns of trigeminal afferent neurons expressing the vanilloid receptor TRPV1 and their coexpression of neuromodulatory peptides. To address these issues, we examined the distribution of TRPV1-positive neurons in the trigeminal ganglion (TG) and trigeminal sensory nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc) in the rat via light and electron microscopy. In addition, we studied the colocalization of TRPV1-positive neurons with substance P (SP) and calcitonin gene-related peptide (CGRP) via confocal microscopy. In TG, only small and medium-sized neurons were immunopositive for TRPV1. The staining for TRPV1 was found in axon collaterals in the dorsal parts of Vp, Vo, and Vi and in terminals and fibers throughout lamina I and the outer zone of lamina II (IIo) of Vc. With electron microscopy, TRPV1-positive fibers in the ascending and descending trigeminal tracts were found to be unmyelinated. Almost all TRPV1-positive terminals in Vc contained numerous large dense-core vesicles and formed synaptic contacts with single small dendrites. Multiple immunofluorescence revealed a high degree of colocalization of TRPV1 with SP and CGRP in TG neurons as well as in fibers and terminals confined to laminae I and IIo of Vc. These results suggest that the central projections of unmyelinated (C) afferents sensitive to noxious heat and capsaicin are organized differently between Vc and the rostral trigeminal nuclei and that Vc may play a role in the development of hyperalgesia.

C1 adrenergic neurons are contacted by presynaptic profiles containing DELTA-opioid receptor immunoreactivity

Ligands of the δ-opioid receptor tonically influence sympathetic outflow. Some of the actions of δ-opioid receptor agonists may be mediated through C1 adrenergic neurons in the rostral ventrolateral medulla. The goal of this study was to determine whether C1 adrenergic neurons or their afferents contain δ-opioid receptors. Single sections through the rostral ventrolateral medulla were labeled for δ-opioid receptor using the immunoperoxidase method and the epinephrine synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) using the immunogold method, and examined at the light and electron microscopic level. Few (∼5% of 903) profiles dually labeled for PNMT and δ-opioid receptor were detected; most of these were dendrites with diameters <1.5 μm. δ-Opioid receptor immunoreactivity was affiliated with multivesicular bodies in dually labeled perikarya, whereas δ-opioid receptor immunoperoxidase labeling appeared as isolated clusters within both singly and dually labeled dendrites. The majority (∼83% of 338) of δ-opioid receptor-immunoreactive profiles were axons and axon terminals. δ-Opioid receptor-immunoreactive terminals averaged 0.75 μm in diameter, contained numerous large dense-core vesicles and usually formed appositions or asymmetric (excitatory-type) synapses with their targets. The majority (>50% of 250) of δ-opioid receptor-immunoreactive axons and axon terminals contacted PNMT-immunoreactive profiles. Most of the contacts formed by δ-opioid receptor-immunoreactive profiles (∼75% of 132) were on single-labeled PNMT-immunoreactive dendrites with diameters <1.5 μm. The prominent localization of δ-opioid receptors to dense-core vesicle-rich presynaptic profiles suggests that δ-opioid receptor activation by endogenous or exogenous agonists may modulate neuropeptide release. Furthermore, the presence of δ-opioid receptors on axon terminals that form excitatory-type synapses with PNMT-immunoreactive dendrites suggests that δ-opioid receptor ligands may modulate afferent activity to C1 adrenergic neurons. The observation that some PNMT-immunoreactive neurons contain δ-opioid receptor immunoreactivity associated with multivesicular bodies and other intracellular organelles suggests that some C1 adrenergic neurons may present, endocytose and/or recycle δ-opioid receptors.

Unusual neurofilament composition in cerebellar unipolar brush neurons.

During antibody screening on sections of rat cerebellum, we noticed a group of small neurons which exhibited unusual staining properties. They were robustly immunopositive for the high molecular weight neurofilament protein, moderately immunostained with antibodies to the low molecular weight neurofilament protein and alpha-internexin, but only faintly immunoreactive (in PAP sections) or essentially immunonegative (in immunofluorescent sections) with all members of a panel of antibodies directed against the middle molecular weight neurofilament protein. Since neurons generally react equally well with phosphate-independent, (antibodies to) low, middle and high molecular weight neurofilament protein, we conclude that middle molecular weight neurofilament protein is present in these cells in an unusually low relative amount. These cells are found in the granular layer and appear concentrated in the flocculus, ventral paraflocculus, and vermis, particularly in the ventral uvula and nodulus (lobules IXd and X). Previous studies performed by Hockfield defined a population of neurons of similar appearance and distribution using the monoclonal antibody Rat-302, which recognized an uncharacterized 160 kDa protein. We show here that the cells described by Hockfield are identical to those we have found and furthermore that the Rat-302 antibody specifically recognizes the dephosphorylated form of the lysine-serine-proline repeated sequences of high molecular weight neurofilament protein. These cells were studied by pre-embedding immunoelectron microscopy. The nucleus is deeply indented and shows little condensed chromatin. The cytoplasm contains scattered microtubules and a larger number of neurofilaments than expected in a small cell. There are numerous large dense core vesicles, an unusual organelle consisting of ringlet subunits, and relatively little granular endoplasmic reticulum. A thin axon and a single stout dendritic trunk emanate from the perikaryon. Although the cell body and the dendritic shaft may form either complex contacts with mossy fibres (resembling those previously termed en marron synapses) or simple symmetric synapses with small boutons containing pleomorphic vesicles, most of the synaptic relations are established on the shafts of brush-like branchlets that form at the tip of the dendrite and enter one or two glomeruli. Each branchlet forms an extraordinarily extensive asymmetric synapse with the mossy fibre rosette and the subsynaptic region shows a microfibrillar web connected to the postsynaptic density. In addition to other organelles, the branchlets contain numerous mitochondria and large dense core vesicles. Short, non-synaptic appendages with few cytoplasmic organelles emanated from the cell body, dendritic shaft and branchlets.(ABSTRACT TRUNCATED AT 400 WORDS)

Small granule-containing cells in the heart of the toad (Bufo melanostictus).

This study reveals the presence of small granule-containing cells in the heart of the toad, Bufo melanostictus. The cells were small in size (5-25 microns in diameter) and located in the interatrial septum. The small cells occurred singly or in small clusters within the cardiac ganglia or near the myocardial cells. They were characterized by numerous large dense core vesicles (80-300 nm) in their cytoplasm. With 5-hydroxydopamine treatment, the granularity of the large dense core vesicles was greatly intensified. The large dense core vesicles were variable in size and shape and extended into the long and short processes of the cells. Some of these processes were in close contact with myocardial cells. Other cytoplasmic organelles included rough endoplasmic reticulum, free ribosomes, randomly distributed mitochondria and glycogen particles. Individual cells or cell clusters were usually ensheathed by a thin layer of cytoplasm from sheath cells. The small granule-containing cells in the present study correspond to the catecholamine-containing (SIF) cells described by earlier workers. These cells presumably regulate muscular and ganglionic activities by virtue of their close association.

An intracellular HRP study of the rat globus pallidus. II. Fine structural characteristics and synaptic connections of medially located large GP neurons.

In order to classify the presynaptic elements contacting the principle class of globus pallidus neurons, electron microscopic examination of serial sections made from a medially located large globus pallidus neuron, labeled with intracellular horseradish peroxidase, was undertaken. In addition, the use of labeled and light microscopically reconstructed material allowed us to quantitatively determine the distribution of each bouton type along the soma and dendrites. Six types of presynaptic terminals contacting the labeled cell have been recognized. Type 1 endings, the most numerous (84%), make symmetrical contacts on all portions of the cell, except spines, contain large pleomorphic, and a few large dense-core vesicles. Type 2 endings are filled with small spherical-to-ellipsoidal synaptic vesicles. They make asymmetrical contacts only with higher-order dendrites and account for 12% of synaptic contacts onto the labeled neuron. Type 3 endings are large, contain sparsely distributed large pleomorphic vesicles, and make two symmetrical synapses per bouton, one onto a spine head and the other onto the underlying dendritic shaft. They are infrequent (0.2%), being found only in association with dendritic spines. Type 4 endings contain large pleomorphic synaptic vesicles and no dense-core vesicles. They make symmetrical contacts with the short primary dendrites. Type 5 endings contain a mixture of small clear pleomorphic vesicles and numerous large dense-core vesicles. They contact only the cell body and the short primary dendrites, making up 20% of somatic synaptic contacts but less than 1% of contacts onto dendrites. Type 6 boutons contain oval and flattened synaptic vesicles and establish symmetrical contacts with higher-order dendritic branches and the cell body.

Fine structure of an octopaminergic neuron and its terminals.

The large octopaminergic dorsal unpaired median neuron of the locust that innervates the extensor tibiae muscle, DUMETi, was examined electronmicroscopically. Its soma contains many Golgi complexes apparently making dense-core vesicles similar to those found in peripheral branches and terminals. There are also larger stores of the dense material in the soma, especially near the exit of the principal neurite, that are not in vesicular form. Since the neurons can be penetrated and stimulated by microelectrodes, they form favorable subjects for direct studies of the control of neurosecretion. Preterminal fine branches of the neuron were located in proximal outer bundles of muscle fibers into which they had been traced electrophysiologically. They contain numerous large dense-core vesicles arrayed in rows near microtubules. These fine branches have a thick layer of collagenous connective tissue between the axon and the muscle fiber. Final terminals have varicosities containing many vesicles, lying inside the outer layers of the sarcolemmal complex of muscle fibers. They do not form synaptic structures. Terminals of another DUM neuron, one that innervates the dorsal longitudinal flight muscles (DUMDL), were similar in detail to those of DUMETi. DUMETi swelled about 20-fold in cross-sectional area above a ligature, in a 12-hr period, indicating that there is an extensive centrifugal flow of material in it, and sprouted a branch.

Adrenergic neurons in the spinal cord of the pike (Esox lucius) and their relation to the caudal neurosecretory system.

The lower spinal cord including the caudal neurosecretory system of the pike (Esox lucius) was investigated by means of light and electron microscopy and also with the fluorescence histochemical method of Falck and Hillarp for the visualization of monoamines. A system of perikarya displaying a specific green fluorescence of remarkably high intensity is disclosed in the basal part of the ventrolateral and lateral ependymal lining of the central canal. The area corresponding to the upper half of the urophysis has most cells; their number decreases caudally and cranially. A considerable number of their beaded neurites reach the neurosecretory neurons by different routes but are only occasionally present in the actual neurohemal region. An intensely fluorescent dendritic process is sometimes observed terminating with a bulbous enlargement at the ependymal surface in the central canal. Besides small, electron lucid vesicles in the terminal parts of the axons, the neurons contain numerous large dense-core vesicles which can apparently take up and store 5-hydroxydopa (5-OH-dopa) and 5-hydroxydopamine (5-OH-DA). These neurons are thought to be adrenergic and to contain a primary catecholamine, possibly noradrenaline.