Pleomorphic Agranular Vesicles Research Articles

GABA and glutamate-like immunoreactivity at synapses on depressor motorneurones of the leg of the crayfish, Procambarus clarkii.

To investigate their synaptic relationships, depressor motorneurones of the crayfish leg were impaled with microelectrodes, intracellularly injected with horseradish peroxidase, and prepared for electron microscopy. Post-embedding immunogold labelling with antibodies against gamma-aminobutyric acid (GABA) or glutamate was carried out either alone or together on the same section and allowed the identification of three classes of input synapses: 51% were immunoreactive for glutamate and contained round agranular vesicles, 31% were immunoreactive for GABA and contained pleomorphic agranular vesicles, and the remainder were immunoreactive for neither and also predominantly contained pleomorphic agranular vesicles. Output synapses were abundant in some of the motorneurones but were not seen in others, suggesting that members of the motor pool differ in their connectivity.

Glutamate is a transmitter that mediates inhibition at the rectifying electrical motor giant synapse in the crayfish.

Spike transmission at the electrical synapse between the giant fibres (GFs) and motor giant neurone (MoG) in the crayfish can be blocked by depolarising postsynaptic chemical inhibition, which has previously been shown to be mediated in part by gamma-aminobutyric acid (GABA). The authors show that glutamate applied to the synaptic region of the MoG mimics the depolarisation of the chemical input and can also block spike transmission from the GFs. The glutamate induces an inward current mediated by a conductance increase that is 30-40% of that induced by GABA and that is blocked substantially by picrotoxin. Glutamate has no effect on the presynaptic GF, and the effects in the MoG are maintained in the presence of cadmium, indicating that the glutamate is acting directly on the MoG. Both GABA and glutamate have similar effects on the cell body, where the response reverses 10-20 mV positive to resting potential, is dependent on chloride concentration, and is inhibited by picrotoxin. Joint application of glutamate and GABA induces a nonadditive current under voltage clamp, suggesting that the transmitters can activate the same postsynaptic receptors. Immunocytochemical staining shows that, whereas some synaptic profiles impinging on the MoG contain pleomorphic agranular vesicles and are immunoreactive to GABA and not glutamate (as previously reported), there are at least as many other profiles that contain round, agranular vesicles and that are immunoreactive to glutamate and not to GABA. Thus, the authors conclude that some of the interneurones mediating inhibition of the electrical synapse use glutamate as their neurotransmitter.

The neuronal organization of the supracapsular part of the stria terminalis in the rat: The dorsal component of the extended amygdala

In the present normal anatomical light and electron microscopic study in the rat, histochemical (Nissl, Timm, Golgi) or immunocytochemical (microtubule-associated protein type 2, glutamate decar boxylase, glutamate receptor subunit 1, synaptophysin) stains were used to analyse neurons embedded within the stria terminalis and their associated neuropil. These cells are closely related to the bed nucleus of the stria terminalis and the centromedial amygdala, and have been termed the “supracapsular part of the bed nucleus of the stria terminalis”. The largest part of this neuronal complex is located in the ventrolateral part of the stria, where it appears as a round or oval “lateral pocket” in virtually any type of light microscopic preparation because of its collection of neuronal cell bodies and dense neuropil, in addition to a lacework of unmyelinated axons. A much smaller but still distinct “medial pocket” is located in the medial corner of the stria. The large lateral subdivision of the supracapsular stria terminalis is directly continuous with the lateral bed nucleus of the stria terminalis and extends to the central amygdaloid nucleus, containing a column of neurons that is only broken up into cell clusters at the most caudal levels of the stria as it drops vertically toward the amygdala. The considerably smaller medial subdivision appears, in turn, to be directly continuous with the medial part of the bed nucleus of the stria terminalis. The medial column tapers off more rapidly than the lateral part, so that as the middle levels are approached, only small interrupted clusters of cells are seen. Solitary neurons can also be found in practically every part of the stria terminalis except among the ventrally located axons of the commissural component. Most of the neurons are small to medium in size, as viewed in transverse sections of the stria, but larger neurons are also encountered. In sections parallel to the stria, many neurons are fusiform in appearance. The dendrites are often aligned in a longitudinal fashion; many of the dendrites related to the cells in the lateral pocket are moderately to densely spined, whereas those in the medial pocket are more sparsely spined. The neuropil in both the lateral and medial pockets is characterized by boutons, bundles of unmyelinated axons, and dendrites. Based on their vesicle content, the boutons are divided into three major types: (A) round or slightly oval, agranular vesicles of uniform size; (B) pleomorphic, agranular vesicles, many of which are flattened; and (C) pleomorphic agranular vesicles, some of which are considerably larger than the ones in type B boutons. Type A boutons establish contacts with both dendritic spines and shafts, whereas types B and C usually contact dendritic shafts and sometimes somata. These synaptic components are similar to those described earlier for the central and medial amygdaloid nuclei. Overall, our results support the concentration advanced in 1923 by Johnston [ J. comp. Neurol. 35, 337–481] that the cells accompanying the stria terminalis are interconnecting columns of a macrostructure encompassing the bed nucleus of the stria terminalis and centromedial amygdala. More recently, it has been appreciated that columns of neurons below the globus pallidus also belong to this macrostructure [Alheid G. F. et al. (1995) In The Rat Nervous System, 2nd edn, pp. 495–578, Academic, San Diego; de Olmos J. S. et al. (1985) In The Rat Nervous System, pp. 223–334, Academic, Sydney], which has been named the “extended amygdala”.

Vasoactive intestinal peptide in the hamster seminal vesicle: Distribution, binding sites and possible functions

The presence and functional role of vasoactive intestinal peptide in the hamster seminal vesicle were studied by a combination of structural and functional approaches. The use of an immuno-fluorescence staining technique in both cryostat sections and whole-mount preparations revealed that vasoactive intestinal peptide-immunoreactive nerve fibres were mainly localized in the lamina propria of the mucosal layer. In double-stained preparations, vasoactive intestinal peptide immunoreactivity was found to be localized in nerves also containing acetylcholinesterase activity. At the ultrastructural level, the use of an immunogold staining method showed that vasoactive intestinal peptide immunoreactivity occurred in large granular vesicles (80–150 nm in diameter) in nerve varicosities which also contained small pleomorphic agranular vesicles. In order to evaluate the anatomical distribution of vasoactive intestinal peptide binding sites in the seminal vesicle, we have utilized an in vitro receptor autoradiographic technique. Vasoactive intestinal peptide binding sites were localized in the basal region of the secretory epithelium, in the muscle layer and in the wall of blood vessels. In vitro incorporation of [ 3H] l-leucine into protein by tissue slices revealed that vasoactive intestinal peptide (1 μM) significantly increases the amount of released protein. Vasoactive intestinal peptide (0.1–1 μM) did not affect the resting tension of the muscle but significantly inhibited the increase in muscle tension induced by carbachol. Atropine prevented the effect of carbachol, indicating that the latter is mediated by muscarinic receptors. Our results suggest that in the hamster seminal vesicle, vasoactive intestinal peptide is involved in the modulation of muscarinic function and in the control of secretion.

Fine structure and synaptic architecture of HRP‐labelled primary afferent terminations in lamina IIi of the rat dorsal horn

The fine structure and synaptic architecture of the afferent terminations in dorsal horn lamina II are studied using a combined light and electron microscopic procedure after anterograde labelling with horseradish peroxidase. Vibratome parasagittal sections, stained with heavy metal intensified diaminobenzidine after tracer application to the dorsal roots, were flat-embedded in Epon. The five types of labelled terminal arbors occurring in lamina IIi (Cruz et al., '87: J. Comp. Neurol. 261:221-236) were drawn and relocated in 5-microns sections cut serially from the thick sections. Ultrathin sections were then cut from the 5-microns sections so that the terminal fibers and swellings observed in the light microscope could be traced in the electron microscope. The flame-shaped arbors arose from fine myelinated stem fibers. Terminal strands generated large oval central terminals of type II synaptic glomeruli (CII), which established frequent axoaxonal contacts. Similar terminals have been labelled in the cat after tracer injections into hair-follicle fibers (Réthelyi et al., '82: J. Comp. Neurol. 207:381-393). The other four plexuses arose from unmyelinated stem fibers. The swarms of ultrafine boutons consisted of extremely thin terminal fibers generating very small, round, or polygonal glomerular terminals containing tightly packed agranular synaptic vesicles of variable size and one mitochondrion at best. The terminal strands of the bouquet plexus bore long and scalloped central varicosities of type I synaptic glomeruli (CI) with pleomorphic agranular vesicles and a relative abundance of dendroaxonal contacts. These features, together with the location in dorsal lamina IIi, suggest their belonging to the fluoride resistant acid phosphatase (FRAP)-reactive population. The boutons of the undulating fibers and those of the lateral plexus were, like those of the bouquets, scalloped and elongated rostrocaudally (CI), but contained a few large granular vesicles. The occurrence of the swarm, undulating, and lateral plexuses in ventral lamina IIi, which seems to lack FRAP or peptidergic terminals, suggests an origin from other, still unidentified neurochemical populations of fine primary afferents.

5-Hydroxytryptamine immunoreactive varicosities in the lamprey spinal cord have no synaptic specializations - an ultrastructural study

The distribution and fine structure of 5-hydroxytryptamine (5-HT) immunoreactive cell bodies and axonal varicosities have been studied in the lamprey spinal cord, using the peroxidase-antiperoxidase (PAP) immunohistochemical technique and subsequent analysis of ultrathin serial sections. Immunostained cell bodies were found in the ventral spinal cord close to the central canal. Immunostained varicosities were found throughout the spinal cord with the highest density in the ventromedial plexus and the dorsal horn. Only large granular vesicles could be clearly distinguished in immunostained cell bodies and varicosities, but it was concluded based on a comparison with unstained normal tissue that these boutons also contained small, pleomorphic agranular vesicles. Immunoreactive varicosities were studied in the ventromedial plexus, the dorsal horn, the dorsal column, the dorsolateral and ventrolateral funiculi and the grey matter. No morphological differences could be observed between varicosities in the different loci. The varicosities were in no case seen to make synaptic contact with surrounding neuronal elements, even when followed through serial sections. Consequently, 5-HT released from boutons in all parts of the spinal cord could be expected to act on 5-HT receptors located on nearby as well as distant receptors.

Ultrastructure of the pineal gland of the monkey, Macaca fascicularis, with special reference to the presence of synaptic junctions on pinealocytes

The present study described the normal ultrastructure of the monkey pineal gland. The gland was composed of the principal pinealocytes, intramural neurons and glial cells. The nucleus of the pinealocytes was deeply infolded with evenly distributed chromatin materials. The abundant cytoplasm was rich in organelles including the well-developed Golgi apparatuses, multivesicular bodies, dense-cored vesicles and widely scattered free and polyribosomes. A variety of axon terminals was observed and the majority of them contained pleomorphic agranular vesicles with a few large dense-cored vesicles. A few terminals showed flattened vesicles or small dense cored vesicles. Some of the axon terminals formed synaptic contacts with the cell bodies of pinealocytes. These synapses were mainly concentrated in the posterior third of the gland. The occasional intramural neurons observed were postsynaptic to axon terminals containing round agranular vesicles. The sources of the nerve fibres and terminals forming synaptic junctions with pinealocytes and intramural neurons were discussed.

Antibodies against GABA and glutamate label neurons with morphologically distinct synaptic vesicles in the locust central nervous system

Antibodies raised against GABA and glutamate were used to stain sections through locust thoracic ganglia for light and electron microscopy. Using a peroxidase-antiperoxidase method for light microscopy, the GABA antibody was shown to label inhibitory motor neurons thought to use GABA as their neurotransmitter, and the glutamate antibody to label excitatory motor neurons thought to use glutamate. An immunogold method was used to reveal labelled neuropilar processes in the electron microscope. Each antibody specifically labels a particular population of processes. With the GABA antibody, labelling is equally clear whether the processes concerned contain synaptic vesicles or not and is strongly contrasted against very low background levels. With the glutamate antibody, most processes show some affinity for the antibody, probably reflecting the presence of metabolic glutamate, however one population can be clearly distinguished by the presence of a much greater density of gold particles over synaptic vesicles. In the locust it appears, therefore, that the antibody can distinguish clearly between the metabolic and neurotransmitter pools of glutamate. It has been proposed that synaptic vesicles in GABAergic neurons have a different shape to those in glutamatergic neurons. This was supported by the electron microscope immunocytochemistry. Those showing GABA-like immunoreactivity contain predominantly pleomorphic agranular vesicles approximately 21 × 30 nm in diameter. Those showing glutamate-like immunoreactivity contain round agranular vesicles of about 38 nm in diameter. The GABA antibody appears to label all processes containing pleomorphic agranular vesicles. By contast, some processes containing round agranular vesicles are not labelled by the glutamate antibody, even though the vesicles they contain are statistically identical in size to those in labelled profiles. With neither antibody was the labelling of glial cells greater than the background level.