scholarly journals Immunogold electron microscopic demonstration of glutamate and GABA in normal and deafferented cerebellar cortex: correlation between transmitter content and synaptic vesicle size.

1990 ◽  
Vol 38 (12) ◽  
pp. 1767-1777 ◽  
Author(s):  
J Hámori ◽  
J Takács ◽  
P Petrusz

Selective labeling of mossy fiber terminals and parallel fibers was obtained in rat cerebellar cortex by a glutamate antibody produced and characterized by Hepler et al. The high-resolution electron microscopic immunogold demonstration of this amino acid offered the possibility of determining the size and shape of synaptic vesicles in glutamate-positive mossy endings. Mossy terminals that stained with the glutamate antibody formed two distinct populations, one with spherical synaptic vesicles with an average diameter of 34.0 nm (more than 90% of all mossy fiber endings) and one with pleomorphic and smaller synaptic vesicles which had an average diameter of 28.5 nm. We present experimental evidence that the mossy terminals with large round vesicles are of extracerebellar origin, whereas those with small pleomorphic synaptic vesicles are endings of nucleocortical fibers. The presence of two distinct classes of gamma-aminobutyric acid (GABA)-containing axon terminals within cerebellar glomeruli has also been demonstrated; those originating from the cerebellar nuclei contain large (36.2 nm) synaptic vesicles, whereas the majority of GABA-stained axon terminals that are of local (cortical) origin contain small (29.1 nm) synaptic vesicles. It therefore appears that, at least in the case of glutamate and GABA, morphological characterization of the axon terminals based on the size and shape of synaptic vesicles is not a reliable indicator of their functional nature (i.e., whether they are excitatory or inhibitory); convincing evidence for the identity of the transmitter can be obtained only by electron microscopic immunostaining procedures. Our results also suggest the existence of both inhibitory and excitatory feedback from cerebellar nuclei to cerebellar cortex.

1995 ◽  
Vol 43 (3) ◽  
pp. 337-343 ◽  
Author(s):  
H G Gilerovitch ◽  
G A Bishop ◽  
J S King ◽  
R W Burry

Silver enhancement of small gold particles can be used with pre-embedding immunocytochemistry to analyze the distribution of label over cell organelles. We have developed a method that improves tissue morphology, has good penetration of reagents, and allows greater control of silver enhancement of 1.4-nm gold. In this study we analyzed the distribution of glutamic acid decarboxylase (GAD), a synthetic enzyme for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), in the cerebellar nuclei of the mouse. Pre-embedding immunocytochemistry was carried out on brain sections fixed with high concentrations of glutaraldehyde and sodium metabisulfite. After incubations with a monoclonal antibody to GAD and a 1.4-nm NanoGold-labeled secondary antibody, sections were silver-enhanced with N-propyl gallate as a reducing agent and MES as a new buffer system. In the cerebellar nuclei, GAD label was specifically localized in axon terminals over clusters of synaptic vesicles. These terminals formed axosomatic and axodendritic contacts. The majority of GAD-labeled terminals had cytological characteristics indicating their origin from Purkinje cells, which are known to contain GAD.


1991 ◽  
Vol 6 (1) ◽  
pp. 75-92 ◽  
Author(s):  
R. Ranney Mize ◽  
Chang-Jin Jeon ◽  
Omar L. Hamada ◽  
Robert F. Spencer

AbstractThe inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is found in the superior colliculus (SC) of many mammalian species. In cat, several distinct classes of putative GABAergic neuron have been identified using antibodies directed against the neurotransmitter. It is not known whether these classes are found in other species. To study this, we examined the distribution, morphology, ultrastructure, and synaptic organization of GABA immunoreactive neurons in the SC of the Rhesus monkey (Macaca mulatta). Antibody-labeled neurons were distributed throughout the monkey SC, but were most densely concentrated within the zonal and superficial gray layers (32.5% of the total). These neurons were all small cells ranging from 6.6–16.3 μm in average diameter, and had granule, pyriform, and horizontal morphologies. Four types of labeled profile were identified in single ultrathin sections with the electron microscope. Presynaptic dendrites (PSDs) contained pleomorphic vesicles, received synaptic input from unlabeled axon terminals, and sometimes formed symmeytric synaptic contacts with postsynaptic profiles. Two subtypes were found. One type contained loose accumulations of synaptic vesicles throughout the profile and had a distinctive varicose shape. The other type contained small discrete clusters of synaptic vesicles near the site of synaptic apposition. The former were much more common. Profiles with typical axon terminal morphology were also found. These profiles usually contained numerous flattened vesicles and formed symmetric synapses with postsynaptic profiles, both dendrites and cell bodies. Some conventional dendrites and myelinated axons were also labeled. Serial ultrathin section reconstructions revealed that PSDs formed complex synaptic relationships with other elements. Retinal terminals, identified by their characteristic pale mitochondria, established synaptic contacts with both types of PSD. These PSDs also established contact with each other, providing a possible anatomical substrate for disinhibition. We conclude that the monkey SC has multiple GABAergic cell types, similar to those found in cat may represent an organization common to both mammals and some other vertebrate species. The circuitry established by these cell types may provide a mechanism for disinhibition as well as inhibition in the mammalian SC.


An electron microscopic study has been made of the normal ventrolateral and centre-median nuclei of the thalamus of the monkey and, in experimental material, of the mode of termination in the nuclei of afferent fibres from the motor cortex, the globus pallidus and the deep cerebellar nuclei. There are striking similarities but also a few subtle differences in the ultrastructure of the centre-median and ventrolateral nuclei. Three classes of cell are present: a large multipolar cell with much cytoplasm filled with many organelles is probably the relay cell; a small fusiform cell with a thin rim of cytoplasm and light vacuolated mitochondria, which may contain discoid vesicles, gives rise to P profiles and axon initial segments; the third type is intermediate between the other two in size and other features. There are four types of vesicle-containing synaptic profiles. The most frequent are SR axon terminals, which are small, with spherical synaptic vesicles and are pre-synaptic at asymmetrical contacts with small and medium dendrites and their spines and P profiles; SR terminals are found mainly in the interglomerular neuropil. LR terminals, the largest synaptic profiles, are found in all glomeruli; they contain many round synaptic vesicles and form numerous asymmetrical synapses, being presynaptic to the main dendrite and all its spines and many of the P profiles in a glomerulus. P profiles are irregular pale processes, which occur in considerable numbers outside and within glomeruli (70% of intra-glomerular profiles) and contain pleomorphic synaptic vesicles which are discoid. They arise from tiny unmyelinated profiles and they may have synaptic interaction in several glomeruli. Larger processes, similar in character to proximal dendrites, but with discoid vesicles and synaptic features like those of smaller P profiles are also found, and may be in continuity with a cell soma. P profiles are post-synaptic to LR, SR and F axons, both pre- and postsynaptic to other P profiles and pre-synaptic to conventional dendrites; there are reciprocal synapses between pairs of P profiles. F axons, the least common profiles, are usually extra-glomerular and synapse with proximal dendrites and cell somata; they contain cylindrical synaptic vesicles and are pre-synaptic to P profiles and dendrites at symmetrical synapses. Cell somata in the centre-median nucleus possess spines, and in both nuclei dendrites have spines, both in glomeruli and the neuropil. Multivesicular bodies are frequently present in the parent dendrite subjacent to the spine. Spines in the neuropil are post-synaptic to SR and P profiles and, in the centre-median nucleus, also to F axons. Some spines appear to be post-synaptic only to F axons. At least half the extraglomerular spines are associated with two synapses, a dyadic arrangement with one synapse on the spine and one on the parent dendrite close to the base of the spine, while many dyads have only a single presynaptic profile. Some spines have a more complex synaptology including serial synapses and triads. In glomeruli, spines are always post-synaptic to the LR bouton and always dyadic as the LR terminal also contacts the dendritic shaft close to the base of the spine. Spines account for half the contacts between an LR terminal and the main dendrite in a glomerulus. Glomeruli are found in both nuclei and serial sections have shown the multiplicity of profiles and the complexity of synaptic organization within them. Each glomerulus contains one LR bouton, one main dendrite, and a large number of P profiles, with an occasional SR or F axon terminal at the periphery of the aggregation. The dendrites are of medium calibre, often with several spines, around which the other profiles are situated. An analysis is presented of the profiles and synaptic arrangements in thirty glomeruli from the centre-median and ventrolateral nuclei, with a reconstruction of one large glomerulus. The LR bouton makes numerous synaptic contacts with the main dendrite, half the contacts being upon spines, and a considerable number with P profiles. The latter also have many synapses, being approximately equally pre-synaptic to P profiles and the main dendrites. Various specific types of synaptic array are present within the glomeruli: serial synapses; sequential arrays, where the third profile of a serial synapse is again pre-synaptic; reciprocal synapses; triads which are similar to serial synapses but with an additional contact, profile 1 also being pre-synaptic to profile 3. The complexity of the glomerular synaptology is accentuated by the overlapping of several different types of synaptic array in a given glomerulus. Afferent fibres from the motor cortex and globus pallidus terminate in both nuclei as SR boutons, and in the ventrolateral nucleus the mode of termination of both groups of fibres is very similar. In the centre-median nucleus the terminals of fibres from the cortex have a higher proportion of multisynaptic contacts, are more frequently pre-synaptic to P profiles, and the dendrites upon which they end are less often in receipt of other synapses. The termination of fibres from the globus pallidus in the centre-median nucleus differs from terminals of fibres from the cortex and globus pallidus in the ventrolateral nucleus in being pre-synaptic to fewer P profiles. When the somatic sensory cortex was destroyed as well as the motor area degenerating axon terminals were present in the ventroposterior nucleus; their mode of termination was similar to those in the ventrolateral nucleus, but they were far greater in number. Axons from the cerebellar deep nuclei end in the ventrolateral nucleus as LR boutons within glomeruli. There was no evidence of degeneration in the centre-median nucleus after damage of the cerebellum.


The nature and immediate postoperative course of experimental degeneration of axon terminals have been studied in the somatic sensory cortex. The first somatic sensory area was examined at intervals of 2 to 6 days following lesions in the thalamus, opposite cortex or ipsilateral second somatic sensory area. There is a characteristic sequence of degenerative changes which affects the terminals of each of the afferent fibre systems studied. This commences as a simple, though marked, increase in electron density of the axoplasm with no loss of synaptic vesicles and little alteration in the size or shape of the terminal. Following this, there is a progressive loss of vesicles and disruption of the mitochondria with shrinkage of the terminal and its compression, invasion and fragmentation by astroglial processes. There is evidence that many fragments are phagocytosed by the invading astroglia but a thin sliver always remains attached at the synaptic contact zone. Within the range of survival periods used, no changes affect the synaptic region nor the postsynaptic profile and if the latter is a dendritic spine, it is not detached from the parent dendrite. Changes in degenerating axons are similar, except that the largest thalamo-cortical fibres show a stage of neurofilamentous hyperplasia. In the cortex at a distance from the lesion only smaller astrocytic processes are involved in breaking down the degenerating products; close to a lesion, however, all astrocytic processes and perikarya become involved and many atypical glial cells which are difficult to classify as astrocytes or oligodendrocytes become visible; the vascular pericytes also display large heterogeneous dense bodies and other inclusions.


Author(s):  
Werner J. Niklowitz

In a previous publication we have shown a useful application of phase microscopy in cytological studies of nervous tissue. Using this method it has been possible to identify the axon terminals of the hippocampal mossy fiber layer with phase microscopy and to corroborate its identification with electron microscopy using the same material Further combined phase and electron microscopic investigations of this specific region of the hippocampus give additional insight into the possible chemical nature of the transmitters in the mossy fiber boutons in rabbits.


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