Selective neuronal vulnerability during experimental scrapie infection: insights from an ultrastructural investigation.

The goal was to test whether all neurons are equally susceptible to degeneration in response to PrP(Sc) scrapie infection. We tested this by immunogold GABA labeling. Our ultrastructural results indicates that GABAergic neurons are less vulnerable than other neuronal populations. This conclusion is supported by our findings: (1) reversal of the normal ratio of non-GABAergic to GABAergic neurons in the terminal stages, which implies that non-GABAergic neurons degenerated earlier, and (2) that the degeneration of GABAergic neurons occurs late in the disease after reactive astrogliosis, a response to nerve cell death.

Electron microscopic imaging of multiple markers in glutaraldehyde fixed CNS tissue of Macaca fascicularis: maximizing information from a single experimental animal.

Sensory and motor pathways in the central nervous system (CNS) of macaque monkeys were visualized by anterograde or retrograde axonal transport of wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) reacted with the chromagen tetramethylbenzidine (TMB), or by the use of anterograde degeneration after specific ablation lesions. To maximize information from each animal we combined the results of the anterograde and retrograde axonal transport with several pre- and post-embedding markers at both the light and electron microscopic levels while maintaining good preservation of tissue. Pre-embedding techniques included those for cytochrome oxidase activity and the calcium-binding proteins calbindin D-28k and parvalbumin. Post-embedding techniques included immunocytochemistry for gamma-aminobutyric acid (GABA) or other amino acid neurotransmitters. We believe that the methods described here provide superior tissue preservation, thus permitting a more detailed analysis of tissue prepared after experiments concerned with neural circuitry.

Analysis of gamma-aminobutyric acidergic synaptic contacts in the thalamic reticular nucleus of the monkey.

Gamma-aminobutyric acidergic (GABAergic) neurons in the thalamic reticular nucleus (TRN) spontaneously generate a synchronous bursting rhythm during slow-wave sleep in most mammals. A previous study at the electron microscopic level in cat anterior TRN has suggested that synchronous bursting activity could result from the large number of presumably GABAergic dendrodendritic synaptic contacts. However, little is known about the synaptology of the monkey thalamic reticular nucleus and whether it contains dendrodendritic contacts. To address this issue, we examined tissue obtained from Macaca fascicularis that was prepared for electron microscopy using postembedding techniques to demonstrate GABA immunoreactivity. Examination of the anterior (motor) and posterior (somatosensory) portions of the TRN disclosed the following: The majority of synaptic contacts (87.5% of 958) were formed by axon terminals showing no GABA immunoreactivity and making asymmetric synaptic contacts on dendrites or cell bodies. A further 6.4% of synaptic contacts was composed of GABA-immunoreactive presynaptic terminals making symmetric contacts with the dendrites of TRN neurons. The majority resembled the pleomorphic vesicle containing F-terminals seen in the dorsal thalamus and known to originate from axons of TRN. A subset or possible second class did not resemble any previously described class of GABA-immunoreactive terminals in the TRN. Both classes of these terminals making symmetric contacts may originate wholly or partially within the nucleus. There was one dendrodendritic synaptic contact and only a small number (3.2%) of axodendritic contacts with synaptic vesicles visible both pre- and postsynaptically. We conclude that dendrodendritic contacts are probably not responsible for the synchronized bursting neuronal activity seen in the slow-wave sleep of monkeys, and that, if TRN neurons are coupled synaptically, the most likely mechanism is through the synapses formed by recurrent axon collaterals of TRN neurons onto TRN dendrites.

Inhibitory synaptic input to identified rubrospinal neurons in Macaca fascicularis: an electron microscopic study using a combined immuno-GABA-gold technique and the retrograde transport of WGA-HRP.

Rubrospinal neurons of the magnocellular division of the red nucleus of Macaca fascicularis were retrogradely labeled following spinal cord microinjections of wheat germ agglutinin-horseradish peroxidase, as demonstrated by the chromagen tetramethylbenzidine, identifying the mesencephalic cells of origin of this descending motor pathway. The tissue was processed for electron microscopy and subsequently tested on the electron microscope grid for immunoreactivity of gamma aminobutyric acid (GABA) in presumed local circuit neuronal somata, in dendrites, and in axonal terminals. Results demonstrate the presence of retrogradely labeled rubrospinal neurons of medium and large diameters (30-90 microns) and immunoreactive neurons of small size (less than 20 microns in diameter) within the nucleus. In addition, there are substantial numbers of GABAergic, presumably inhibitory, synaptic structures contacting somata and primary, medium, and small sized dendrites, as well as spineheads of rubrospinal neurons. The immunoreactive presynaptic profiles exhibit two different morphological appearances: one axonal and the other dendritic. Axonal terminals contain densely packed pleomorphic to flattened vesicles and form primarily symmetrical synapses with somata and all regions of the dendritic arbor. GABAergic profiles resembling presynaptic dendrites (PSDs) are also present. These profiles possess scattered flattened to pleomorphic synaptic vesicles in a translucent cytoplasm and are often postsynaptic to axonal terminals of unknown origin, or to GABAergic profiles. GABAergic local circuit neurons (LCNs), the neurites of which remain within the confines of the nucleus, appear to be contacted primarily by cortical and cerebellar afferents. These LCNs may or may not possess axons and thus may represent both the source of the GABAergic axonal terminals as well as that of the PSDs. Inhibitory afferents from other sources, such as the mesencephalic reticular formation, may also account for GABAergic terminals involved in this inhibition. We propose that the level of excitability of rubrospinal neurons and their subsequent activation of spinal motor neurons and interneurons is significantly regulated by the local circuit GABAergic inhibitory interneuronal population of the nucleus proper and probably by axons entering the nucleus from an extranuclear source.

Red nucleus of Macaca fascicularis: an electron microscopic study of its synaptic organization.

The parvicellular and magnocellular divisions of the red nucleus of the old world monkey, Macaca fascicularis, were analyzed at an electron microscopic level to examine the morphology of the synaptic profiles terminating on rubral neurons and to categorize them by their individual characteristics. The parvicellular division, or anterior two-thirds of the nucleus, is composed of small (10-15 microns) and medium-size (20-30 microns) cells, which are uniformly distributed with high packing density throughout this portion of the nucleus. These cells have invaginated nuclei and are often indented by blood vessels and glial cell somata (satellite cells) that lie in close proximity. The magnocellular portion, occupying the caudal one-third of the nucleus, is composed of an additional population of large cells, ranging from 50-90 microns in diameter, which often contain prominent lipofuscin granules and are frequently indented by blood vessels. Satellite glial cells are not a prominent feature in the magnocellularis portion of the nucleus. The large cells are separated one from the other by fields of myelinated axons either coursing through the nucleus or projecting to and from the nucleus itself. Although the divisions of the nucleus in the Macaca fascicularis are spatially distinct, each possesses a morphological similarity in regard to the categories of synaptic profiles seen at the electron microscopic level. These synaptic profiles are classified as follows: large terminals containing numerous, predominantly rounded vesicles (LR), which can often be seen to form the central profile in a synaptic glomerular arrangement; terminals of similar size with predominantly rounded vesicles but with a pale axoplasmic matrix (LRP); small profiles with rounded vesicles (SR); profiles containing granular dense-cored vesicles (DCV); profiles with numerous flattened vesicles (F); profiles containing pleomorphic vesicles (PL), some of which can be interpreted as presynaptic dendrites (PSD) because they are seen to be postsynaptic and contain ribosomes; and profiles with rounded synaptic vesicles, which are associated with subsynaptic Taxi bodies (T). Most of the various synaptic profile types were found to have similar distributions on the dendritic arbors of rubral neurons in both divisions of the nucleus. However, the LRP-type terminal predominates on the cell bodies and proximal dendrites of the large neurons in magnocellularis. Unlike other regions in the nervous system, F type terminals are rarely seen to contact neuronal somata. This study provides a basis for future experimental studies of afferents to the nucleus in this species.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructural evidence for direct monosynaptic rubrospinal connections to motoneurons in Macaca mulatta.

The magnocellularis division of the red nucleus of the Macaca mulatta, a midbrain structure involved in processing motor information, is known by light microscopic analysis to project, via the rubrospinal tract, to the contralateral intermediate horn of the spinal cord. Physiological studies, however, provide additional evidence for direct monosynaptic connections to motoneurons subserving distal musculature. This electron microscopic study demonstrates, by analyzing the anterograde transport of 5% wheatgerm agglutinin-horseradish peroxidase injected into the red nucleus, the presence of labeled terminals synapsing upon somata and proximal dendrites of motoneurons in the lateral portion of the ventral horn of the cervical enlargement of the spinal cord. We conclude that this anatomical evidence confirms the presence of direct monosynaptic connections to spinal motoneurons in the primate.

The use of specimen tilt in transmission electron microscopy of the central nervous system.

Thin sections of nervous tissue were viewed at different tilt angles using a transmission electron microscope equipped with a eucentric goniometer stage. In a comparison study of various degrees of tilt, one can observe additional morphological features within synaptic profiles, define subsynaptic structures such as Taxi-bodies, and clearly see the crystalline formation of cytochemical tracers. This study demonstrates the value of tilting thin-sections in the analysis of synapses and other biological material at the ultrastructural level.

Non-myelinated axons are rare in the medullary pyramids of the macaque monkey.

Previous electron microscopic studies of the medullary pyramids have concluded that non-myelinated axons constitute about 30-60% of all axons in the pyramid of the rat, and about 8-15% in the cat and monkey. Physiological studies of pyramidal tract axons have not found fibers conducting in the range predicted for non-myelinated axons, less than 1 m/s. This present study of the primate pyramid demonstrates that most of the profiles which could be interpreted as being non-myelinated axons when viewed in cross-section, are actually astroglial processes when examined in longitudinal section. We conclude that non-myelinated axons constitute less than 1% of the pyramidal tract axons in the old world adult primate.