The contribution of Camillo Golgi to our understanding of the structure of the nervous system.

A hundred years ago Camillo Golgi and Santiago Ramón y Cajal were awarded the Nobel Prize for Physiology or Medicine for their investigations on the structure of the nervous system. The work of Cajal is universally acknowledged, whereas Golgi's contribution is less well known. This article reviews the main achievements of Golgi in that field. In addition to Golgi's most important results, the errors he made in interpreting his own findings are examined. These errors contributed notably to a widespread neglect and underestimation of his important contributions to our understanding of the structure of the nervous system.

The perineuronal glial tissue of spinal ganglia. Quantitative changes in the rabbit from youth to extremely advanced age.

The volumes of the nerve cell bodies and those of the enveloping satellite cell sheaths from spinal ganglia were determined by morphometric methods applied to electron micrographs in young, adult, old and very old rabbits. The mean volume of the nerve cell bodies increased progressively with age; this is probably related to the increase with age of the body size of the rabbits studied. The mean volume of the satellite cell sheaths did not differ significantly in young, adult and old animals, but was significantly smaller in very old animals. It is extremely unlikely that this marked reduction in the volume of the satellite cell sheath is the result of a pathological process. The mean value of the volume ratio between the satellite cell sheaths and the related nerve cell bodies did not differ significantly in young and adult animals, but was significantly smaller in old and very old animals. This ratio was particularly low in very old animals. Our analysis showed that in each age group the volume of the satellite cell sheath is linearly related to the volume of the related nerve cell body. This result suggests that in rabbit spinal ganglia the quantitative relations between glial and nervous tissue are tightly controlled throughout life. It is suggested that ganglionic neurons release signals to influence and control the volume of their associated glial tissue. Since satellite cells have important support roles for the neurons they surround, it is likely that the marked reduction in the volume of perineuronal sheaths in the extremely advanced age is accompanied by a reduction of those roles, with negative consequences for neuronal activity.

A study of mitochondria in spinal ganglion neurons during life: quantitative changes from youth to extremely advanced age.

In view of the central role that mitochondria are thought to play in the ageing process, we investigated changes in mitochondria of spinal ganglion neurons in rabbits aged 1, 3.6, 6.7, and 8.8 years (the latter extremely old). Mitochondrial size increased significantly with age, while mitochondrial structure did not change. The total volume of mitochondria within the perikaryon did not change significantly during life. This indicates that in these neurons mitochondrial degradation was completely compensated by the production of new mitochondria even in the extremely advanced age. We also found that the mean volume of neuronal perikaryon increased markedly with age, so that the mean percentage of perikaryal volume occupied by mitochondria decreased significantly with a difference of about 33% between the youngest and the oldest animals. This decrease is only in very small part due to lipofuscin accumulation, so that the ratio of the total volume of mitochondria to the volume of functionally active cytoplasm decreased with age. The mitochondria of the neurons studied seem therefore unable to adapt their total volume to the volume of functionally active cytoplasm, that increases with age. This result is consistent with the observation that the neurons of old animals have a reduced ability to respond to high energy demands.

Aging is associated with an increase in dye coupling and in gap junction number in satellite glial cells of murine dorsal root ganglia.

Glial cells in both central and peripheral nervous systems are connected by gap junctions, which allow electrical and metabolic coupling between them. In spite of the great current interest in aging of the nervous system, the effect of aging on glial cell coupling received little attention. We examined coupling between satellite glial cells in murine dorsal root ganglia using the dye coupling technique and electron microscopy. We studied mice at ages of postnatal 90-730 days. Dye coupling incidence between satellite glial cells associated with a single neuron increased from 24.2% at postnatal day 90 to 50.5% at postnatal day 730. Dye coupling between satellite glial cells that are in contact with two or more neurons increased from 2.7% at postnatal day 90 to 18.6% at postnatal day 730 (P<0.05). Examination of the ganglia with the electron microscope showed that the number of gap junctions per 100 microm2 of surface area of satellite glial cells increased from 0.22 at postnatal day 90 to 1.56 at postnatal day 730 (P<0.01). The mean length of individual gap junctions did not change with age. These results provide strong evidence for an increase of functional coupling between satellite glial cells during life. This increase is apparently due to an increase in the total area of the system of gap junctions connecting these cells.

The Golgi apparatus of spinal ganglion neurons: quantitative changes with aging.

The Golgi apparatus of spinal ganglion neurons was studied in 1, 3.6, 6.7, and 8.8-year-old rabbits. The structure of this organelle did not differ in the four age groups examined. While the mean volume of the neuronal perikaryon increased progressively with age, the total volume of the Golgi apparatus remained stable throughout life. As a consequence, the mean percentage of perikaryal volume occupied by this organelle decreased significantly with age. Since the percentage of perikaryal volume occupied by lipofuscin remained at low levels throughout life, the ratio of the total volume of the Golgi apparatus to the functionally active volume of cytoplasm decreased with age. It is possible that this decrease is related to the reduction in neuronal metabolism that occurs in senescence. The age-related quantitative changes in the Golgi apparatus were very similar in large light and in small dark neurons. Finally, neither fragmentation, nor peripheral displacement of the Golgi apparatus was observed with advancing age.

Increase in number of the gap junctions between satellite neuroglial cells during lifetime: an ultrastructural study in rabbit spinal ganglia from youth to extremely advanced age.

This study investigated quantitative aspects of the gap junctions between satellite neuroglial cells that envelope the spinal ganglion neurons in rabbits aged 1 year (young), 3.6 years (adult), 6.7 years (old), and 8.8 years (very old). Both the total number of gap junctions present in 30,000 microm2 of surface area occupied by perineuronal satellite cells, and the density of these junctions increased throughout life, including the extremely advanced age. By contrast, the mean length of individual gap junctions did not change with age. Thus, the junctional system which provides morphological support for the metabolic cooperation between satellite cells in rabbit spinal ganglia becomes more extensive as the age of the animal increases. These results support the hypothesis that the gap junctions between perineuronal satellite cells are involved in the spatial buffering of extracellular K+ and in neuroprotection.

Gap junctions between perineuronal satellite cells increase in number with age in rabbit spinal ganglia.

The gap junctions between perineuronal satellite cells were studied in the spinal ganglia of 12, 42, and 79-month-old rabbits. The mean number of gap junctions per 100 microm2 of surface of the section occupied by satellite cells was significantly greater in old rabbits than young adults. Since the mean length of individual gap junctions did not change with age, the increase in number of gap junctions cannot be due to fragmentation of pre-existing gap junctions but is very likely due to the formation of new gap junctions. The increase in number of gap junctions cannot be related to an increase in number of perineuronal satellite cells since the mean number of these cells is significantly smaller in aged rabbits than in young adults. It is suggested that the increase in number of gap junctions with age may enhance the suggested neuroprotective role of satellite cells towards ganglionic neurons. The present findings, together with previous observations, suggest that the gap junctions between perineuronal satellite cells are dynamic structures, able to adapt to varying neuronal demands and varying environmental conditions.

Ratios between number of neuroglial cells and number and volume of nerve cells in the spinal ganglia of two species of reptiles and three species of mammals.

We studied the ratios between number of neuroglial (=satellite) cells and number and volume of neurons with which they are associated in the spinal ganglia of two species of reptiles (lizard and gecko) and three species of mammals (mouse, rat, and rabbit). In all five species, we found that the number of satellite cells associated with a nerve cell body increased with increasing volume of the latter. This result shows that there is a quantitative balance between neuroglia and nerve tissue in spinal ganglia. This balance seems to be maintained by a tight regulation of the number of satellite cells. We also found that the mean volume of nerve cell body corresponding to a satellite cell was lower for small neurons than for large ones. Since satellite cells metabolically support spinal ganglion neurons, the metabolic needs of small neurons are better satisfied than those of large ones. For a nerve cell body of a given size, the number of associated satellite cells did not differ between the lizard and gecko, nor between the mouse, rat, and rabbit. However, this number was significantly smaller in the reptiles than in the mammals. This result could be explained by the lower metabolic rate in the nervous system of poikilotherms than mammals, or could have a phylogenetic significance. These two interpretations are not mutually exclusive.

The Golgi apparatus of satellite cells associated with spinal ganglion neurons: changes with age in the rabbit.

We studied the Golgi apparatus in the satellite cell sheaths enveloping spinal ganglion neurons of rabbits aged 12, 42, and 79 months. We found neither structural changes nor indications of peripheral displacement of this organelle with advancing age. The mean percentage of cytoplasmic volume occupied by the Golgi apparatus decreased significantly passing from young adult to old rabbits. This decrease was only in very minor part a consequence of lipofuscin accumulation, so that the ratio between the total volume of the Golgi apparatus and the functionally active volume of cytoplasm decreased with age. The mean cytoplasmic volume of perineuronal satellite cell sheaths did not change significantly with increasing age, whereas the total volume of the Golgi apparatus within these sheaths decreased significantly with age. This decrease strongly suggests that the activity of satellite cells diminishes in old age, further suggesting that these cells are unable to compensate for the decrease in the neuronal metabolism that a number of authors have described in old age.

Age-related quantitative changes in mitochondria of satellite cell sheaths enveloping spinal ganglion neurons in the rabbit.

We studied mitochondria in the satellite cell sheaths which envelope the spinal ganglion neurons of rabbits aged 12, 42, and 79 months. While the mean cytoplasmic volume of satellite cell sheaths did not change significantly with age, the mean percentage of cytoplasmic volume occupied by mitochondria decreased with age. This decrease is mainly due to a reduction in the total mitochondrial mass and only in minor part is a consequence of lipofuscin accumulation. Mitochondrial structure did not change, while mitochondrial size increased with age. Comparison between mitochondria in nerve cell bodies and those in satellite cell sheaths showed that: (1) the mean percentage of cytoplasmic volume occupied by mitochondria was greater in nerve cell bodies than satellite cell sheaths and the ratio between these two percentages remained constant with advancing age; (2) the total mitochondrial mass was much greater in nerve cell bodies than satellite cell sheaths and the ratio between these two values increased with age; (3) the extent of increase of mitochondrial size with age was similar in nerve cell bodies and satellite cell sheaths. The results of the present study suggest that: (1) the ability of satellite cell sheaths to produce energy decreases with age; (2) the decreased ability of sensory neurons in old animals to meet high energy demands may be partly due to the diminished contribution of their associated satellite cell sheaths.

Satellite cell reactions to axon injury of sensory ganglion neurons: increase in number of gap junctions and formation of bridges connecting previously separate perineuronal sheaths.

This study investigated satellite cell changes in mouse L4 and L5 spinal ganglia 14 days after unilateral transection of sciatic and saphenous nerves. The ganglia were studied under the electron microscope in single and serial sections, and by dye injection. Satellite cell responses to axon injury of the neurons with which they are associated included the formation of bridges connecting previously separate perineuronal sheaths and the formation of new gap junctions, resulting in more extensive cell coupling. Some possible consequences of these satellite cell reactions are briefly discussed.

Glial cell plasticity in sensory ganglia induced by nerve damage.

Numerous studies have been done on the effect of nerve injury on neurons of sensory ganglia but little is known about the contribution of satellite glial cells (SCs) in these ganglia to post-injury events. We investigated cell-to-cell coupling and ultrastructure of SCs in mouse dorsal root ganglia after nerve injury (axotomy). Under control conditions SCs were mutually coupled, but mainly to other SCs around a given neuron. After axotomy SCs became extensively coupled to SCs that enveloped other neurons, apparently by gap junctions. Serial section electron microscopy showed that after axotomy SC sheaths enveloping neighboring neurons formed connections with each other. Such connections were absent in control ganglia. The number of gap junctions between SCs increased 6.5-fold after axotomy. We propose that axotomy induces growth of perineuronal SC sheaths, leading to contacts between SCs enveloping adjacent neurons and to formation of new gap junctions between SCs. These changes may be an important mode of glial plasticity and can contribute to neuropathic pain.

Changes with age in the Golgi apparatus of rabbit spinal ganglion neurons.

We studied the Golgi apparatus in spinal ganglion neurons of rabbits aged 12, 42 and 79 months. We found no structural changes, no indications of fragmentation, no indications of peripheral displacement affecting this organelle with advancing age. The volume of the perikaryon increased significantly with age, whereas the total volume of the Golgi apparatus remained essentially constant. Hence the mean percentage of perikaryal volume occupied by the Golgi apparatus decreased with age. This decrease was only in very minor part a consequence of lipofuscin accumulation, so that the ratio between the total volume of the Golgi apparatus and the functionally active volume of cytoplasm decreased with age. This decrease could be related to the reduced neuronal metabolism that occurs during ageing. It is possible that the delivery to the axon of newly synthesized proteins destined for fast transport is one of the Golgi apparatus roles that decrease with age. Finally, we found that the age-related quantitative changes in the Golgi apparatus did not differ between large light and small dark neurons.

Quantitative changes in mitochondria of spinal ganglion neurons in aged rabbits.

Within the context of our research on the age-related structural changes in spinal ganglia, we studied the mitochondria of the neuronal perikaryon in the spinal ganglia of 12-, 42-, and 79-month-old rabbits. Both the volume of the perikaryon and the total mitochondrial mass within the perikaryon increased significantly passing from young adult to old animals. Hence, there is no net loss of mitochondria in these neurons with age. Since, however, the volume of the perikaryon increased by more than 63% while the total mitochondrial mass within the perikaryon increased by only 18%, the mean percentage of perikaryal volume occupied by mitochondria decreased with age. This decrease is only in very minor part a consequence of lipofuscin accumulation, so that the ratio between the total mitochondrial mass and the functionally active volume of cytoplasm decreased with age. Possible causes of this decrease are discussed briefly. Moreover, while the mitochondrial structure did not change, mitochondrial size increased with age. Finally, in each of the three age groups both the mean percentage volume of mitochondria and the mean mitochondrial size were very similar in large light and in small dark neurons.

Decrease in the nucleo-cytoplasmic volume ratio of rabbit spinal ganglion neurons with age.

Nuclear and perikaryal volumes, as well as the mean percentage of perikaryal volume occupied by lipofuscin, were estimated in spinal ganglion neurons of 12, 42 and 79 month-old rabbits. The volume ratio between nucleus and perikaryal cytoplasm (N/C volume ratio) decreased progressively and significantly with increasing age. This was not a consequence of the perikaryal enlargement due to the age-related lipofuscin accumulation since the same result was obtained when the volume occupied by lipofuscin was subtracted from the perikaryal volume. The decrease in the N/C volume ratio may depend on an age-related loss of DNA, a cytoplasmic influence on nuclear size, or other unknown causes.

Amount and distribution of lipofuscin in nerve and satellite cells from spinal ganglia of young adult and aged rabbits.

The quantitative aspects of the age-related accumulation of lipofuscin were studied in the rabbit spinal ganglia by stereological methods using the electron microscope. Both neurons and their associated satellite cells were examined. In neurons, the shape and structure of lipofuscin bodies differed in young adults (12-months-old) compared to aged rabbits (79-months-old), whereas substantial changes were not observed in satellite cells. Both in nerve and satellite cells, lipofuscin bodies were scattered singly in young adults, but were often clustered in old animals. Lipofuscin occupied an average of 0.36% of neuronal perikaryal volume in the young adults and 2.55% in the aged rabbits; these percentages are much lower than those observed in the same neurons of other species. In the satellite cells, the corresponding values were 0.29% and 2.02%. In the young adults, the mean size of lipofuscin bodies was significantly greater in neurons than in satellite cells; the size of these bodies increased with age by about 2.7 times in neurons and by about 1.7 times in satellite cells. Consequently, in the old rabbits the mean size of lipofuscin bodies was about 2.2 times greater in neurons than in satellite cells. It has been suggested that lipofuscin bodies are transferred from the neuronal perikaryon to the surrounding satellite cells and then removed via the capillaries. However, the present findings suggest that lipofuscin located in satellite cells represents pigment formed by these cells rather than being transferred from neuronal perikaryon. It would appear that the age-related accumulation of lipofuscin in the rabbit spinal ganglia has little effect on neuronal metabolism.

The perikaryal surface of spinal ganglion neurons: differences between domains in contact with satellite cells and in contact with the extracellular matrix.

The perikaryal surface of spinal ganglion neurons undergoes dynamic changes throughout life. In particular, numerous slender projections develop and retract continuously from this surface. We showed previously that the outgrowth of these projections, while an intrinsic property of spinal ganglion neurons, is also influenced by the surrounding microenvironment. Since the latter consists of satellite cells and the extracellular matrix, we sought to determine the relative contributions of each of these components to the outgrowth of perikaryal projections. To this end, we took advantage of a little known characteristic of the satellite cell sheaths: in the rabbit, these sheaths can exhibit gaps that leave the nerve cell body surface directly exposed to the extracellular matrix. We compared the surface domains covered by satellite cells with those in direct contact with the extracellular matrix. We found that the perikaryal projections are abundant in the former domains but are absent in the latter. We also found that the perineuronal extracellular matrix of rabbit spinal ganglia contains laminin and fibronectin, two glycoproteins that have been reported to promote the growth of axonal processes from sensory ganglion neurons. Laminin and fibronectin were also present at the level of the gaps in the satellite cell sheath. These results: (1) provide additional evidence that environmental factors influence the outgrowth of perikaryal projections from spinal ganglion neurons; (2) suggest that satellite cells permit the outgrowth of these projections; (3) suggest that in the spinal ganglia of adult rabbits the perineuronal extracellular matrix is not in itself able to promote the outgrowth of these projections. This study provides a further example of the influence that supporting neuroglial cells have on sensory ganglion neurons.

The Golgi Stain: invention, diffusion and impact on neurosciences.

The black reaction, invented in 1873 by Camillo Golgi (1843-1926, was the first technique to reveal neurons in their entirety, i.e. with all their processes. This important development passed unnoticed at first and only received wide international attention after a long delay. The Golgi stain was widely employed for almost thirty years and was directly responsible for major advances in our knowledge of the microscopic anatomy of the nervous system, as well as in other fields of study. In the hands of other researchers, the black reaction provided vital evidence that helped to establish the neuron theory. The Golgi stain was almost forgotten in the period between the two World Wars, but the introduction of the electron microscope to neurocytological resarch revived its use around the middle of the twentieth century. Today, the black reaction is still used extensively not only in combination with electron microscopy, but also as an autonomous technique in studies on the evolution, ontogeny, and organization of the nervous system.

Structural changes in spinal ganglion neurons of lizards after cold-exposure.

We studied structural changes in spinal ganglion neurons that occur in lizards exposed to the cold, both at the light and electron microscope levels. Two types of perikaryal changes were found in the cold-exposed animals: (a) In 25% of all ganglion neurons, the central region of the perikaryon was devoid of Nissl bodies and a narrow peripheral zone stained deeply basophilic. Electron microscopic examination of these cells showed that mitochondria, Golgi complexes and other organelles were assembled in the central region of the perikaryon, while most cisternae of granular endoplasmic reticulum and free polysomes were confined to the periphery of the perikaryon. These changes seem to take place mainly in dark neurons. (b) In 8.6% of all ganglion neurons, Nissl bodies were present throughout the perikaryon, but separated by large, clear spaces. Under the electron microscope, these clear spaces were filled with large numbers of densely packed filaments. It seems that mainly light neurons undergo this type of structural change. The degree of nuclear eccentricity was significantly greater in the neurons of cold-exposed animals than in controls. The nucleolar volume was significantly increased and both the percentages of nuclei with two nucleoli and of nuclei with 'vacuolated' nucleoli were significantly greater in neurons displaying structural changes than in the other neurons. The structural modifications observed in spinal ganglion neurons of cold-exposed lizards closely resemble those seen in the same lizard neurons following axonal section. They could be due to a) metabolic changes induced by low temperature and fasting, b) alterations in the flow of nerve impulses from the periphery, or c) impaired retrograde transport of trophic substances from the periphery to the cell body.

Age-related decrease in the overall extent of perikaryal projections in rabbit spinal ganglion neurons.

The overall extent of the perikaryal projections of sensory neurons from spinal ganglia of young adult and aged rabbits was estimated by a stereological method using the electron microscope. The extent of perikaryal projections was significantly smaller in the aged animals. This age-related decrease did not seem to depend on factors intrinsic to the neuron, but on the absence of a satellite cell covering over extensive portions of the nerve cell body surface. This decrease may influence the organization of the subplasmalemmal cytoskeleton, metabolic exchange between the nerve cell body and its environment and perhaps also neuronal metabolism.