dorsal root ganglia, axotomy, ultrastructure, coated pit, gap junction
INTER-CELLULAR COMMUNICATIONS IN THE RAT DORSAL ROOT GANGLIA
Jun Mizutani, Eisuke Sakuma, Hong-Jian Wang,
Tsuyoshi Soji, and Nobuo Matsui
Address of Correspondence: Dr. Jun Mizutani,
Department of Orthopaedic Surgery,
Nagoya City University Medical School,
1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi, 467-8601, Japan
Tel: +52-853-8236; Fax: +52-842-0266
Nagoya Medical Journal 43:223-233(2000)
The histological and ultrastructural changes of nerve cells in dorsal root ganglia (DRGs) following axotomy have been studied intensively (1-8). Most of these reports mention gross ultrastructural changes, but do not analyze ultrastructural alterations quantitatively.
All cell-to-cell interactions and cellular exchange of substances take place via the cytoplasmic membrane. As changes in substance intake into the cytoplasm and cell-to-cell interaction should take place following axotomy, we took interest in two special components of the cytoplasmic membrane usually seen in DRGs, namely, the pinocytotic vesicle (coated pit) in the nerve cell and the gap junction between the satellite cells. Both the pinocytotic vesicles (coated pits) in nerve cells and the gap junctions between satellite cells are specialized apparatuses of the cytoplasmic membrane essential for cellular transmission of information.
Although electron microscopic details following axotomy have been intensively studied, as mentioned above no report has focused on the alteration of coated pits and gap junctions after axotomy. This report discusses changes of the number of coated pits in nerve cells and gap junctions which connect satellite cells as a syncytium.
MATERIALS AND METHODS
All experiments were performed according to the animal handling regulations of Nagoya City University. Twelve 60 day-old Wistar rats were used. Under Nembutal (pentobarbital) anesthesia, central axotomy was done 5mm proximal to the L6 dorsal root ganglion (DRG) after L5 laminectomy. At 1, 4, 7 and 14 days after axotomy, three animals each were again anesthetized with Nembutal and perfused with fixative for 5 minutes through the left ventricle of the heart. The fixative used was 2.5% glutaraldehyde and 2% sucrose in 0.05 M cacodylate buffer (pH 7.4). After the perfusion, the L6 DRG were excised from each rat, and refixed by immersion for 1 hr in the same solution used for perfusion. The sections were then postfixed for 1 hr in 1% osmium tetroxide and 2% sucrose buffered by 0.05 M sodium cacodylate (pH 7.4). After postfixation, they were rinsed in ice-cold water for 5 min and dehydrated in a graded series of ethanol. Following two rinses in 100% ethanol for 10 min each, samples were embedded in epoxy resin (9). Ultrathin sections were prepared, placed on copper grids, stained with uranyl acetate and lead citrate, and observed using a Hitachi H-7000 transmission electron microscope. Three rats were used as controls. The DRGs were excised just after laminectomy and ultrathin sections were made in the same way as mentioned above.
The specimens were studied for: 1) coated pits in the nerve cells, and 2) gap junction between satellite cells. To enable the quantitative assessment of the numbers of coated pits, we first defined pinocytotic vesicles as an invaginating of the cytoplasmic membrane whose opening at the cell surface was less than the diameter of the vesicle. Therefore the pinocytotic vesicles in this series were identical to so-called coated pits.
Only nerve cells with distinct nucleoli were included in the pinocyte count. On x6000 magnification views, all coated pits along the circumference of the membrane were counted in 60 nerve cells in each sample and analyzed for statistical differences using SheffeŽÕs test.
1. Control group
In the control group satellite cells outnumbered nerve cells. Each nerve cell was completely enveloped by its own group of satellite cells. The outer side of the plasma membrane of the satellite cells was smoother and outlined by a basement membrane, while the boundary between the nerve cell and its satellite cells, which is composed of both plasma membrane of the satellite cells and the nerve cell, had a very complicated structure owing to the presence of multiple projections from both the nerve cell and the satellite cells. (Fig. 1). Gap junctions were only observed between the cytoplasmic expansions of satellite cells belonging to the same nerve cell. No gap junctions could be found between a nerve cell and a satellite cell. Coated pits were observed in the nerve cell body, with a mean number of 8.4 per nerve cell (Figs. 1a, 1b and 7).
2. Axotomy group
2.1: One day after axotomy
The cell bodies of the satellite cells displayed the same thin outlines as controls. The gap junctions between cytoplasmic expansions of the satellite cells were observed (Figs. 2 and 3). The mean number of the coated pits was 7.2 per nerve cell (Fig. 7).
2.2: Four days after axotomy
Some satellite cell bodies had lost their thin morphology and become hypertrophied. Direct contact between some satellite cell bodies could be observed. This was rarely seen in the control group (Fig. 4). Gap junctions were observed. The mean number of coated pits was 8.9 per nerve cell (Fig. 7).
2.3: Seven days after axotomy
Thickened satellite cells were frequently seen, and direct contact between satellite cell bodies was a common finding. Only limited areas showed the normal features seen in controls. Gap junctions were still observed (Fig. 5). The mean number of coated pits was 10.9 per nerve cell (Fig. 7).
2.4: 14 days after axotomy.
Interestingly, the gap junctions which were observed in the former specimens were never observed at this point. Nissl substances in the nerve cell were smaller, more dispersed, and had become unclear. The cell bodies and cytoplasmic expansions of the satellite cells had hypertrophied still further (Fig. 6). The mean number of coated pits was 6.0 per nerve cell (Fig. 7).
Summary of statistical analysis of coated pits
The numbers of coated pits counted were 8.4¡Þ2.8 for the control group, 7.2¡Þ2.2 one day after axotomy, 8.9¡Þ3.2 three days after axotomy, 10.9¡Þ3.7 seven days after axotomy, and 6.0¡Þ3.3 14 days after axotomy (mean¡ÞSD). The coated pits increased at seven days after axotomy, and later markedly decreased. The increase on day seven and the decrease on day 14 were statistically significant from control (p<0.01) (Fig. 7).
Known histological and ultrastructural changes in nerve cells of DRGs following axotomy include dispersed Nissl substances, irregularities of the nuclear membrane, eccentric location of the nucleus, and hypertrophied perikaryon. The gross ultrastructural changes of nerves and satellite cells in this study were in agreement with these reports.
The cell membrane is the frontline through which all transmission of information and exchange of substances between cells take place. Though no previous report has studied these ultrastructural changes quantitatively, changes in cell activity inevitably result in ultrastructural alterations which are quantifiable. Coated pits and gap junctions thus are valuable indicators of such alterations.
Pinocytotic vesicles (coated pits) are frequently found along the neuronal plasma membrane in all developmental stages (10). In this study we confirmed the existence of coated pits in adult rat DRGs. The function of the coated pit is the uptake of proteins from the extracellular space (7,11,12). Some of the proteins taken up by the ganglionic nerve cells are believed to be synthesized by the satellite cells.
There are two kinds of histologically distinct pinocytotic vesicles (11), the fluid-phase pinocytotic vesicles and the receptor-mediated one. Fluid-phase pinocytotic vesicles have smooth surfaces, contain ions and molecules in the same concentration as in the extracellular fluid, and their uptake of substances into the cytoplasm is nonselective. In contrast, receptor-mediated pinocytotic vesicles have coated membranes, internalize specific substances from the extracellular space by their binding receptors selectively (13). In previous reports the pinocytotic vesicles observed in nerve cells were mainly of the coated type(10). Likewise, in this study, the pinocytotic vesicles, defined as vesicles with so-called coated pit morphology, were of the receptor-mediated type.
The statistically significant findings in this study were an increase of coated pits at seven days following axotomy and a later marked decrease at 14 days. We believe these changes reflect adaptive alteration of activity of the nerve cells following central axotomy. The increase in the number of the coated pits at seven days after axotomy indicates requirement of high metabolic activity to maintain the nerve cell itself. And the decrease in number of the vesicles at 14 days after axotomy indicates the collapse of cellular function when the nerve cells succumb to chromatolysis.
In the literature, gap junctions between satellite cells have been studied by freeze-fracture technique (14,15). However no detailed study has been done on the changes in gap junctions with elapsed time following axotomy. Gap junctions allow the free transcellular exchange of small molecules such as ions, promoting cell-to-cell coupling or "electronic synapse," and are known to exist in excitable tissue such as heart muscle 5). The cell complex coupled by gap junctions is able to form a kind of synchronized system in which each cell can share the rapid distribution of information. Soji and Herbert (16) demonstrated the presence of gap junctions in the adult rat hypophysis, noting that they were associated only with the folliculo-stellate cells and not with any of the granular cell types. They also pointed to their role in the rapid dissemination of information through a complex system of interconnecting follicules.
We found that gap junctions existed between satellite cells up to seven days after axotomy, but were not observed at 14 days after surgery. Although the numbers were not adequate for statistical analysis, these facts indicate the synchronized unit between satellite cells is maintained up to seven days after central axotomy, but breaks down by 14 days. This is the first report of such phenomena.
The nerve cells are completely enveloped by several satellite cells whose outer cytoplasmic membrane possesses a basement membrane. The basement membrane isolates the nerve and satellite cell complex from the extracellular space. Therefore, to gain an understanding of this functional unit, investigation must include both the interaction between nerve cell and satellite cell and the interaction between satellite cells.
Coated pits are evidence of the interaction between nerve cell and satellite cell. Because of its envelope of satellite cells and basement membrane, the nerve cell has no direct contact with the surrounding matrix of connective tissue and blood vessels. Therefore, at least some of the proteins taken up by the pinocytotic vesicles into nerve cells are from the satellite cells. Satellite cells may thus play a regulatory or supportive role in nerve cell physiology and pathology.
As to the interaction between satellite cells, we hypothesize that in the normal DRG the several satellite cells belonging to a nerve cell act as a unit, synchronized by gap junctions (Fig.8). Once a satellite cell recognizes the precise status of a nerve cell, a certain signal is transmitted to other satellite cells through gap junctions, enabling the satellite cells to function as a syncytium.
Following central axotomy, each functional unit of the DRGs, composed of the nerve cell and its satellite cells, responds at the ultrastructural level. The axotomized nerve cell transmits a signal to its satellite cells which respond in synchronicity by action of the gap junctions, in a neurotrophic manner. Increased pinocytotic vesicles are ultrastractural evidence of the increase of metabolic activity in the nerve cell.
When this reactive process fails to maintain the functional integrity of the unit, the coated pits decrease and the nerve cells undergoes chromatolysis. In this situation, the syncytium of satellite cells is no longer justified, and the gap junctions can no longer be observed by electron microscopy.
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