Developmental and Growth Changes in Neuron Differentiation, Dark and Light Neurons, and Age-related Neuronal Death in the Cranial Nerve Ganglia and in the Autonomic Nervous System with Reference to their Functional Significance: A Contribution to the Neurosensory and Motor Control of Living, Habits, Behaviour and Aging Process

MATERIALS AND METHODS

The chicks gallus gallus domesticus, Whit leghorn breed were used. Fertilized eggs were incubated at 37.5^o C, after every 24 hours, it was considered as Embryonic day 1 (E1), Embryonic day 2 (E2) etc till hatching (H). Embryos / Fetuses till hatching were removed carefully under anesthesia and aseptic conditions, and fixed in 10% formaldehyde solution (HCHO) solution at least for two weeks. Large (older) fetuses were cut transversely into suitable smaller pieces and labelled serially for future orientation. The tissues of older fetuses (i.e., E 15 and onwards till adult) were usually decalcified after fixation. Serial sections of 8 B 10 μ were stained by Cresyl Fast Violet for Nissl granules. Only a few selected stages that showed some remarkable changes i.e., E 6, E 8,E 10, E 13 E 15, E 18, chicks on the day hatching (H) and adult are included in this investigation. In all three animals in each group, with a total of twenty-four animals were used.Ganglia of both sides were used for examination. Every section of the ganglion was examined, drawn and the cells were plotted in a diagram with the help of a light microscope having acamera lucida attachment. Different categories of neurons were classified into Dark and Light neurons according to the difference in the intensity of cytoplasmic stain (Pillay, 1999a). Only those cells having a clear nucleus, and a nucleolus were counted and measured with the help of an eyepiece graticule. The following categories of cells were classified.

Tiny (< 5 μ), very small (6B10 μ), small (11B15 μ), medium sized (16B20 μ), big (21B25 μ), very big (26B30 μ), large (31B35 μ), very large (36B40 μ), giant (41B45 μ) (Pillay, 1999a). The categorization of cells on the basis of size with uniform difference of 5 μ was initially maintained just for the sake of convenience. However, this proved to be very useful in that, the behaviour, especially that of the very small cells, is very interesting on the day of hatching (H) (uniformly) in all the ganglia studied. This explains that this particular stage of cellular growth (very-small cell stage) is a critical period during development, indication a stage of active cell-process formation (axon-formation), beginning to establish functional connections with the target tissues.

RESULTS

The results of cell-counts of different categories of cells (Fig: 1) are presented in the form of tables in order to avoid too many descriptions, for various ganglia as illustrated below.

Fig: 1:	Shows dark neurons (with dark cytoplasmic stain) and light neurons (with light cytoplasmic stain) in the developing ganglion

Table 1:	Illustrates the total number of dark and light cells in the trigeminal ganglion in different age groups of animals in the ontogeny of the chick. D = Dark cells, L = Light cells, E = Embryonic age, H = Day of hatching, A = Adult

Table 2:	Illustrates the total number of dark and light cells in the geniculate ganglion in different age groups of animals in the ontogeny of the chick D = Dark cells, L = Light cells, E = Embryonic age, H = Day of hatching, A = Adult

Table 3:	Illustrates the total number of dark and light cells in the vestibular ganglion in different age groups of animals in the ontogeny of the chick D = Dark cells, L = Light cells, E = Embryonic age, H = Day of hatching, A = Adult

Table 4:	Illustrates the total number of dark and light cells in the acoustic ganglion in age groups of animals in the ontogeny of the chick D = Dark cells, L = Light cells, E = Embryonic age, H = Day of hatching, A = Adult

Table 5:	Illustrates the total number of dark and light cells in the proximal ganglionic complex of cranial nerves IX and X in different age groups of animals in the ontogeny of the chick. D = Dark cells, L = Light cells, E = Embryonic age, H = Day of hatching, A = Adult

Table 6:	Illustrates the total number of dark and light cells in the petrous ganglion in different age groups of animals in the ontogeny of the chick D = Dark cells, L = Light cells, E = Embryonic age, H = Day of hatching, A = Adult

Table 7:	Illustrates the total number of dark and light cells in the nodose ganglion in different age groups of animals in the ontogeny of the chick D = Dark cells, L = Light cells, E = Embryonic age, H = Day of hatching, A = Adult

Table 8:	Illustrates the total number of dark and light cells in the ciliary ganglion in different age groups of animals in the ontogeny of the chick D = Dark cells, L = Light cells, E = Embryonic age, H = Day of hatching, A = Adult

Table 9:	Illustrates the total number of dark and light cells in the superior cervical ganglion in different age groups of animals in the ontogeny of the chick. D = Dark cells, L = Light cells, E = Embryonic age, H = Day of hatching, A = Adult

The following evidences are presented in support of this assumption about the significance of the dark and light groups of cells. However, these statements are given in the form of brief points in order to avoid unnecessary long descriptions which might also need repetitions for every ganglion studied. Later, the facts are described in relation to some of the relevant available literature. Whenever necessary, for more details, the results of that particular ganglion may be verified and the facts be confirmed. The percentage or ratio of the dark and light cells in the ganglion in any age group may be found in the description of the results.

Exceptionally, in certain stages of development, in some of these ganglia (given below), the number of cells are reduced instead of a continuous increase, indicating probably the presence of an active phagocytic process which would remove the inactive or dead cells (so-called light cells) during developmental period. However, the light-cell stage is not represented in them probably because the phagocytosis is so active and so-fast to leave this light-cell stage for clear observation. These developmental stages probably represent some critical periods (in that particular ganglion) in their attempt to establish functional projection to their innervation fields. It may be noticed that most of these periods of cellular loss are just before the occurrence of light cells in the ganglion.

The descriptions of the dark and light neurones by different investigators in different animals vary greatly in available literature. Some of these conflicting views from the literature which are considered more useful are described below. Some investigators (Peach 1972, Gaik and Farbman 1973) have described in the trigeminal ganglion of the chick that the large neurones contain many isolated clumps of basophilic material in a neurofilament-rich cytoplasmic matrix having a diameter ranging from 19 - 40 microns and that the smaller neurones having a diameter ranging from 11 - 30 microns tend to be multipolar and have a greater concentration of ribosomes and granular endoplasmic reticulum which are dispersed through a dense matrix. Based on their staining properties, these larger ones are called light neurones and the smaller ones are called dark neurones. In the adult rats, trigeminal neurones have been classified into dark and light types on the basis of distribution of cytoplasmic organelles within the neurones and on the relative density of cytoplasm (Dixon 1963, Carmel and Stein 1969, Matsura et al 1969). In a comparative ultra-structural study of the trigeminal ganglion (Moses et al 1965) small dark cells were densely packed with rough endoplasmic reticulum and ribosomes, and large light cells with a dispersed and occasionally clumped ergastoplasm. It may be noticed that the classification (Peach 1972, Gaik and Farbman 1973) of dark (small) and light (large) neurones overlap in size. However, all the above investigators have found that the dark (smaller) neurones have a greater concentration of ribosomes and granular endoplasmic reticulum, which is suggestive of a more active role than that of their lighter (larger) counterparts. This observation also supports the present results that the dark cells represent a group of active cells and the light cells represent a group of resting, inactive, dying, dead or degenerating cells.

However, in subsequent reports on human autopsied trigeminal ganglia, it was not easy for these workers (Moses 1967) to readily identify dark and light neurones. The majority of cells were intermediate in appearance. On rare occasions when they did observe these two neuronal types, the cells differed only in their cytoplasmic density and not in the arrangement of any of their organelles. These observations led these investigators to conclude that the dark and light cells were not real entities, but resulted from fluid-shifts between the cells in question and the surrounding extra-cellular spaces. By the same token, other investigators (Pineda et al 1967, Maxwell 1967) working with cat and monkey trigeminal ganglia found that if proper tissue fixation techniques were used, there were no base for classifying the trigeminal neurones into dark and light cell types. It is possible that these conflicting reports given by different investigators may be attributed to the species difference of the experimental animals used.

There has been suggestions (Gaik and Farbman 1973) that shortly before hatching (E18 onwards), there appears two classes of interposed neurones characteristic of the mature trigeminal ganglion : large light types and small dark type. Neither of these two populations corresponds uniquely to either of the two segregated populations (large peripherally and distally located cells, and small centrally and proximally located groups) found in the embryo. In the present study in the chick, the dual cytology of neurones (dark and light cells) is found in all the ganglia studied. They were observed not only in the mature ganglia (from 18th day of incubation to adult) as stated by earlier investigators (Peach 1972, Gaik and Farbman 1973, Noden 1978, Yates 1961, Hess 1955, Finkelbrand and Siberman 1977), but also in earlier developmental stages (please refer to the results). It is not clear whether the above workers failed to observe these classes of cells in earlier developmental stages because of unreliable staining techniques or whether they did not attempt to investigate their presence during early embryonic periods. It is also noticed in the present investigation that in the early stages of development, only dark cells are found in all the ganglia. The light cells begin to appear only after certain period of embryonic growth which varies from ganglion to ganglion : probably the time of their occurrence coincides with their failure to establish proper functional projection to their innervation fields. For example, in the present study in the trigeminal ganglion, the light cells have appeared for the first time on E8 which seems to coincide with the day (E8) of establishment of functional connections as observed on the basis of the presence of reflexogenic responses to tactile stimulus of the beak (Hamburger and Narayanan 1969).

It is frequently suggested (Hamburger 1961, Gaik and Farbman 1973, Noden 1978, Yates 1961, D= Amico-Martel and Noden 1980, Johnston 1966, Noden 1975) that the large light and small dark neurones found in the trigeminal ganglion are correlated with the dual embryonic origin as derived from both the epidermal placode and neural crest. Through most of the second week of development the large cells are found in the distal and ventro-lateral parts of the ganglion and the small cells are found in the proximal (core) and medio-dorsal parts (Hamburger 1962, Ebendal and Headlund 1975). Even though the present study is not aimed at finding out the embryological origin of these two categories of cells, the segregation of large cells in the distal and ventro-lateral parts and the small cells in the proximal (core) and medio-dorsal parts of the ganglion is evident from the present results similar to these findings.

Also there is both circumstantial and direct evidence that cytological dichotomy in the adult ganglion is not based on separate embryological origin. First, other sensory ganglia that are exclusively of neural crest origin such as trunk dorsal root ganglia, or of placodal origin such as acoustic ganglion have both dark and light types of neurones in the present study. Similar views have been advocated by earlier investigators (Rosenbluth 1962, Spoendlin 1974) also. In addition, the light and dark neurones are found interspersed at random throughout the ganglion during embryonic development and post-hatching periods in the present study, whereas the segregation of crest and placode-derived neurones (small dark and large light cells respectively) are found only in the embryo. Both transplantation experiments (Noden 1978) and birth-date analysis (D= Amico-Martel and Noden 1980) have proved that neurones from each of these anlagen retain their original separate locations during later stages of development and maturation. Thus the dual cytology of the mature trigeminal ganglion is not based on separate embryonic origins.

Since there is no clear evidence of trigeminal neuron projection to the solitary nuclear complex that normally receives only visceral input (Dubbeldam 1980, Dubbeldam and Karten 1978, Arends and Dubbeldam 1984), there should be no visceral neurones in the trigeminal ganglion. The work on trigeminal ganglion (Kishida et al 1985) have demonstrated that the trigeminal ganglion had the smallest proportion of dark cells (46.8 %) whereas in the proximal ganglionic complex of the cranial nerves IX and X and the distal ganglion of cranial nerve IX the proportion of the dark cells was much greater (84.7 % and 70.3 % respectively). Since these two ganglia have both somatic and visceral neurones (Dubbeldam et al 1979) the large proportion of small dark cells suggests that at least some of the dark cells are visceral neurones. In a work on spinal ganglia (Cervero et al 1984), the visceral neurones had a tendency to be smaller than somatic neurones. If this is true, then the larger portion of dark cells in the proximal ganglionic complex of cranial nerves IX and X observed by the above workers suggests that this ganglion has more visceral neurones than the distal ganglion of cranial nerve IX. The specific functional attribution for the neurones, based only on size difference (small and large cells) or staining properties (dark and light cells) as suggested by the above-mentioned workers, however, could not be confirmed in the present study because of the following reasons:

It is also thought useful to quote the views of a few earlier investigators on Cell Death and Degeneration, and Removal of Dead Cells in order to complete the full evaluation of the dark and light cells. In this view, the following points are given for relevant reference.

Cell death and degeneration
One feature of development of many parts of the nervous system is the occurrence of two opposing processes: cellular proliferation which leads to the production of large numbers of neurones and massive cellular degeneration which results in the loss of many of these same neurones. These two processes ultimately control the final number of neurones of a neural centre. Even though no attempt was taken to investigate the purpose, reasons or ways of causing cell death which occur in different ganglia studied in the present series of investigation, the suggestions of some of the earlier investigators which are found suitable are given below in order to supplement the present assumption derived from a critical analysis of all these results. In the present study, cell death occurs mainly around E10 - E13 after which the ganglion prepares itself for a massive phagocytic activity which nearly comes to an end by the day of hatching. This resembles the descriptions (Hamburger 1934) that there are corresponding and parallel changes in the brain stem auditory nuclei and their peripheral ganglionic projections.

The observations (Hamburger 1934, 1958, Levi-Montalcini 1949, Cowan 1973) support the idea that it is the events in the target tissue (i.e., the periphery) which normally regulate cell survival. They demonstrated that the periphery both controls the proliferation and initial differentiation of undifferentiated cells and also provides the conditions necessary for continued growth and maintenance of neurones in stages following the first outgrowth of neurites. Several experiments (Hamburger 1958, Hamburger and Levi-Montalcini 1949, 1975, Prestige 1967, 1976, Kelly and Cowan 1972, Landmesser and Cowan 1974, Sohal 1976) have led to a general acceptance of the idea that neuronal death regulates nerve cell numbers in response to peripheral demands by eliminating those nerve cells whose fibres fail to establish proper peripheral connections. There are evidences (Cowan 1973, Hamburger 1975, Prestige 1970) suggesting that events at the target tissue controlling normal cell-death, involve the notion of either a competition between neurones for a limited number of synaptic sites and / or for a limited amount of trophic substances supplied by the target. Failure of the neurones to either make or receive the appropriate synaptic connections has been attributed to most neuronal deaths during embryogenesis (Cowan 1971, 1973). It also seems more likely that the main function of cell death in this system is probably to remove redundant neurones which though being in the correct muscle, have failed to form a contact (Landmesser and Morris 1975). According to this argument, the nervous system is programmed to over-produce cells in order to saturate the target and insure that all muscle fibers become innervated. The cell death presumably plays a role in the normal formation of orderly connections.

In some experiments (Hamburger and Levi-Montalcini 1949, Prestige 1970) in which it has been possible to experimentally enlarge the projection fields of the neuronal population, or to increase the number of afferents which it receives (a technique known to experimental neuro-embryologists as “ peripheral over-loading or peripheral enlargement”) the number of neurones found in the population at later stages of development has been significantly greater than normal. In contrast to these experiments, peripheral ablation experiments (Hamburger 1934, Kelly and Cowan 1972, Landmesser and Pilar 1974, Prestige 1970) have shown that the severity of the normal neuronal degeneration is much increased resulting in the additional cell death usually occurring over the same period as the naturally occurring neuronal loss. That is, natural cell death is known to be greatly enhanced by peripheral depletion.

From the available evidence, it is convincing to accept the idea that the peripheral influences, peripheral demands, trophic factors supplied by the target organs etc are important factors in controlling the neuronal death and the number of functional neurones available in the ganglia and that the neuronal death plays a role in the normal formation of orderly connections.

Removal of dead cells
Several investigators have suggested that macrophages are important in removing debris of dead cells (Levi-Montalcini 1950). Different sources of these macrophages have been suggested. One possibility is the transformation of mononuclear leucocytes in the circulatory system into tissue macrophages (Marchasi 1964, Sulton and Weiss 1966, Furth and Cohn 1968, Kitamura et al 1972). It has also been demonstrated (Adrian and Smotherman 1970) many radioactively labelled mononuclear leukocytes can infiltrate into nervous system and aggregate around damaged nervous tissue. There is also electron-microscopic evidence that the leukocytes invade nervous system by crossing through the wall of the blood capillaries (Matthews and Kruger 1973). It has also been demonstrated in an ultra-structural study (Wang-Chu and Oppenheim 1978) that the phagocytic cells contain neuronal debris that exhibit most of the characteristics of mononuclear leukocytes. The phagocytic activity of the satellite cells in the chick embryonic spinal ganglia are attributed to the removal of cellular debris of degenerated cell (Tennyson 1970) during early development. There are also reports (O=Connor and Wyttenbach 1974, Pilar and Landmesser 1976) that phagocytosis is accomplished by glial cells. However, some investigators (Michaels et al 1971) believe that the degenerating cells produce hydrolytic enzymes for their own degeneration. Therefore, there is obvious reason to believe that the leukocytes from the blood stream can penetrate through the wall of the capillaries into the nervous system and function as tissue macrophages to remove the neuronal debris during cell death and that the glial and satellite cells also can act as phagocytes.

Research summary
The chicks, Gallus gallus domesticus, White Leghorn breed were used in this study. The fertilised eggs were incubated at a temperature of 37.5 degree Centigrade. Embryos from the 3rd embryonic day onwards up to hatching were taken carefully without causing damage. They were immediately fixed in 10% formaldehyde solution and kept in the fixative for at least two weeks. The pieces having the head, neck and thorax were processed separate and paraffin blocks were prepared. The head of the adult animal rostral to the level of the 6th cervical vertebrate was separated and fixed in the fixative. Wherever necessary, the tissues were decalcified after fixation. The nodose ganglion in the adult was dissected out carefully fixed in the fixative and processed like other tissues. Serial sections having a thickness ranging from 8-10 microns were taken and stained by cresyl fast violet. In the embryos of 3-5 days, even though the ganglionic configuration of the different ganglia was noticed, their exact identification could not be done with accuracy. Therefore, the present study will include only the following stages of the chick which are of value of some critical interest in one ganglion or the other - E6, E8, E10, E13, E15, E18, The day of hatching, and the adult. The ganglia used for observation are ciliary, trigeminal, geniculate, vestibular, acoustic, superior ganglionic complex of glossopharyngeal and vagus nerves, petrous, nodose, superior cervical and 4th cervical spinal ganglia. Different types of cell were classified according to the intensity of stain in the cytoplasm and their size. Counting and categorizing the different types of cell were based on the camera lucida drawings of individual sections. Wherever possible, immunocyto-chemistry and microwave techniques were employed. The behaviour of different cell groups supposedly originating from the epidermal placodes and the neural crest were followed.

On the evidence available, the dark cells are considered as active ones; the light cells are considered as those which have failed to establish proper functional projections and are therefore destined to die and disappear from the vicinity of the ganglion and are classified as inactive, dying, dead or degenerating ones.

Even though the cell death and degeneration take place during all stages of cellular growth and maturation, it is most prominent and common among the small and medium sized one. Therefore, the small and medium sized ones. Therefore, the small and medium sized cells appear to be critical ones in the development of the ganglion; probably it is during these stages of cell growth the peripheral and central processes begin to grow from the cell body and attempt to get established in their projection fields. If they succeed in their attempt they continue to function and remain as dark cells, but if they fail in this attempt they lose their activity, tend to die and disappear, and change to a light coloured cell on staining. It is quite possible that some cells might lose their capacity and become light cells even after their processes grow into larger classes.

Since the small and medium sized cells are the youngest stages in which the light cells make their first appearance, it might indirectly mean that the establishment of connections might begin immediately after the very-small-celled stage, i.e., around small-cell stage. Successive larger classes of cells might represent the degree of development and maturation of their processes and continuation of functional capacity of the cell. However, the inherent nature of the cells also forms a basis for them to grow larger in size.

The cells which have started to die or degenerate and change into light cells might regain their capacity under favourable conditions and turn into dark active cells again. This might add to the existing number of dark cells in the ganglion. This may be possible either by recovery of the defects which have earlier developed either within the cells themselves or in the micro environment or by developing new collateral branch in order to substitute or supplement the previous functional loss. Such behaviour is frequently observed in the vestibular ganglion and also occasionally in some developmental stages in other ganglia.

It is presumed that the cell death plays a role in the normal formation of orderly connections.

The tiny cells are always found to be dark type. At no time light cells are found among them.

The very small type of cells are also usually dark during embryonic development till E18. The light cells have appeared among the very small type of cells also just on the day of hatching which may or may not continue in the adult situation. This probably signifies the possible attempt to eliminate the growing cells since they are no longer needed to replace larger categories of cells which have already well-developed neuronal connections at this stage by the day of hatching.

The cellular proliferation in the early stages of development might concern mainly with neuronal elements even though a proportion of these cells might be phagocytes being necessary to eliminate dead cells at every stage of development, and those formed around E15 - E18 might be concerned with mainly phagocytes which help remove the unsuccessful or redundant neuronal elements which probably fail to establish (to give or receive) proper functional connections.

It is assumed that the phagocytic cells might develop either from macrophages, glial cells, satellite cells and/or blood borne leukocytes.

Even after hatching, a few cells might continue to have the potentiality to act as phagocytes, to be protective for the ganglion even in later stages of life.

It is reasonable to presume that the optimum number of functional neurons must have been formed by the day of hatching since the animal is prepared and ready for an independent living by then.

From the evidences available, it is possible to presume that the optimum number of functional neurons and connections must have been established even before E18.

Usually the number of light cells are reduced very much on E18 even though the cell population is the highest in the whole ontogeny indicating probably a stage of faster and active removal of dead cells by the tremendously increased phagocytic cells. The phagocytosis is too fast that the inactive, dying or dead cells (i.e., so-called light cells) are removed immediately as soon as they are formed, to leave such a light celled stage for clear observation.

The light cell stage becomes clearly observable only when the phagocytic process is slow and become prominent at a time when some of the important connections are being actively established.

The volume of the ganglion is the greatest on E18 during the whole embryonic period as observed in all the ganglia studied. However, the ganglion shows its greatest volume in the adult situation in the whole ontogeny except the vestibular and acoustic ganglia. In most ganglia, the volume shows a reduction before and after E18 (in which the ganglion is larger in size, and has a tremendously increased phagocytic activity) i.e., around E15 and on the day of hatching.

The behaviour of cells in the vestibular ganglion as well as in the acoustic ganglion appears to be nearly similar in many respects.

Volume of the adult ganglion is lesser than that found on E18 even though its rostrocaudal length is greatest in the adult situation.

There is the appearance of larger proportion of light cells on the day of hatching while these cells are reduced in the adult situation.

There is an increased number of tiny cells in the adult ganglion than that seen on the day of hatching, probably indicating the importance of a renewal system of older cells which becomes functionally weak, in order to keep a balanced function.

However, the appearance of a few light cells in the vestibular ganglion on E6 might indirectly indicate the beginning of an early establishment of a functional connection, since the light cells are presumed to show up once these cells fail to form proper functional projection.

In the vestibular ganglion on the day of hatching the number of light cells is more than that of the dark cells, while in the adult situation the reverse is the case. In the adult, the proportion of these cells changes where the larger proportion is dark and smaller proportion is light types; probably among the enormous number of light cells formed on the day of hatching which are presumed to undergo death and degeneration, the major portion has recovered from this tendency possibly gaining the normal activity and function again by a process of re-activation of the original fibres before it is too late or by the development and establishment of new collateral branches which becomes functional and substitute the original unsuccessful ones.

In the vestibular ganglion the appearance of an increased number of tiny cells in the adult animal is comparison to that seen on the day of hatching (different and peculiar from other ganglia) probably provides a chance to replace or substitute the inactive (or dead) cells during ageing process by their growth, maturation and establishing new functional connections in view of the importance of an efficient vestibular function for normal behaviour of the animal.

The acoustic ganglion is also peculiar in that the larger classes of cells which have appeared in several stages following E6 have at last have disappeared in the adult situation, leaving a condition similar to that observed in the embryo as early as E6, whereas in most ganglia studied the larger classes of cells appear a new as the age increases. Probably the larger classes of cells are functionally unimportant in this ganglion and are eliminated in the adult situation.

The acoustic neurones are more sensitive to damage by ageing process compared to the cells in other ganglia studied and the functional reduction might become more prominent by increasing age. A renewal system to replace older cells by growth of new tiny cells might be in action, peculiar and similar to the vestibular ganglion.

The cell population and its density in most ganglia become low in the adult situation.

The rostrocaudal length of the ganglion is usually the greatest in the adult situation in the whole ontogeny except the vestibular and acoustic ganglia.

On the day of hatching there is always a reduction in the total number of cells than that found on E18 but having an increased number and increased proportion of light cells. The volume of the ganglion is also usually found to be reduced during this period.

In the adult situation the trigeminal ganglion is the largest one among all cranial nerve ganglia. The next one, greater in size is the nodose ganglion of the vagus nerve.

The behaviour of cells in the genicular ganglion also appears to be peculiar by being irregular in that they do not follow any of the clearly defined formula or regulation which are seen in other ganglia.

At least in some situation the nerve cells are found distributed in the nerve trunk in longitudinal rows between the nerve fibres, e.g., facial and vestibulocochlear nerves.

While a fluctuation in the cell population is taking place in the ganglion during development and growth, there are usually one, two or three stages which show population increase through the whole ontogeny as described below.

Cellular population of the 4th cervical spinal ganglion has only one raise, i.e., on E18 and a single decrease on the day of hatching and later a further reduction of cells in the adulthood.

Some ganglia have two raises in the cell population - one on E8 and another on E18 and a reduction on the day of hatching and later a continued reduction in the adult situation. e.g., trigeminal ganglion, proximal ganglionic complex of nerve IX and X, and nodose ganglion.

Some ganglia have the first population raise on E10 and another on E18 and later reduces on the day of hatching and also in the adult. e.g., cilliary ganglion, vestibular ganglion, acoustic ganglion and superior cervical ganglion.

Still others have three periods of population raise, on E8, E13 and E18 and later reduces on the day of hatching and also in the adult. e.g., genicular ganglion and petrous ganglion.

The nodose ganglion is the only ganglion which contains frank light cells even as early as E6. This indicates that the extensive establishment of connections takes place even before this stage, probably due to its functional importance supplying the vital organs such as heart, lungs and alimentary canal. Similarly the vestibular ganglion also shows a few light cells on E6 even though no light cells are observed on E8, E10 etc which also probably implies the importance of vestibular function.

It may be thought that the time of appearance of light cells in the ganglion might be indirectly related to the onset of establishment of active functional connections and to the functional importance of the organs which it supplies.

The increased volume of the ganglion on E18 might be due to a combined effect of tissue reaction resulted from the toxic substances of dead cells along with the phagocytic activity which are necessary to protect the young and delicate animal from the toxic effects.

The cell-space available for every cell in the ganglion is the greatest in the adult situation while the cell number is greatly reduced and the ganglionic volume is greatly increased.

The reduction or loss in the number of neurones in the adult ganglion in comparison to that found on the day of hatching might indicate a functional reduction of the ganglion probably as a result of ageing process.

The fluctuations in the number of cells during embryonic development may be considered as a normal process for the purpose of re-arrangement and better organization in order to perform an efficient function.

It is seen that there appears to be an initial small increase in the volume of the ganglion approximately upto E10, then there is a fall during the period of rapid cell loss around E8-E15 and then a rapid increase around E13-E18. However, these periods vary from ganglion to ganglion. The major period of cellular degeneration is usually around E11-E15 of incubation even though its intensity, amount and duration differ in different ganglia. It is also noteworthy that cell loss has essentially ended around E15 by the time the ganglion begins to show the greatest expansion so as to reach its maximum size on E18 during embryonic development.

It is assumed that the period of accelerated degeneration (or cell loss) is the period of active establishment of proper connections of the ganglion cells.

There is usually a great increase in the volume of the ganglion between E15 of incubation and the day of hatching which is due in part to the increased size of the cell bodies, and to the increased size of the neuropil lying between them because of the development of a network of nerve fibres and blood vessels.

It may be agreed that several convergent intrinsic and extrinsic factors might play a role in the regulative and/or stimulative control of behaviour of the ganglion and its cells during proliferation, aggregation of cells into ganglion, growth, maturation, establishment of functional projections, cell death, degeneration, disintegration, removal of dead cells and ageing processes.