Axonal Numbers and Sizes in the Connectives and Peripheral Nerves of the Leech J O Y C E M. W I L K I N S O N A N D RICHARD E. COGGESHALL Department of A n a t o m y a n d Marine Biomedicul Institute, University of T e x a s Medical Branch, Galveston, T e x a s 77550
ABSTRACT The present study is an electron microscopic analysis of the numbers and sizes of axons in the connectives and nerves of the medicinal leech. In either the right or left connectives for ganglia 14-18, there are approximately 2860 ( & 294 S.D.) axons. Ninety-seven percent of these axons are less than one micron in diameter. The median connective, Faivre’s nerve, contains 97 ( & 3 S.D.) axons, and 94% of these fibers are smaller than one micron. In the peripheral nerve roots for ganglia 14-18, there are approximately 2351 ( k 3 1 1 S.D.) axons. Ninety-eight percent of the axons in the nerves are less than one micron in diameter. Thus, there are approximately 20,000 axons associated with each of the segmental ganglia 14-18 in the leech, and the vast miority of these fibers are less than one micron in diameter.
Each segmental ganglion of the leech contains approximately 350 nerve cells (Apathy, 1897), and many of these cells are individually identified (Nicholls and Baylor, ’68; Stuart, ’70). However, the number of axons in the connectives and peripheral nerves of the leech is not known, and such information is necessary to gain further insight into the complexity of this simple nervous system. Furthermore, there is a remarkable similarity of cell numbers and types from one ganglion to the next and one animal to the next, and it would be desirable to see if this rigidity of organization also applies to axonal numbers and sizes in the connectives and nerves. However, because of the small size of most of the axons in this animal, it is necessary to use the electron microscope to see the axons clearly. Thus, the present study is an electron microscopic analysis of the connectives and nerves of certain ganglia in the leech, and its primary purpose is to ascertain the number and sizes of the axons contained therein. MATERIALS A N D METHODS
ganglia are numbered sequentially until the large anal ganglion is reached) with their associated nerves and connectives were exposed and flooded with fixative consisting of 2 % glutaraldehyde and 0.1% picric acid in 0.1 M cacodylate buffer at pH 7.4. After a few seconds, the tissue was removed and placed in fresh fixative for two hours and then into 1% osmic acid in 0.1 M cacodylate buffer €or two hours. The tissue was then stained “en bloc” by immersion in a solution of 0.5% magnesium uranyl acetate in distilled water for 16 hours. Dehydration was in ethanol and the tissue was embedded in a mixture of epon and araldite. Sections were cut with glass or diamond knives, stained with lead citrate (Venable and Coggeshall, ’65), and examined in a Philips 300 electron microscope. Axons were counted and measured from electron micrographs of the connectives and nerves. Small axons having circular profiles had their diameters measured directly. Larger axons, which tended to be irregular, had their largest and smallest diameters measured and these values were averaged.
Leeches (Hirudo medicinalis) were anesthetized in 10% ethanol, pinned ventral RESULTS side up on paraffin plates, and opened by Beautiful gross and light microscopic an incision down the mid-ventral line. Segmental ganglia 14-18 (ganglion 1 is the descriptions of the leech nervous system first segmental ganglion caudal to the sub- were in existence before the end of the esophageal ganglion and the 21 segmental nineteenth century (e.g., Retzius, 1891; J. COMP. NEUR., 162. 387-396
38 7
388
JOYCE M. WILKINSON AND RICHARD E. COGGESHALL
Rohde, 1891; Apathy, 1897) and these observations were extended to the level of the e l e c t m microscope by Gray and Guillery ('63) and Coggeshall and Fawcett ('64). The early studies and many others showed that the leech central nervous system consists of a supra-esophageal ganglion, a subesophageal ganglion, 21 segmental ganglia, and an anal ganglion. The ganglia are joined to each other by connectives and innervate the body wall by means of peripheral nerves. The Connectives Each leech segmental ganglion is united to the next by three connectives, two large lateral ones and a small median one often referred to as Faivre's nerve (e.g., Coggeshall and Fawcett, '64) (fig. 1). The connectives contain longitudinally directed axons which have various cross sectional areas and shapes. It is important to be able to distinguish the axons from other cell processes in the connective. The other major cellular components of each connective are the processes of the giant glial cells of the connective which surround and infold the axons (Gray and Guillery, '63; Coggeshall and Fawcett, '64). The axons can be distinguished from the glial processes by their longitudinal orientation as opposed to the predominately radial orientation of the glial processes and by their content of neurotubules and neurofibrillar bundles (Gray and Guillery, '63), which are distinct from the tonofilaments which are the main constitutents of the glial cytoplasm. The numbers of axons in the three connectives are presented in table 1. Note that the right and left connectives contain slightly less than 3,000 axons (right 2,908 % 305 S.D.; left 2,813 t 289 S.D.). By contrast, the much smaller Faivre's nerve contains slightly less than 100 fibers (97 -+ 3 S.D.). Thus, the total number of axons passing from one ganglion to the next is approximately 6,000. A histogram of the size distribution of the axOnS in a lateral connective is shown in figure 2.4. Note that approximately 80% of the fibers are less than 0.5 p in diameter and that onlv 3% of the fibers are larger than 1 LL. ~ h the ~two lateral ~ , connectives are predominately =On pathways.
Fig. 1 A cross section of a leech connective. Note the two large lateral connectives and the small median one, Faivre's nerve. The connectives are surrounded by fibrous tissue which has muscle cells within it. An endothelium encircles the entire connective. The larger axons are located at the periphery of the connective, and the majority of small axons are located near the center. Note that the largest axon of the connective is present in Faivre's nerve. X 3,825.
389
AXONAL NUMBERS AND SIZES IN THE LEECH
By contrast, the small median connective, Faivre’s nerve, has less than 50% of its fibers under 0.5 p in diameter, and it contains the largest axon in the leech connective (fig. 2B). The average axonal diameter in either the right or left connective is approximately 60% that of the average diameter in Faivre’s nerve. The leech is a contractile animal and the animals from which the above counts and histograms were taken were fixed in extension. To determine how much the diameters of the axons change when the connectives and peripheral nerves shorten, the connective and nerve were allowed to contract and then fixed. The connectives shortened from approximately 5 mm to 1.5 mm in length and the peripheral nerves a similar amount. When the leech was fixed in extension, the diameter of the large axon is Faivre’s nerve was, on the average, 4.3 p, and the same axon was approximately 9 p in diameter when the nerves and connectives were fully contracted. The size of the other axons in the nerves and connectives changed proportionately. The peripheral nerve roots Two peripheral nerves, an anterior and posterior, pass from either side of each segmental ganglion to enter the body wall. The nerve roots, which are those portions of the nerves between the ganglion and the wall of the ventral blood sinus, consist of bundles of axons wrapped in fibrous tissue (fig. 3). All these bundles are surrounded by a well developed perineurium which, in turn, is overlain by the endothelium. Within the perineurium are occasional muscle cells and cells similar to the small flattened cells around the ganglion. Thus, the organization of the peripheral nerve root is very similar to that of the connective, the major difference being that there are numerous bundles of axons in the nerve as opposed to the three bundles that make up the connectives. Although a full description of the peripheral distribution of the nerves is beyond the scope of this paper, it might be pointed out that when the peripheral nerve passes from the ganglion across the ventral sinus into surrounding connective tissue, it loses its endothelial covering, acquires another flattened cellular lining, and begins to
TABLE 1
Numbers of axons in the right, left and median (Faivre’s nerve) connectives f o r ganglia 1 4 through I 8 Connective
Between G.14 & G.15 Leech 3 Leech 7 Leech 10 Between G.15 & G.16 Leech 7 Leech 10 Leech 12 Between G.16 & G.17 Leech 7 Leech 1 0 Leech 1 1 Between G.17 & G.18 Leech 5 Leech 6 Leech 12 Mean Range of axonal numbers Lateral connectives Median connective Mean number of axons per connective Later a1 connectives Median connective
Right
Left
Faivre’s nerve
2804 2697 3204
2582 2401 3202
99 96 98
3085 2712 241 9
2753 3006 24 76
93 93 97
3166 2889 2447
98 96 97
3246 3231 2958
2900 3152 2777
104 97 95
2908
2813
97
3104 . ~ 306 1 2373
.
2373-3246 93-104 2860 f 294 S . D . 97 t 3 S.D.
break up into the various branches characteristic of that nerve. All of the axon counts for this paper were done on the nerve roots and are thus proximal to any nerve branches. There is some segregation of axons according to size in the various axon bundles in the nerve roots. The large axons tend to be located at the periphery of the nerve root (fig. 4). Medium sized axons tend to congregate in small fascicles (fig. 5), whereas the small axons occur in great numbers in individual fascicles (fig. 6). As in the connectives, the small axons are wrapped in large bundles and surrounded by thin glial processes (fig. 6). Some of the medium sized axons contain granules that resemble those found in the Retzius cells, which are two large 5-hydroxytryptamine containing neurons in each segmental ganglion (fig. 5). The numbers of axons in the four peripheral nerve roots for each ganglion are summarized in table 2. The average number of axons in each root is 2,351 311 S.D. with a range of 1,724-2,792. The histogram of the size distribution
*
390
%I
JOYCE M. WILKINSON AND RICHARD E. COGGESHALL
loo--
BlOO, lo01
8060-
%
-
1004
4 20
40~
W
20~
%L.
80-
I"%
2
ol L 0
2
U
4
6
'
8
diameter in M
Histograms of the diameters of axons in (A) a lateral connective, (B) Faivre's Fig. 2A-C nerve and (C) a nerve root. More than 90% of the axons are less than 1 p in diameter. The insets are histograms of the axons that are less than 1 f i in diameter. Note that the large majority of axons are less than 0.5 p in diameter.
of the axons in peripheral nerve are shown in figure 2C. Note that more than 95% of the axons in the nerves are less than 0.5 p in diameter. Approximately 1% of the axons in these nerves are above 1 in diameter, and several of these are prominent. Unfortunately, the size and location of these large fibers is not distinctive enough to allow these fibers to be individually identified from one animal to the next. DISCUSSION
One of the important thrusts of recent comparative neurobiology is the attempt to work out enough of the wiring diagram of a simple system so that facets of behavior can be explained in terms of the electrical activity and synaptic interconnections
of individually identified neurons. The segmental ganglion of the leech is one of the most favorable preparations for this type of work because it has relatively few neurons, most of which can be penetrated by microelectrodes. To gain a greater understanding of this system, however, it is necessary to know not only the number of neurons in a ganglion but the number of axons associated with that particular ganglion. The present study shows that there are approximately 12,000 axons (6,000 anteriorly and 6,000 posteriorly) in the connectives associated with each of the segmental ganglia 14-18 and approximately 9,500 axons in the four peripheral nerves associated with these same ganglia. The 20,000+ axons found in the nerves and connectives are far more than the number
AXONAL NUMBERS AND SIZES IN THE LEECH
391
Fig. 3 A cross section of a nerve root. Note that the axon bundles are surrounded by fibrous sheath. Some bundles contain many small axons and others large single axons. X 3,700.
of cell bodies in each ganglion. It is true that there are neurons scattered along the peripheral nerves in the leech (Gaskell, '14), but these are not numerous and do not explain the discrepancy between axonal and neuronal numbers. Another pos-
sible explanation for the preponderance of axonal numbers over neuronal numbers would be that the central neurons branch repeatedly and send many processses into a connective or peripheral nerve. Although the evidence is far from complete, this
392
JOYCE M. WILKINSON AND RICHARD E. COGGESHALL
Fig. 4 An electron micrograph of some of the large axons seen in the preceding picture. Note that axons are separated from the fibrous tissue by supporting cell processes. X 14,400.
AXONAL NUMBERS A N D SIZES I N T H E LEECH
393
Fig. 5 Medium sized axons in a fascicle of the peripheral nerve. T w o of these axons contain electron opaque granules (arrows). These granules resemble those in neurons known to contain biogenic amines in the leech. X 35,600.
does not seem to be the case. Methylene blue studies, which reveal whole ganglion cells, show that although the neurons have an extensive arborization within the neuropil and may send out several axons, they almost always send only one axon into any particular nerve or connective (Retzius, 1891; Nicholls and Baylor, ’68). More recent studies using injected materials to outline identified cells are in agreement with these findings (e.g., Nicholls and Purves, ’70; Lent, ’71). Thus, unless unidentified neurons put out an enormous number of processes into several nerves and connectives, which is unlikely, the source of most of the axons is extraganglionic. For the peripheral nerves, therefore, most of the fibers must come from the periphery and are presumably sensory. For the connectives, however, the origin of the axons is not as apparent, but one presumption is that the axons from a n y neuron pass for long distances up and down the nerve cord and so build up to sizeable numbers in any particular connective. However, the important consideration raised by these findings is that the concept
of “simplicity” for this nervous system relates primarily to the number of cell bodies in a ganglion and not to the number of information carrying channels associated with that ganglion. It is interesting that other invertebrate ganglia that are useful because of their simplicity also have a marked preponderance of axonal numbers over neuronal numbers. Ogawa (’28, ’30, ’34), for example, noted this for the earthworm and other annelids, although he was handicapped by the resolution of the light microscope and could only see the larger fibers. He also emphasized that fibers in the connectives could pass for long distances in the nervous system, a view that has been confirmed in other invertebrates (e.g., Wiersma, ’52). Gasser (’55) first noted that most unmyelinated fibers in the vertebrate were so small that they could only be counted in the electron microscope. Nunnemacher, et. al. (’62) extended this work to invertebrate nerves and noted the very large number of small axons in some nerves and connectives of the crayfish, a number that they compare with the much smaller number
394
JOYCE M. WILKINSON A N D RICHARD E. COGGESHALL
Fig. 6 A bundle containing many small axons in a nerve root. The majority of axons in the nerve are this size. X 32,000.
395
AXONAL NUMBERS AND SIZES IN THE LEECH TABLE 2
Numbers of axons in the right and left anterior a n d p o s t e n o r nerves for g a n g l z u 14-18 Right
Ganglion 14 Leech 5 Leech 6 Leech 8 Leech 10 Leech 12 Ganglion 15 Leech 3 Leech 8 Leech 10 Leech 12 Ganglion 1 6 Leech 1 Leech 2 Leech 4 Leech 6 Leech 8 Leech 9 Leech 10 Leech 1 1 Leech 12 Ganglion 17 Leech 1 Leech 4 Leech 8 Leech 10 Leech 12 Ganglion 18 Leech I Leech 2 Leech 10 Leech 1 1 Mean
Left
Anterior
Posterior
2282
2393
2367 2068
2764
2382 2318
2792 2683
2657
2595
2563 2609 2206
2729 2456 2208
An tenor
Posterior
2210 2271 2218 2786
261 7
1968 2777 2466
2577 1996 2581
2742 2349
2725 2386 2296
1981 2765 2600 2136 2571 1895
1759
2523 1734
2515 1724
1924 2498
2094 2589
2501
2467 2075 2547
2345 1857
1806 1918
2400 2233 1997
2271
2359
2339
Range of axonal numbers Mean number of axons per nervc
2717 2680
1848 2432
1724-2792 2351 t 311 S.D.
of ganglion cell bodies estimated by Wiersma ('61) in this nervous system, exclusive of the optic ganglia. Another invertebrate preparation, the abdominal ganglion of Aplysia, was shown to contain between 1,500-2,500 nerve cells and 1 5 , 0 0 6 25,000 axons in the nerves and connectives associated with the ganglion (Coggeshall, '67). The leech, therefore, is not unique but it is a striking example of this general trend with approximately 350 ganglion cells in and 20,000 axons associated with a segmental ganglion. To demonstrate how small the axons in the leech are, histograms were done, and this procedure shows that the vast majority of axons are under one micron in diameter. However, the leech is a contractile animal and the nervous system shortens and lengthens in proportion to the length
of the animal. In most of the specimens examined in this study, the leech was extended and the nerves and connectives were fully stretched. Thus, the axons would be relatively long and narrow. To see how much the diameter of these axons is affected by contraction of the nerves and connectives, the connectives and peripheral nerves were allowed to contract as far as possible and then they were fixed. The diameter of the large axon in Faivre's nerve and the large axons in the peripheral nerves were increased in diameter approximately 110% over the same axons in the extended nerves and connectives. Thus, there is a change in axonal diameters during contraction and extension of the animal but the general conclusion that the majority of axons in the leech are very fine is not affected.
396
JOYCE M. WILKINSON AND RICHARD E. COGGESHALL
Some nematodes have nervous systems with the number and location of all neurons precisely specified (c.f., Goldschmidt, ’08, ’09), and it is an interesting question as to how far the nervous systems of higher invertebrates are similarly specified. In the leech there is a remarkable similarity in axonal numbers in any particular nerve or connective from one animal to the next but there is not a perfect identity. Thus, the leech does not seem to have as rigid a control over neuronal and axonal numbers as does the nematode. Another invertebrate, Aplysia, has considerable variation in the number of axons and ganglion cells when small animals are compared to large ones. Thus, when necessary comparative data are available, it would be interesting to see if certain inferences can be drawn about the differences between “rigid” and “non-rigid” nervous systems and also it would be desirable to determine if certain types of neurons are responsible for the changes in axonal and neuronal numbers in “nonrigid” nervous systems. ACKNOWLEDGMENTS
We wish to thank Dr. W. D. Willis for a critical review of the manuscript and Dr. H. T. Hutchison for many helpful discussions. This work is supported by NIH grants 3T01 GM 00459 and NS 10161, and a grant from the Moody Foundation of Galveston, Texas. LITERATURE CITED Apathy, S. 1897 Das leitende Element des Nervensystems und sein topographishen Beziehungen zu den Zellen. Mitt. 2001. Stat. Neopol., 12: 495-748. Coggeshall, R. ,E. 1967 A light and electron microscope study of the abdominal ganglion of Aplysia calfornica. J. Neurophysiol., 30: 12631287. Coggeshall, R. E.,and D. W. Fawcett 1964 The fine structure of the central nervous system of the leech, Hirudo medicinalis. J. Neurophysiol., 27: 229-289. Gaskell, J. R. 1914 The c h r o m a f h system of annelids and the relation of this system to the contractile vascular system in the leech, Hirudo medicinalis. Phil. Trans. Roy. SOC. London., Series B., 205: 153-207.
Gasser, H. S. 1955 Properties of dorsal root unmedullated fibers on the two sides of the ganglion. J. Gen. Physiol., 308: 70S728. Goldschmidt, R. 1908 Das Nervensystem von Ascaris lumbricoides und megalocephala. I. 2. w i s s . Zool., 90: 73-136. 1909 Das Nervensystem von Ascaris lumbricoides und megalocephala. 11. 2. wiss. Zool., 92: 3OG-357. Gray, E. G., and R. W. Guillery 1963 An electron microscopial study of the ventral nerve cord of the leech. Z. Zellforsch. mikrosk. Anat., 60: 826-849. Lent, C . M. 1971 A comparative study of the neuronal geometry of Retzius’ cells in leeches. Am. Zoologist, 1 1 : 675. Nicholls, J. G., and D. A. Baylor 1968 Specific modalities and receptive fields of sensoryneurons in the CNS of the leech. J. Neurophysiol., 31: 740-756. Nicholls, J. G., and D. Purves 1970 Monosynaptic chemical and electrical connextions between sensory and motor cells in the central nervous system of the leech. J. Physiol., 209: 647-667. Nunnemacher, R. F.,G. Camougis and J. H. McAlear 1962 The fine structure of the crayfish nervous system. Fifth Int. Cong. Electron Microscopy. Academic Press, New York. Ogawa, F. 1928 On the number of ganglion cells and nerve fibers in some of the ventral nerve cords of the earthworm. I. The number of ganglion cells. Sci. Rep. TBhoku Univ., (4)3: 7 4 5 756. 1930 On the number of ganglion cells and nerve fibers in some of the ventral nerve cords of the earthworm. 11. The number of nerve fibers. Sci. Rep. Tohoku Univ., (4)5: 691716. 1934 The number of ganglion cells and nerve fibers in the nervous system of the earthworm, Pheretima communissima. Sci. Rep. Tohoku Univ., (4)8: 345-368. Retzius, G. 1891 Zur Kenntis des centralen Nervensystems der Hirudineen. Biol. Unters. Neue Folge, 2: 1 3 4 6 . Rohde, E. 1891 Histologische untersuchungen iiber das nervensystem dir Hirudinen. Schneider’s Zoologische Beitrage, 3: 1-68. Stuart, A. E. 1970 Physiological and morphological properties of motoneurons in the central nervous system of the leech. J . Physiol., 209: 627-646. Sutherland, R. M., and R. F. Nunnemacher 1968 Microanatomy of crayfish thoracic cord and roots. J. Comp. Neur., 132: 499-518. Venable, J. H., and R. E. Coggeshall 1965 A simplified lead citrate stain for electron microscopy. J. Cell. Biol., 25: 407-408. Wiersma, C. A. G. 1952 Neurons of arthropods. Cold Spr. Harb. Symp. Quant. Biol., 17: 155163. 1961 Reflexes and the central nervous system. In: The Physiology of Crustacea. T. H. Waterman, ed. Academic Press, New York, ZI: 241-279.
ABSTRACT The present study is an electron microscopic analysis of the numbers and sizes of axons in the connectives and nerves of the medicinal leech. In either the right or left connectives for ganglia 14-18, there are approximately 2860 ( & 294 S.D.) axons. Ninety-seven percent of these axons are less than one micron in diameter. The median connective, Faivre’s nerve, contains 97 ( & 3 S.D.) axons, and 94% of these fibers are smaller than one micron. In the peripheral nerve roots for ganglia 14-18, there are approximately 2351 ( k 3 1 1 S.D.) axons. Ninety-eight percent of the axons in the nerves are less than one micron in diameter. Thus, there are approximately 20,000 axons associated with each of the segmental ganglia 14-18 in the leech, and the vast miority of these fibers are less than one micron in diameter.
Each segmental ganglion of the leech contains approximately 350 nerve cells (Apathy, 1897), and many of these cells are individually identified (Nicholls and Baylor, ’68; Stuart, ’70). However, the number of axons in the connectives and peripheral nerves of the leech is not known, and such information is necessary to gain further insight into the complexity of this simple nervous system. Furthermore, there is a remarkable similarity of cell numbers and types from one ganglion to the next and one animal to the next, and it would be desirable to see if this rigidity of organization also applies to axonal numbers and sizes in the connectives and nerves. However, because of the small size of most of the axons in this animal, it is necessary to use the electron microscope to see the axons clearly. Thus, the present study is an electron microscopic analysis of the connectives and nerves of certain ganglia in the leech, and its primary purpose is to ascertain the number and sizes of the axons contained therein. MATERIALS A N D METHODS
ganglia are numbered sequentially until the large anal ganglion is reached) with their associated nerves and connectives were exposed and flooded with fixative consisting of 2 % glutaraldehyde and 0.1% picric acid in 0.1 M cacodylate buffer at pH 7.4. After a few seconds, the tissue was removed and placed in fresh fixative for two hours and then into 1% osmic acid in 0.1 M cacodylate buffer €or two hours. The tissue was then stained “en bloc” by immersion in a solution of 0.5% magnesium uranyl acetate in distilled water for 16 hours. Dehydration was in ethanol and the tissue was embedded in a mixture of epon and araldite. Sections were cut with glass or diamond knives, stained with lead citrate (Venable and Coggeshall, ’65), and examined in a Philips 300 electron microscope. Axons were counted and measured from electron micrographs of the connectives and nerves. Small axons having circular profiles had their diameters measured directly. Larger axons, which tended to be irregular, had their largest and smallest diameters measured and these values were averaged.
Leeches (Hirudo medicinalis) were anesthetized in 10% ethanol, pinned ventral RESULTS side up on paraffin plates, and opened by Beautiful gross and light microscopic an incision down the mid-ventral line. Segmental ganglia 14-18 (ganglion 1 is the descriptions of the leech nervous system first segmental ganglion caudal to the sub- were in existence before the end of the esophageal ganglion and the 21 segmental nineteenth century (e.g., Retzius, 1891; J. COMP. NEUR., 162. 387-396
38 7
388
JOYCE M. WILKINSON AND RICHARD E. COGGESHALL
Rohde, 1891; Apathy, 1897) and these observations were extended to the level of the e l e c t m microscope by Gray and Guillery ('63) and Coggeshall and Fawcett ('64). The early studies and many others showed that the leech central nervous system consists of a supra-esophageal ganglion, a subesophageal ganglion, 21 segmental ganglia, and an anal ganglion. The ganglia are joined to each other by connectives and innervate the body wall by means of peripheral nerves. The Connectives Each leech segmental ganglion is united to the next by three connectives, two large lateral ones and a small median one often referred to as Faivre's nerve (e.g., Coggeshall and Fawcett, '64) (fig. 1). The connectives contain longitudinally directed axons which have various cross sectional areas and shapes. It is important to be able to distinguish the axons from other cell processes in the connective. The other major cellular components of each connective are the processes of the giant glial cells of the connective which surround and infold the axons (Gray and Guillery, '63; Coggeshall and Fawcett, '64). The axons can be distinguished from the glial processes by their longitudinal orientation as opposed to the predominately radial orientation of the glial processes and by their content of neurotubules and neurofibrillar bundles (Gray and Guillery, '63), which are distinct from the tonofilaments which are the main constitutents of the glial cytoplasm. The numbers of axons in the three connectives are presented in table 1. Note that the right and left connectives contain slightly less than 3,000 axons (right 2,908 % 305 S.D.; left 2,813 t 289 S.D.). By contrast, the much smaller Faivre's nerve contains slightly less than 100 fibers (97 -+ 3 S.D.). Thus, the total number of axons passing from one ganglion to the next is approximately 6,000. A histogram of the size distribution of the axOnS in a lateral connective is shown in figure 2.4. Note that approximately 80% of the fibers are less than 0.5 p in diameter and that onlv 3% of the fibers are larger than 1 LL. ~ h the ~two lateral ~ , connectives are predominately =On pathways.
Fig. 1 A cross section of a leech connective. Note the two large lateral connectives and the small median one, Faivre's nerve. The connectives are surrounded by fibrous tissue which has muscle cells within it. An endothelium encircles the entire connective. The larger axons are located at the periphery of the connective, and the majority of small axons are located near the center. Note that the largest axon of the connective is present in Faivre's nerve. X 3,825.
389
AXONAL NUMBERS AND SIZES IN THE LEECH
By contrast, the small median connective, Faivre’s nerve, has less than 50% of its fibers under 0.5 p in diameter, and it contains the largest axon in the leech connective (fig. 2B). The average axonal diameter in either the right or left connective is approximately 60% that of the average diameter in Faivre’s nerve. The leech is a contractile animal and the animals from which the above counts and histograms were taken were fixed in extension. To determine how much the diameters of the axons change when the connectives and peripheral nerves shorten, the connective and nerve were allowed to contract and then fixed. The connectives shortened from approximately 5 mm to 1.5 mm in length and the peripheral nerves a similar amount. When the leech was fixed in extension, the diameter of the large axon is Faivre’s nerve was, on the average, 4.3 p, and the same axon was approximately 9 p in diameter when the nerves and connectives were fully contracted. The size of the other axons in the nerves and connectives changed proportionately. The peripheral nerve roots Two peripheral nerves, an anterior and posterior, pass from either side of each segmental ganglion to enter the body wall. The nerve roots, which are those portions of the nerves between the ganglion and the wall of the ventral blood sinus, consist of bundles of axons wrapped in fibrous tissue (fig. 3). All these bundles are surrounded by a well developed perineurium which, in turn, is overlain by the endothelium. Within the perineurium are occasional muscle cells and cells similar to the small flattened cells around the ganglion. Thus, the organization of the peripheral nerve root is very similar to that of the connective, the major difference being that there are numerous bundles of axons in the nerve as opposed to the three bundles that make up the connectives. Although a full description of the peripheral distribution of the nerves is beyond the scope of this paper, it might be pointed out that when the peripheral nerve passes from the ganglion across the ventral sinus into surrounding connective tissue, it loses its endothelial covering, acquires another flattened cellular lining, and begins to
TABLE 1
Numbers of axons in the right, left and median (Faivre’s nerve) connectives f o r ganglia 1 4 through I 8 Connective
Between G.14 & G.15 Leech 3 Leech 7 Leech 10 Between G.15 & G.16 Leech 7 Leech 10 Leech 12 Between G.16 & G.17 Leech 7 Leech 1 0 Leech 1 1 Between G.17 & G.18 Leech 5 Leech 6 Leech 12 Mean Range of axonal numbers Lateral connectives Median connective Mean number of axons per connective Later a1 connectives Median connective
Right
Left
Faivre’s nerve
2804 2697 3204
2582 2401 3202
99 96 98
3085 2712 241 9
2753 3006 24 76
93 93 97
3166 2889 2447
98 96 97
3246 3231 2958
2900 3152 2777
104 97 95
2908
2813
97
3104 . ~ 306 1 2373
.
2373-3246 93-104 2860 f 294 S . D . 97 t 3 S.D.
break up into the various branches characteristic of that nerve. All of the axon counts for this paper were done on the nerve roots and are thus proximal to any nerve branches. There is some segregation of axons according to size in the various axon bundles in the nerve roots. The large axons tend to be located at the periphery of the nerve root (fig. 4). Medium sized axons tend to congregate in small fascicles (fig. 5), whereas the small axons occur in great numbers in individual fascicles (fig. 6). As in the connectives, the small axons are wrapped in large bundles and surrounded by thin glial processes (fig. 6). Some of the medium sized axons contain granules that resemble those found in the Retzius cells, which are two large 5-hydroxytryptamine containing neurons in each segmental ganglion (fig. 5). The numbers of axons in the four peripheral nerve roots for each ganglion are summarized in table 2. The average number of axons in each root is 2,351 311 S.D. with a range of 1,724-2,792. The histogram of the size distribution
*
390
%I
JOYCE M. WILKINSON AND RICHARD E. COGGESHALL
loo--
BlOO, lo01
8060-
%
-
1004
4 20
40~
W
20~
%L.
80-
I"%
2
ol L 0
2
U
4
6
'
8
diameter in M
Histograms of the diameters of axons in (A) a lateral connective, (B) Faivre's Fig. 2A-C nerve and (C) a nerve root. More than 90% of the axons are less than 1 p in diameter. The insets are histograms of the axons that are less than 1 f i in diameter. Note that the large majority of axons are less than 0.5 p in diameter.
of the axons in peripheral nerve are shown in figure 2C. Note that more than 95% of the axons in the nerves are less than 0.5 p in diameter. Approximately 1% of the axons in these nerves are above 1 in diameter, and several of these are prominent. Unfortunately, the size and location of these large fibers is not distinctive enough to allow these fibers to be individually identified from one animal to the next. DISCUSSION
One of the important thrusts of recent comparative neurobiology is the attempt to work out enough of the wiring diagram of a simple system so that facets of behavior can be explained in terms of the electrical activity and synaptic interconnections
of individually identified neurons. The segmental ganglion of the leech is one of the most favorable preparations for this type of work because it has relatively few neurons, most of which can be penetrated by microelectrodes. To gain a greater understanding of this system, however, it is necessary to know not only the number of neurons in a ganglion but the number of axons associated with that particular ganglion. The present study shows that there are approximately 12,000 axons (6,000 anteriorly and 6,000 posteriorly) in the connectives associated with each of the segmental ganglia 14-18 and approximately 9,500 axons in the four peripheral nerves associated with these same ganglia. The 20,000+ axons found in the nerves and connectives are far more than the number
AXONAL NUMBERS AND SIZES IN THE LEECH
391
Fig. 3 A cross section of a nerve root. Note that the axon bundles are surrounded by fibrous sheath. Some bundles contain many small axons and others large single axons. X 3,700.
of cell bodies in each ganglion. It is true that there are neurons scattered along the peripheral nerves in the leech (Gaskell, '14), but these are not numerous and do not explain the discrepancy between axonal and neuronal numbers. Another pos-
sible explanation for the preponderance of axonal numbers over neuronal numbers would be that the central neurons branch repeatedly and send many processses into a connective or peripheral nerve. Although the evidence is far from complete, this
392
JOYCE M. WILKINSON AND RICHARD E. COGGESHALL
Fig. 4 An electron micrograph of some of the large axons seen in the preceding picture. Note that axons are separated from the fibrous tissue by supporting cell processes. X 14,400.
AXONAL NUMBERS A N D SIZES I N T H E LEECH
393
Fig. 5 Medium sized axons in a fascicle of the peripheral nerve. T w o of these axons contain electron opaque granules (arrows). These granules resemble those in neurons known to contain biogenic amines in the leech. X 35,600.
does not seem to be the case. Methylene blue studies, which reveal whole ganglion cells, show that although the neurons have an extensive arborization within the neuropil and may send out several axons, they almost always send only one axon into any particular nerve or connective (Retzius, 1891; Nicholls and Baylor, ’68). More recent studies using injected materials to outline identified cells are in agreement with these findings (e.g., Nicholls and Purves, ’70; Lent, ’71). Thus, unless unidentified neurons put out an enormous number of processes into several nerves and connectives, which is unlikely, the source of most of the axons is extraganglionic. For the peripheral nerves, therefore, most of the fibers must come from the periphery and are presumably sensory. For the connectives, however, the origin of the axons is not as apparent, but one presumption is that the axons from a n y neuron pass for long distances up and down the nerve cord and so build up to sizeable numbers in any particular connective. However, the important consideration raised by these findings is that the concept
of “simplicity” for this nervous system relates primarily to the number of cell bodies in a ganglion and not to the number of information carrying channels associated with that ganglion. It is interesting that other invertebrate ganglia that are useful because of their simplicity also have a marked preponderance of axonal numbers over neuronal numbers. Ogawa (’28, ’30, ’34), for example, noted this for the earthworm and other annelids, although he was handicapped by the resolution of the light microscope and could only see the larger fibers. He also emphasized that fibers in the connectives could pass for long distances in the nervous system, a view that has been confirmed in other invertebrates (e.g., Wiersma, ’52). Gasser (’55) first noted that most unmyelinated fibers in the vertebrate were so small that they could only be counted in the electron microscope. Nunnemacher, et. al. (’62) extended this work to invertebrate nerves and noted the very large number of small axons in some nerves and connectives of the crayfish, a number that they compare with the much smaller number
394
JOYCE M. WILKINSON A N D RICHARD E. COGGESHALL
Fig. 6 A bundle containing many small axons in a nerve root. The majority of axons in the nerve are this size. X 32,000.
395
AXONAL NUMBERS AND SIZES IN THE LEECH TABLE 2
Numbers of axons in the right and left anterior a n d p o s t e n o r nerves for g a n g l z u 14-18 Right
Ganglion 14 Leech 5 Leech 6 Leech 8 Leech 10 Leech 12 Ganglion 15 Leech 3 Leech 8 Leech 10 Leech 12 Ganglion 1 6 Leech 1 Leech 2 Leech 4 Leech 6 Leech 8 Leech 9 Leech 10 Leech 1 1 Leech 12 Ganglion 17 Leech 1 Leech 4 Leech 8 Leech 10 Leech 12 Ganglion 18 Leech I Leech 2 Leech 10 Leech 1 1 Mean
Left
Anterior
Posterior
2282
2393
2367 2068
2764
2382 2318
2792 2683
2657
2595
2563 2609 2206
2729 2456 2208
An tenor
Posterior
2210 2271 2218 2786
261 7
1968 2777 2466
2577 1996 2581
2742 2349
2725 2386 2296
1981 2765 2600 2136 2571 1895
1759
2523 1734
2515 1724
1924 2498
2094 2589
2501
2467 2075 2547
2345 1857
1806 1918
2400 2233 1997
2271
2359
2339
Range of axonal numbers Mean number of axons per nervc
2717 2680
1848 2432
1724-2792 2351 t 311 S.D.
of ganglion cell bodies estimated by Wiersma ('61) in this nervous system, exclusive of the optic ganglia. Another invertebrate preparation, the abdominal ganglion of Aplysia, was shown to contain between 1,500-2,500 nerve cells and 1 5 , 0 0 6 25,000 axons in the nerves and connectives associated with the ganglion (Coggeshall, '67). The leech, therefore, is not unique but it is a striking example of this general trend with approximately 350 ganglion cells in and 20,000 axons associated with a segmental ganglion. To demonstrate how small the axons in the leech are, histograms were done, and this procedure shows that the vast majority of axons are under one micron in diameter. However, the leech is a contractile animal and the nervous system shortens and lengthens in proportion to the length
of the animal. In most of the specimens examined in this study, the leech was extended and the nerves and connectives were fully stretched. Thus, the axons would be relatively long and narrow. To see how much the diameter of these axons is affected by contraction of the nerves and connectives, the connectives and peripheral nerves were allowed to contract as far as possible and then they were fixed. The diameter of the large axon in Faivre's nerve and the large axons in the peripheral nerves were increased in diameter approximately 110% over the same axons in the extended nerves and connectives. Thus, there is a change in axonal diameters during contraction and extension of the animal but the general conclusion that the majority of axons in the leech are very fine is not affected.
396
JOYCE M. WILKINSON AND RICHARD E. COGGESHALL
Some nematodes have nervous systems with the number and location of all neurons precisely specified (c.f., Goldschmidt, ’08, ’09), and it is an interesting question as to how far the nervous systems of higher invertebrates are similarly specified. In the leech there is a remarkable similarity in axonal numbers in any particular nerve or connective from one animal to the next but there is not a perfect identity. Thus, the leech does not seem to have as rigid a control over neuronal and axonal numbers as does the nematode. Another invertebrate, Aplysia, has considerable variation in the number of axons and ganglion cells when small animals are compared to large ones. Thus, when necessary comparative data are available, it would be interesting to see if certain inferences can be drawn about the differences between “rigid” and “non-rigid” nervous systems and also it would be desirable to determine if certain types of neurons are responsible for the changes in axonal and neuronal numbers in “nonrigid” nervous systems. ACKNOWLEDGMENTS
We wish to thank Dr. W. D. Willis for a critical review of the manuscript and Dr. H. T. Hutchison for many helpful discussions. This work is supported by NIH grants 3T01 GM 00459 and NS 10161, and a grant from the Moody Foundation of Galveston, Texas. LITERATURE CITED Apathy, S. 1897 Das leitende Element des Nervensystems und sein topographishen Beziehungen zu den Zellen. Mitt. 2001. Stat. Neopol., 12: 495-748. Coggeshall, R. ,E. 1967 A light and electron microscope study of the abdominal ganglion of Aplysia calfornica. J. Neurophysiol., 30: 12631287. Coggeshall, R. E.,and D. W. Fawcett 1964 The fine structure of the central nervous system of the leech, Hirudo medicinalis. J. Neurophysiol., 27: 229-289. Gaskell, J. R. 1914 The c h r o m a f h system of annelids and the relation of this system to the contractile vascular system in the leech, Hirudo medicinalis. Phil. Trans. Roy. SOC. London., Series B., 205: 153-207.
Gasser, H. S. 1955 Properties of dorsal root unmedullated fibers on the two sides of the ganglion. J. Gen. Physiol., 308: 70S728. Goldschmidt, R. 1908 Das Nervensystem von Ascaris lumbricoides und megalocephala. I. 2. w i s s . Zool., 90: 73-136. 1909 Das Nervensystem von Ascaris lumbricoides und megalocephala. 11. 2. wiss. Zool., 92: 3OG-357. Gray, E. G., and R. W. Guillery 1963 An electron microscopial study of the ventral nerve cord of the leech. Z. Zellforsch. mikrosk. Anat., 60: 826-849. Lent, C . M. 1971 A comparative study of the neuronal geometry of Retzius’ cells in leeches. Am. Zoologist, 1 1 : 675. Nicholls, J. G., and D. A. Baylor 1968 Specific modalities and receptive fields of sensoryneurons in the CNS of the leech. J. Neurophysiol., 31: 740-756. Nicholls, J. G., and D. Purves 1970 Monosynaptic chemical and electrical connextions between sensory and motor cells in the central nervous system of the leech. J. Physiol., 209: 647-667. Nunnemacher, R. F.,G. Camougis and J. H. McAlear 1962 The fine structure of the crayfish nervous system. Fifth Int. Cong. Electron Microscopy. Academic Press, New York. Ogawa, F. 1928 On the number of ganglion cells and nerve fibers in some of the ventral nerve cords of the earthworm. I. The number of ganglion cells. Sci. Rep. TBhoku Univ., (4)3: 7 4 5 756. 1930 On the number of ganglion cells and nerve fibers in some of the ventral nerve cords of the earthworm. 11. The number of nerve fibers. Sci. Rep. Tohoku Univ., (4)5: 691716. 1934 The number of ganglion cells and nerve fibers in the nervous system of the earthworm, Pheretima communissima. Sci. Rep. Tohoku Univ., (4)8: 345-368. Retzius, G. 1891 Zur Kenntis des centralen Nervensystems der Hirudineen. Biol. Unters. Neue Folge, 2: 1 3 4 6 . Rohde, E. 1891 Histologische untersuchungen iiber das nervensystem dir Hirudinen. Schneider’s Zoologische Beitrage, 3: 1-68. Stuart, A. E. 1970 Physiological and morphological properties of motoneurons in the central nervous system of the leech. J . Physiol., 209: 627-646. Sutherland, R. M., and R. F. Nunnemacher 1968 Microanatomy of crayfish thoracic cord and roots. J. Comp. Neur., 132: 499-518. Venable, J. H., and R. E. Coggeshall 1965 A simplified lead citrate stain for electron microscopy. J. Cell. Biol., 25: 407-408. Wiersma, C. A. G. 1952 Neurons of arthropods. Cold Spr. Harb. Symp. Quant. Biol., 17: 155163. 1961 Reflexes and the central nervous system. In: The Physiology of Crustacea. T. H. Waterman, ed. Academic Press, New York, ZI: 241-279.
