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Degeneration of olfactory receptors induced by colchicine
EXPERIMENTAL
NEUROLOGY
Degeneration
483-492
11,
of
Olfactory by
RICHARD Department
of Biological
T.
(1963)
Received
Induced
Colchicine
JACKSON
Science,
Receptors
AND
Lojlola November
CHIN-CHIU University,
LEE’ New
Orleans,
Louisiana
4, 1964
Colchicine evokes drastic degenerative effects on the olfactory receptors and bulb when a small amount of the drug is infused directly into one olfactory bulb of the rabbit. Infusion of 20 lambda of 0.1 M colchicine evoked severe ipsilateral, transneuronal cell degeneration in the olfactory epithelium. Slight alterations were observed in the contralateral bulb and epithelium. Upon intraperitoneal injection of colchicine in doses as high as 10 mg/kg, only slight degeneration was observed in the olfactory bulbs and epithelium. In spite of the severity of degenerative changes evoked by infusion, some olfactory receptors were free from disintegration and persisted up to 8 weeks. Introduction
The olfactory system has the ability to distinguish among an astonishingly large number of odors. In an effort to explain this and other phenomena, such as selective adaptation, it has been postulated that some olfactory receptors are essentially different from other olfactory receptors. This view was made more seductive by LeGros Clark ( 11, 12) when he demonstrated selective degeneration of olfactory receptors. He removed one of the olfactory bulbs in a rabbit and observed that only half the ipsilateral olfactory receptors degenerated. One would expect all or none. We repeated this experiment, and in addition, removed both bulbs in rabbits. The same general results were obtained (13). Matthews and Powell (14) demonstrated an ascending transneuronal change in the olfactory bulb upon the removal of the olfactory epithelium. In an effort to corroborate and 1 This investigation the National Institutes of Medicine, Laboratory
was supported by research grants NB 02213 and AI 06094 from of Health. Dr. Jackson is presently at Emory University School for Ophthalmic Research, Atlanta, Georgia 30322. 483
484
JACKSON
AND
LEE
amplify these studies of mechanically induced, transneuronal degeneration, we decided to attempt selective, chemical degeneration with colchicine. It has been noted many times in the early literature that colchicine caused systemic disorders by acting upon the nervous system. While Ferguson and Theodore (8) showedthe physiological evidence of the action of colchicine upon the central nervous system, Singer et al. (16) were able to demonstrate for the first time the destructive morphological changesin following colchicine the peripheral nerves of the amphibian, Trituuus, treatment. Their findings were confirmed by the work of Angevine ( 1) on mammalian sciatic nerve.
Materials
and
Methods
Young rabbits, weighing 1-2 kg were used in all experiments becausethe olfactory epithelium of older rabbits has usually been damaged by inflammatory rhinitis. Intraperitoneal injections of colchicine”-doses ranged from 0.05 mg/kg to 10 mg/kg. Injected animals were allowed to survive for 2 days through 8 weeks,and killed by an overdose of Nembutal. The animal was exsanguinated, the frontal bones removed and the nasal septum-ethmoturbinate area removed in toto for fixation in alcohol-acetic formalin. Paraffin sections of 8 u were stained according to Bodian’s protargol technique (3, 9) and routinely with hematoxylin and eosin. For bulbar infusion (0.02 ml of 0.05 and 0.1 M) colchicine infusates were employed (which correspondsto 2% and 470 colchicine, respectively). The method of microinfusion was adapted from the design of Singer ( 15). The infusion apparatus consistedof a driving device and an injector whosemain body is a 0.25ml tuberculin syringe fitted with a 26-gauge needle. Polyethylene tubing connected the latter, metal needle to a glassneedle with a tip of about 100 lo in diameter. Under Nembutal anesthesia,a puncture hole was madewith a dental bur in the frontal bone just above the olfactory bulb. The glassneedle was inserted through the hole and about 1 mm deep into the dorsal portion of the bulb. In some cases,the opposite bulb was infused with the samevolume (0.02 ml) of saline solution, otherwise only a puncture wound was made. After an appropriate time has elapsed, the tissuewas removed and stained as described above. 2 Obtained
from
Nutritional
Biochemicals.
OLFACTORY
485
RECEPTORS
Efiects of B&bar Infusion of Colchicbe on the Olfactory Bulb and Olfactory Receptors. The results presented here were obtained from the four animals shown in Table 1. TABLE VARIARONS
OF PARAMETERS
EFFECTS
OF BULBAR
Colchicine concentration Animal No.
1 EMPLOYED
INFUSION
OF
Amount infused
Infusion time
Survival
(min)
(weeks)
120 45
6
15
8
15
2
1
0.05
2 3 4
0.1 0.1
(ml) 0.02 0.02 0.02
0.1
0.01
(M)
IN ASSESSMENT OF COLCHICINE
1
The olfactory bulbs of animal No. 1 are shown in Fig. 1. Not only shrinkage, but also immensealteration occurs in the laminar pattern of the bulb. Each of the bulbar layers is diminished in thickness. The layer of pr’imary olfactory fibers is almost replaced by connective tissue. The glomeruli become small and are poorly impregnated with silver stain due to the destruction of the ramifications, whereas the periglomerular cells are seemingly left intact. The external and internal plexiform layers are almost deprived of nerve fibers and most of the mitral and tufted cells are absent. Most cells in the granular layer also disappear. The latter become indistinguishable from the periventricular layer. In addition, blood vessel dilation and gliosis are notable all over the bulb in H & E preparations. The bulb is about 30% smaller than the control side. The control bulb was not entirely normal in appearance. Comparison with normal bulbs from other animals revealed subtle changes.The radial fibers in the external plexiform layer appear very short and the number of mitral, tufted, and small granular cells seemedreduced. Bulbar degeneration of the same severity was found when higher doses of colchicine infusate were used and longer postoperative survival periods were allowed. A 20-30s bulb shrinkage was observed in animals No. 2 and No. 3. The most obvious changeswere a decreasednumber of granular and mitral cells and radial fibers in the plexiform layer (Fig. 2). The changesseenin rabbit No. 4 were much lesssevere. Slight cell degeneration is represented by the diminished defective silver impregnation in the plexiform
mitral cell population layer (Fig. 3).
and
486
FIG.
colchicine
JACKSON
1.
Rabbit (left).
olfactory bulb The destructive
1 week changes
AND
LEE
following are more
infusion intensive
with 0.02 ml of 0.05 M and shrinkage of the
OLFACTORY
RECEPTORS
487
In all cases, whenever the olfactory bulb shows degenerative changes, a similarly intense secondary degeneration is found in the olfactory epithelium. In animal No. 1, the receptors in the olfactory epithelium of the infused side almost completely disappear in most regions. The thickness of the mucosa is also reduced. In the control epithelium, degeneration of receptors is also observed in approximate accordance to the severity of change between bulbs. Many olfactory rods have disappeared leaving gaps, and many receptor nuclei aer pyknotic. (See illustrations in Ref. 12.) Efiects of Intraperitoneal Injection of Colchicine on the Olfactory Bulb and the Olfactory Receptors. The minimal dose of colchicine employed in the present experiment was 0.05 mg/kg. This dose elicits barely detectable degenerative signs in the epithelium after 3 days. As little as 0.1 mg/kg elicits blood vessel dilation, inflammatory cells, unusual shaped rods and poor silver impregnation of nuclei after 48 hours. A more serious change in the nuclei of the receptors is found in sections taken 2 weeks after injection of 0.2 mg/kg colchicine. Many nuclei are missing and most of the residual cells display pyknotic granules in a pale cytoplasm. Surprisingly, increasing the dose (2, 3, 5 and 10 mg/kg) did not bring about more severe changes in the epithelium. At the 10 mg/kg dose, the bulb alterations are moderate. Neither the laminar pattern nor the bulbar size is changed. Upon close examination, however, notable are the fragmentation of the ramifications in some of the glomeruli and the reduced layer of mitral cells, some of which are in the course of chromatolysis. In addition, the dendritic fibers of the external plexiform layer appear to have been swollen and the number of granular cells is diminished. Nonspecific Pathological Reaction. Certain symptoms in the treated animals are due to the nonspecific poisoning effect of colchicine. At lower doses, by both routes, the animals often had respiratory difficulties and diarrhea. At higher doses (especially 5 and 10 mg/kg) paralysis of the hind limbs occurred in some instances. Discussion
In assessing the degree of degeneration of receptors one is thwarted by the fact that, in sections of normal olfactory epithelium, it is quite common to observe areas that display a few poorly stained receptors bounded by bulb more obvious as compared to other cases. A slight alteration can also be seen on the control side (right), which was infused simultaneously with physiological saline solution. Protargol; 150 X. Abbreviations: nf, layer of sensory nerve fibers; gl, plexiform layer; inp, internal plexiform layer; m, mitral glomeruli ; exp, external cells; gr, granular layer; and pv, periventricular layer.
FIG. 2. Rabbit olfactory bulb 8 weeks following infusion with 0.02 ml of 0.1 M colchicine (left). The numbers of mitral cells and their dendrites are diminished. Fewer granular layers are seen in contrast with those in the control (right) bulb. Protargol; 150 X. Abbreviations same as in Fig. 1.
FIG. 3. Rabbit olfactory bulb 2 weeks after infusion with 0.01 ml of 0.1 M colchicine (left). The disappearance of the small cells in the granular layer, the diminished mitral cell population, and defective silver impregnation in the external plexiform layer and glomeruli are notable as compared with the control (right) bulb. Protargol; 150 X. Abbreviations same as in Fig. 1. 489
FIG. 4. Olfactory 450 x.
Normal rabbit olfactory epithelium epithelium after infusion of the 490
(A). bulb
Note with
receptor elements colchicine (B).
(arrows). Protargol;
OLFACTORY
RECEPTORS
491
very densely packed receptor areas. With experience, however, one can evaluate damage. The relatively mild degeneration caused by intraperitoneal injection is not surprising if one realizes that several previous experiments with systemic colchicine failed to demonstrate neuropathological effects or the presence of labelled drug in nervous tissue (2, 4, 5). Also, it appears that neuropathological concentrations are much higher than those required to elicit systemic or mitotic effects ( 1) . Since infusion of 0.02 ml of 0.05 M colchicine evokes drastic destruction in both the olfactory bulb and receptors, it can be assumed that the threshold is much lower. On the other hand, very large doses do not obliterate all the receptors in spite of severe destruction in the bulb. We observed degenerative receptor losses of between 20-8070 which bracket the quotation of LeGros Clark (11). Regardless of the number, it would seem that, again, some receptors are resistant and some are not. This is not inconsistent with the findings of Eayrs (6) who observed rather less that half of the ganglion cells, whose axons comprise the optic nerve, persisted following optic nerve transection. It is doubtful that colchicine exerts a specific effect on nervous tissue. It has been found to inhibit dephosphorylation and deamination of desoxyribonucleotides at 1.2 X lo-” M (7). Hell and Cox (10) and Truhaut and Bohuon (17) have demonstrated inhibition of desoxyribonuclease activity. References 1.
2.
3. 4.
5. 6. 7.
1956. A study of toxic and neuropathological effects of J, B., JR. colchicine in rodents following direct infusion of nervous tissues. Ph. D. thesis, Cornell Univ. (L. C. Card No. Mic 56-975), 201 pp. University Microfilms, Ann Arbor, Michigan (Dissertation Abstr. 16: 625). BACK, A., E. WALASZEK, and E. UYEKI. 1951. Distribution of radioactive colchicine in some organs of normal and tumor-bearing mice. Proc. Sot. Exptl. Biol. Med. 77: 667-669. BODIAN, D. 1937. The staining of paraffm sections of nervous tissues with activated protargol. The role of fixative. Anat. Record 69: 153-162. BROWN, W. O., and L. SEED. 1945. Effect of colchicine on human tissue. Am. J. czin. Pathol. 16: 189-195. BRUES, A. M. 1942. The fate of colchicine in the body. /. Clin. Invest. 21: 646-647. EAYRS, J. T. 1952. Observations of the retina following optic nerve section. Brit. J. Ophthalmol. 36: 453-459. “Colchicine in agriculture, medicine, EIGSTI, 0. J., and P. DUSTIN, JR. 1955. biology and chemistry.” Iowa State College Press, Ames, Iowa.
ANCEVINE,
492
8. 9. 10.
11. 12. 13. 14.
15.
16.
17.
JACKSON
AND
LEE
F. C., JR., and P. THEODORE. 1952. On colchicine. I. General Pharmacology. J. Pharmacol. Enptl. Than@. 106: 261-270. FOLEY, J. 0. 1943. A protargol method for staining nerve fibers in frozen or celloidin sections. Stain Technol. 18: 27-33. HELL, E., and D. E. Cox. 1963. Effects of colchicine and colchemid on synthesis of desoxyribonucleic acid in the skin of guinea pig’s ear in vitro. ,Vatz~e 197: 287-289. LEGROS CLARK, W. E. 1956a. Observations on the structure and organization of olfactory receptors in the rabbit. Yale J. Biol. Med. 29: 83-95. LEGROS CLARK, W. E. 1956b. Inquiries into the anatomical basis of olfactory discrimination. Proc. Roy. Sot. London, Ser. B 146: 299-319. 1960. Olfactory nerve degeneration. MARTINEZ, I. R., and R. T. JACKSON. Assoc. Sozltheast. Biol. Btdl. 7: 34. MATTHEWS, M. R., and T. P. S. POWELL. 1962. Some observations on transneuronal cell degeneration in the olfactory bulb of the rabbit, J. Bnut. 96: 89-102. SINGER, M. 1954. .4pparatus ior continuous infusion of microvolumes of solution into organs and tissues. Pmt. Sot. Exptl. Biol. Med. 86: 378-380. SINGER, M., D. FLINKER, and R. L. SIDMAN. 1956. Nerve destruction by colchicine resulting in suppression of limb regeneration in adult Triturzrs. J. Exptl. Zool. 131: 459-491. TRUHAUT, R., and C. BOHUON. 1963. Influences of colchicine and N-desacethythiocolchicine on the desoxyribonuclease activity of the blood plasma in the mouse. Cornfit. Rend. 256: 162%163C.
FERGUSON,
NEUROLOGY
Degeneration
483-492
11,
of
Olfactory by
RICHARD Department
of Biological
T.
(1963)
Received
Induced
Colchicine
JACKSON
Science,
Receptors
AND
Lojlola November
CHIN-CHIU University,
LEE’ New
Orleans,
Louisiana
4, 1964
Colchicine evokes drastic degenerative effects on the olfactory receptors and bulb when a small amount of the drug is infused directly into one olfactory bulb of the rabbit. Infusion of 20 lambda of 0.1 M colchicine evoked severe ipsilateral, transneuronal cell degeneration in the olfactory epithelium. Slight alterations were observed in the contralateral bulb and epithelium. Upon intraperitoneal injection of colchicine in doses as high as 10 mg/kg, only slight degeneration was observed in the olfactory bulbs and epithelium. In spite of the severity of degenerative changes evoked by infusion, some olfactory receptors were free from disintegration and persisted up to 8 weeks. Introduction
The olfactory system has the ability to distinguish among an astonishingly large number of odors. In an effort to explain this and other phenomena, such as selective adaptation, it has been postulated that some olfactory receptors are essentially different from other olfactory receptors. This view was made more seductive by LeGros Clark ( 11, 12) when he demonstrated selective degeneration of olfactory receptors. He removed one of the olfactory bulbs in a rabbit and observed that only half the ipsilateral olfactory receptors degenerated. One would expect all or none. We repeated this experiment, and in addition, removed both bulbs in rabbits. The same general results were obtained (13). Matthews and Powell (14) demonstrated an ascending transneuronal change in the olfactory bulb upon the removal of the olfactory epithelium. In an effort to corroborate and 1 This investigation the National Institutes of Medicine, Laboratory
was supported by research grants NB 02213 and AI 06094 from of Health. Dr. Jackson is presently at Emory University School for Ophthalmic Research, Atlanta, Georgia 30322. 483
484
JACKSON
AND
LEE
amplify these studies of mechanically induced, transneuronal degeneration, we decided to attempt selective, chemical degeneration with colchicine. It has been noted many times in the early literature that colchicine caused systemic disorders by acting upon the nervous system. While Ferguson and Theodore (8) showedthe physiological evidence of the action of colchicine upon the central nervous system, Singer et al. (16) were able to demonstrate for the first time the destructive morphological changesin following colchicine the peripheral nerves of the amphibian, Trituuus, treatment. Their findings were confirmed by the work of Angevine ( 1) on mammalian sciatic nerve.
Materials
and
Methods
Young rabbits, weighing 1-2 kg were used in all experiments becausethe olfactory epithelium of older rabbits has usually been damaged by inflammatory rhinitis. Intraperitoneal injections of colchicine”-doses ranged from 0.05 mg/kg to 10 mg/kg. Injected animals were allowed to survive for 2 days through 8 weeks,and killed by an overdose of Nembutal. The animal was exsanguinated, the frontal bones removed and the nasal septum-ethmoturbinate area removed in toto for fixation in alcohol-acetic formalin. Paraffin sections of 8 u were stained according to Bodian’s protargol technique (3, 9) and routinely with hematoxylin and eosin. For bulbar infusion (0.02 ml of 0.05 and 0.1 M) colchicine infusates were employed (which correspondsto 2% and 470 colchicine, respectively). The method of microinfusion was adapted from the design of Singer ( 15). The infusion apparatus consistedof a driving device and an injector whosemain body is a 0.25ml tuberculin syringe fitted with a 26-gauge needle. Polyethylene tubing connected the latter, metal needle to a glassneedle with a tip of about 100 lo in diameter. Under Nembutal anesthesia,a puncture hole was madewith a dental bur in the frontal bone just above the olfactory bulb. The glassneedle was inserted through the hole and about 1 mm deep into the dorsal portion of the bulb. In some cases,the opposite bulb was infused with the samevolume (0.02 ml) of saline solution, otherwise only a puncture wound was made. After an appropriate time has elapsed, the tissuewas removed and stained as described above. 2 Obtained
from
Nutritional
Biochemicals.
OLFACTORY
485
RECEPTORS
Efiects of B&bar Infusion of Colchicbe on the Olfactory Bulb and Olfactory Receptors. The results presented here were obtained from the four animals shown in Table 1. TABLE VARIARONS
OF PARAMETERS
EFFECTS
OF BULBAR
Colchicine concentration Animal No.
1 EMPLOYED
INFUSION
OF
Amount infused
Infusion time
Survival
(min)
(weeks)
120 45
6
15
8
15
2
1
0.05
2 3 4
0.1 0.1
(ml) 0.02 0.02 0.02
0.1
0.01
(M)
IN ASSESSMENT OF COLCHICINE
1
The olfactory bulbs of animal No. 1 are shown in Fig. 1. Not only shrinkage, but also immensealteration occurs in the laminar pattern of the bulb. Each of the bulbar layers is diminished in thickness. The layer of pr’imary olfactory fibers is almost replaced by connective tissue. The glomeruli become small and are poorly impregnated with silver stain due to the destruction of the ramifications, whereas the periglomerular cells are seemingly left intact. The external and internal plexiform layers are almost deprived of nerve fibers and most of the mitral and tufted cells are absent. Most cells in the granular layer also disappear. The latter become indistinguishable from the periventricular layer. In addition, blood vessel dilation and gliosis are notable all over the bulb in H & E preparations. The bulb is about 30% smaller than the control side. The control bulb was not entirely normal in appearance. Comparison with normal bulbs from other animals revealed subtle changes.The radial fibers in the external plexiform layer appear very short and the number of mitral, tufted, and small granular cells seemedreduced. Bulbar degeneration of the same severity was found when higher doses of colchicine infusate were used and longer postoperative survival periods were allowed. A 20-30s bulb shrinkage was observed in animals No. 2 and No. 3. The most obvious changeswere a decreasednumber of granular and mitral cells and radial fibers in the plexiform layer (Fig. 2). The changesseenin rabbit No. 4 were much lesssevere. Slight cell degeneration is represented by the diminished defective silver impregnation in the plexiform
mitral cell population layer (Fig. 3).
and
486
FIG.
colchicine
JACKSON
1.
Rabbit (left).
olfactory bulb The destructive
1 week changes
AND
LEE
following are more
infusion intensive
with 0.02 ml of 0.05 M and shrinkage of the
OLFACTORY
RECEPTORS
487
In all cases, whenever the olfactory bulb shows degenerative changes, a similarly intense secondary degeneration is found in the olfactory epithelium. In animal No. 1, the receptors in the olfactory epithelium of the infused side almost completely disappear in most regions. The thickness of the mucosa is also reduced. In the control epithelium, degeneration of receptors is also observed in approximate accordance to the severity of change between bulbs. Many olfactory rods have disappeared leaving gaps, and many receptor nuclei aer pyknotic. (See illustrations in Ref. 12.) Efiects of Intraperitoneal Injection of Colchicine on the Olfactory Bulb and the Olfactory Receptors. The minimal dose of colchicine employed in the present experiment was 0.05 mg/kg. This dose elicits barely detectable degenerative signs in the epithelium after 3 days. As little as 0.1 mg/kg elicits blood vessel dilation, inflammatory cells, unusual shaped rods and poor silver impregnation of nuclei after 48 hours. A more serious change in the nuclei of the receptors is found in sections taken 2 weeks after injection of 0.2 mg/kg colchicine. Many nuclei are missing and most of the residual cells display pyknotic granules in a pale cytoplasm. Surprisingly, increasing the dose (2, 3, 5 and 10 mg/kg) did not bring about more severe changes in the epithelium. At the 10 mg/kg dose, the bulb alterations are moderate. Neither the laminar pattern nor the bulbar size is changed. Upon close examination, however, notable are the fragmentation of the ramifications in some of the glomeruli and the reduced layer of mitral cells, some of which are in the course of chromatolysis. In addition, the dendritic fibers of the external plexiform layer appear to have been swollen and the number of granular cells is diminished. Nonspecific Pathological Reaction. Certain symptoms in the treated animals are due to the nonspecific poisoning effect of colchicine. At lower doses, by both routes, the animals often had respiratory difficulties and diarrhea. At higher doses (especially 5 and 10 mg/kg) paralysis of the hind limbs occurred in some instances. Discussion
In assessing the degree of degeneration of receptors one is thwarted by the fact that, in sections of normal olfactory epithelium, it is quite common to observe areas that display a few poorly stained receptors bounded by bulb more obvious as compared to other cases. A slight alteration can also be seen on the control side (right), which was infused simultaneously with physiological saline solution. Protargol; 150 X. Abbreviations: nf, layer of sensory nerve fibers; gl, plexiform layer; inp, internal plexiform layer; m, mitral glomeruli ; exp, external cells; gr, granular layer; and pv, periventricular layer.
FIG. 2. Rabbit olfactory bulb 8 weeks following infusion with 0.02 ml of 0.1 M colchicine (left). The numbers of mitral cells and their dendrites are diminished. Fewer granular layers are seen in contrast with those in the control (right) bulb. Protargol; 150 X. Abbreviations same as in Fig. 1.
FIG. 3. Rabbit olfactory bulb 2 weeks after infusion with 0.01 ml of 0.1 M colchicine (left). The disappearance of the small cells in the granular layer, the diminished mitral cell population, and defective silver impregnation in the external plexiform layer and glomeruli are notable as compared with the control (right) bulb. Protargol; 150 X. Abbreviations same as in Fig. 1. 489
FIG. 4. Olfactory 450 x.
Normal rabbit olfactory epithelium epithelium after infusion of the 490
(A). bulb
Note with
receptor elements colchicine (B).
(arrows). Protargol;
OLFACTORY
RECEPTORS
491
very densely packed receptor areas. With experience, however, one can evaluate damage. The relatively mild degeneration caused by intraperitoneal injection is not surprising if one realizes that several previous experiments with systemic colchicine failed to demonstrate neuropathological effects or the presence of labelled drug in nervous tissue (2, 4, 5). Also, it appears that neuropathological concentrations are much higher than those required to elicit systemic or mitotic effects ( 1) . Since infusion of 0.02 ml of 0.05 M colchicine evokes drastic destruction in both the olfactory bulb and receptors, it can be assumed that the threshold is much lower. On the other hand, very large doses do not obliterate all the receptors in spite of severe destruction in the bulb. We observed degenerative receptor losses of between 20-8070 which bracket the quotation of LeGros Clark (11). Regardless of the number, it would seem that, again, some receptors are resistant and some are not. This is not inconsistent with the findings of Eayrs (6) who observed rather less that half of the ganglion cells, whose axons comprise the optic nerve, persisted following optic nerve transection. It is doubtful that colchicine exerts a specific effect on nervous tissue. It has been found to inhibit dephosphorylation and deamination of desoxyribonucleotides at 1.2 X lo-” M (7). Hell and Cox (10) and Truhaut and Bohuon (17) have demonstrated inhibition of desoxyribonuclease activity. References 1.
2.
3. 4.
5. 6. 7.
1956. A study of toxic and neuropathological effects of J, B., JR. colchicine in rodents following direct infusion of nervous tissues. Ph. D. thesis, Cornell Univ. (L. C. Card No. Mic 56-975), 201 pp. University Microfilms, Ann Arbor, Michigan (Dissertation Abstr. 16: 625). BACK, A., E. WALASZEK, and E. UYEKI. 1951. Distribution of radioactive colchicine in some organs of normal and tumor-bearing mice. Proc. Sot. Exptl. Biol. Med. 77: 667-669. BODIAN, D. 1937. The staining of paraffm sections of nervous tissues with activated protargol. The role of fixative. Anat. Record 69: 153-162. BROWN, W. O., and L. SEED. 1945. Effect of colchicine on human tissue. Am. J. czin. Pathol. 16: 189-195. BRUES, A. M. 1942. The fate of colchicine in the body. /. Clin. Invest. 21: 646-647. EAYRS, J. T. 1952. Observations of the retina following optic nerve section. Brit. J. Ophthalmol. 36: 453-459. “Colchicine in agriculture, medicine, EIGSTI, 0. J., and P. DUSTIN, JR. 1955. biology and chemistry.” Iowa State College Press, Ames, Iowa.
ANCEVINE,
492
8. 9. 10.
11. 12. 13. 14.
15.
16.
17.
JACKSON
AND
LEE
F. C., JR., and P. THEODORE. 1952. On colchicine. I. General Pharmacology. J. Pharmacol. Enptl. Than@. 106: 261-270. FOLEY, J. 0. 1943. A protargol method for staining nerve fibers in frozen or celloidin sections. Stain Technol. 18: 27-33. HELL, E., and D. E. Cox. 1963. Effects of colchicine and colchemid on synthesis of desoxyribonucleic acid in the skin of guinea pig’s ear in vitro. ,Vatz~e 197: 287-289. LEGROS CLARK, W. E. 1956a. Observations on the structure and organization of olfactory receptors in the rabbit. Yale J. Biol. Med. 29: 83-95. LEGROS CLARK, W. E. 1956b. Inquiries into the anatomical basis of olfactory discrimination. Proc. Roy. Sot. London, Ser. B 146: 299-319. 1960. Olfactory nerve degeneration. MARTINEZ, I. R., and R. T. JACKSON. Assoc. Sozltheast. Biol. Btdl. 7: 34. MATTHEWS, M. R., and T. P. S. POWELL. 1962. Some observations on transneuronal cell degeneration in the olfactory bulb of the rabbit, J. Bnut. 96: 89-102. SINGER, M. 1954. .4pparatus ior continuous infusion of microvolumes of solution into organs and tissues. Pmt. Sot. Exptl. Biol. Med. 86: 378-380. SINGER, M., D. FLINKER, and R. L. SIDMAN. 1956. Nerve destruction by colchicine resulting in suppression of limb regeneration in adult Triturzrs. J. Exptl. Zool. 131: 459-491. TRUHAUT, R., and C. BOHUON. 1963. Influences of colchicine and N-desacethythiocolchicine on the desoxyribonuclease activity of the blood plasma in the mouse. Cornfit. Rend. 256: 162%163C.
FERGUSON,