Developmental Brain Research, 1 (1981) 283-286 283 © Elsevier/North-Holland Biomedical Press

Short Communications

Cell survival in chick embryo ciliary ganglion is reduced by chronic ganglionic blockade

LINDA WRIGHT*

Neuroembryology Laboratory, N.C. Division of Mental Health Research Section, Anderson Hall, Dorothea Dix Hospital, Raleigh, N.C. 27611 (U.S.A.)

(Accepted November 20th, 1980)

Key words: ciliary ganglia - - chick embryo - - chlorisondamine - - cell death

Changes in the activities of enzymes involved in the synthesis or degradation of transmitters has been used as an index of maturation in autonomic neuronsL In some cases, a reduction in enzyme activity during normal development may reflect decreased cell survival rather than, or in addition to, changes in the rate of development ~,6. Similarly, changes in enzyme activity following experimental manipulation during development may also reflect cell loss. In the ciliary ganglion, biochemical indexes of development are altered after denervation 14, and after treatment with a ganglionic blocker, chloris- ondamine 4. Surgical removal of afferents to the ciliary ganglion in the early chick embryo results in a virtually complete loss of ganglion cells 1°. Chiappinelli et alA have reported that choline acetyltrans- ferase (CHAT) is reduced in chick iris and ciliary ganglion after chronic treatment with chlorisondamine (on days 5, 8, 10 and 13 of incubation). This suggests that the development of transmitter enzymes may be regulated via interaction at the presynaptic site. Since they also report that chlorisondamine treat- ment reduced ganglionic dry weight 4, which could reflect a loss of cells, it may be that the same synaptic interaction governs cell survival as well as enzyme maturation. The present study provides evidence that treatment with chlorisondamine does, in fact, reduce cell survival in the ciliary ganglion.

Ch lo r i sondamine was admin is te red into the yolk sac o f chick embryos on days

6, 8, 10 and 12 o f incubat ion, a t a dose o f 1.87 mg in 20/A saline. This is the same dose

tha t was given in the previously ment ioned s tudy 4, with a slight modif ica t ion in the days

o f adminis t ra t ion . Thus, the dose given is slightly higher than the one tha t Chiappinel l i

et ah found to p roduce reduced volume and lowered C h A T activi ty in chick cil iary

ganglia. The embryos were sacrificed at 14 days o f incubat ion, and the entire heads were

fixed in Carnoy ' s solution. Subsequently, bra in stems and ci l iary gangl ia were dissected

out, embedded in parap las t , sect ioned at 12 and 8 #m, respectively, and s tained with

thionin. Cell counts were made f rom every fifth or tenth section at a magnif icat ion o f

500 × using a drawing tube. Only cells with a dist inct ch romat in mass were counted,

and Abe rc romb ie ' s cor rec t ion 1 was appl ied. The pe r iod o f na tu ra l cell death in the

gangl ion occurs between embryon ic days 8 and 15 (see ref. 9), so these counts were made

af ter the m a j o r pe r iod o f cell death. Cell counts (Table I) show tha t chronic t rea tment

with ch lor i sondamine dur ing the pe r iod o f cell dea th reduces the survival o f ci l iary

* Present address: Department of Anatomy, The Medical College of Pennsylvania, 3300 Henry Avenue, Philadelphia, Pa. 19129, U.S.A.

284

T A B L E I

('ell numbers in chick ciliary ganglia

Control Chlorisondamine

Choroid Ciliary Total Choroid Ciliary Total

2298 2441 2502

Z ::= 2414

%ofcontrol:

1518 3816 1778 1216 2994 1260 3701 1833 1072 2905 1356 3858 1661 1114 2775

1920 1011 2931 1880 1069 2949 1872 960 2832

1378 3797 1824 1074 2898

75.6 77.9 76.4

ganglion neurons by 24 ~ as compared to controls; ciliary and choroid cell populations were reduced by the same amount.

The question then arose as to whether ganglionic blockade and subsequently reduced ganglion cell survival would affect survival of cells in the accessory oculomotor nucleus (AOM), which is the sole source of afferents to the ciliary ganglion 11. Natural cell death in the AOM occurs over approximately the same developmental period as does cell death in the ciliary ganglion 12, although onset of cell death in the AOM may lag somewhat behind the onset in the ganglion 5. Cell counts (Table II) reveal little in- dication of a reduction in cell numbers in the AOM by day 14 after postsynaptic receptor blockade by chlorisondamine. This effect is markedly different from that seen in the lateral motor column motorneurons after neuromuscular blockade with curare, where this treatment prevents much of the natural cell death among the motorneuronslL Unlike the situation in the neuromuscular system, however, in the present case we have no independent verification of the extent of receptor blockade. Thus, it is conceivable that higher doses of chlorisondamine might give different results. Regrettably, the dose used here was the highest nonlethal dose possible with the chick embryo. Alternatively, one might expect that the reduced numbers of ganglion cells present following chloris- ondamine treatment to constitute a reduced periphery, thus resulting in the survival of fewer AOM cells, similar to the situation reported in the lateral motor column of the chick and in many other systems following reduction of the periphery 17. One possible explanation for the finding of no differences in AOM cell numbers in this study is that the trophic influence factor that supports the AOM cells, and which is presumably supplied by the ganglion cells, is not blocked by postsynaptic blockade, and that even a somewhat reduced ganglion is capable of supporting all of the cells of the AOM. Black 2 has commented that only severe postsynaptic lesions in the sympathetic nervous system interfere with presynaptic ontogeny. This may also be the case with the ciliary ganglion-AOM system. Since normally there are twice as many cells in the ciliary ganglion as there are in the AOM, it seems likely that each AOM cell innervates more than one ganglion cellL If under the present experimental condition a single ganglion cell can provide adequate 'trophic' support for an AOM cell, then the 24 ~o reduction in ganglion neurons might not be reflected by a cell loss in the AOM, even if the latter are dependent upon the ganglion cells for survival.

TABLE II

Cell numbers in chick accessory oculomotor nucleus

285

Control Chlorisondamine

1724 1739 1770 1562 1693 1646 1755 1631

1701 1562

Z = 1735.5 z = 1640 (94.5~oofcontrol)

To further test the presynaptic effects of chlorisondamine, cell counts were made on the dorsal motor nucleus of the vagus (DMV), another source ofpreganglionic para- sympathetic fibers s. Table III shows that there were no differences in cell number between controls and chlorisondamine-treated embryos. At the time the cell counts were done, on day 14 of incubation, 29 ~o of the original DMV cells have been lost in the process of natural cell death. Natural cell death in the DMV continues until sometime after hatching, resulting in a loss of 49 ~o of the cells. It is not known what effect chloris- ondamine treatment has on visceral parasympathetic ganglia. Preliminary data on the effects of chronic treatment with ganglionic blockers (chlorisondamine and hexame- thonium) on cell survival in the spinal sympathetic preganglionic nucleus of Terni indicate that this population is also unaffected by receptor blockade (R.W. Oppenheim, personal communication).

In summary, treatment of chick embryos with chlorisondamine, which blocks ganglionic receptors, during the period of cell death produces a 24 ~o decrease in the number of cells surviving in the ciliary ganglion, but has no apparent effect on cell survival in preganglionic neurons of the accessory oculomotor nucleus, in preganglionic neurons of the spinal nucleus of Terni, or on the dorsal motor nucleus of the vagus. Thus, the present results indicate that receptor interactions may be a critical factor in the mediation of afferent influences on cell survival in the ciliary ganglion 10. Moreover, further comparisons between presynaptic systems which respond to receptor blockade by changes in cell survival (e.g. the neuromuscular system15), and those which do not, may lead to a better understanding of the mechanisms involved in naturally occurring neuronal cell death.

TABLE llI

Cell numbers in the dorsal motor nucleus o f the vagus

Control Chlorisondamine

5630 5579 5775 5541 5724 5534 5720 5341

X=5712 5499 (96~ofcontrol)

286

This research was supported in part by N I H postdoctoral fellowship 1 F32

NS06422-01.

1 would like to thank Dr. Ron Oppenheim for his valuable comments on this

manuscr ipt and his helpful suggestions th roughout the course of this research.

1 Abercrombie, M.,Estimation of nuclear population from microtome sections, Anat. Rec.,94(1946) 239-247.

2 Black, I. B., Regulation of autonomic development, Ann. Rev. Neurosci., l (1978) 183-214. 3 Caserta, M. T., Johnson, E. M. and Ross, L. L., Effect of destruction of the postganglionic sym-

pathetic neurons in neonatal rats on development of choline acetyltransferase and survival of preganglionic cholinergic neurons, Neurosci. Abstr., 2 (1976) 295.

4 Chiappinelli, V. A., Fairman, K. and Giacobini, E., Effects of nicotinic antagonists on the develop- ment of the chick lumbar sympathetic ganglia, ciliary ganglion and iris, Develop. Neurosci., 1 (1978) 191-202.

5 Cowan, W. M. and Wenger, E., Degeneration of the cells of origin of the ciliary ganglion after early removal of the optic vesicle, J. exp. ZooL, 168 (1968) 105-124.

6 Giacobini, G., Filogamo, G., Weber, M., Bouquet, P. and Changeux, J. P., Effects of a snake a- neurotoxin on the development of innervated skeletal muscles in chick embryo, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 1708-1712.

7 Jacobson, M., Developmental Neurobiology, 2nd Edn., Plenum, New York, 1978, pp. 281-293. 8 Krause, R., Mikroskopische Anatomie der Wirbeltiere in Einzeldarstellung, Walter de Gruyter

Berlin and Leipzig, 1921. 9 Landmesser, L. and Pilar, G., Synaptic transmission and cell death during normal ganglionic

development, J. Physiol. (Lond.), 241 (1974) 737-750. 10 Levi-Montalcini, R., Regressione secondaria del ganglio ciliare dopo aspotazione della vesciola

mesencepfalica in embrione di polio, R. Acad. Naz. Lincei, 3 (1947) 144-146. 11 Narayanan, C. H. and Narayanan, Y., An experimental i~*quiry into the central source of pre-

ganglionic fibers to the chick ciliary ganglion, J. comp. Neurol., 166 (1976) 101-110. 12 Narayanan, C. H. and Narayanan, Y., Neuronal adjustments in developing nuclear centers of the

chick embryo following transplantation of an additional optic primordium, J. Embryol. exp. Morphol., 44 (1978) 53 70.

13 Oppenheim, R. W., Neuronal cell death and some related regressive phenomena during neuro- genesis. A selective historical review and progress report. In W. M. Cowan (Ed.), Studies in De- velopmental Neurobiology : Essays in Honor of Viktor Hamburger, Oxford University Press, in press.

14 Pilar, G., Jenden, D. J. and Campbell, B., Distribution of acetylcholine in the normal and denervated pigeon ciliary ganglion, Brain Research, 49 (1973).

15 Pittman, R. H. and Oppenheim, R. W., Cell death of motoneurons in the chick embryo spinal cord. IV. Evidence that a functional neuromuscular interaction is involved in the regulation of naturally occurring cell death and the stabilization of synapses, J. comp. Neurol., 187 (1979) 53-70.