232 Brain Researc& 384 ( 19861 232-239 Elsevier

BRE 12046

Autoradiographic Evidence for Receptor Cell Renewal in the Olfactory Epithelium of a Snail

RONALD CHASE and JANINE RIELING

Department of Biology, McGill University; Montreal, Que. (Canada)

(Accepted 4 March 1986)

Key words: Olfaction - - Cell renewal - - Chemoreceptor cell - - [~H]Thymidine - - Autoradiography - - Gastrolx~d mollusc

The tentacles of the terrestrial snail Achatinafulica contain an epithelium at their tips which is specialized for olfaction. The histolo- gy of the snail's olfactory organ bears a striking resemblance to that of the olfactory mucosa in the nose of vertebrates, where the re- ceptor cell population is known to undergo a continuous process of renewal. In the present experiments, [3H]thymidine was delivered as a single pulse that was determined to have a maximum duration of about 1 h, Thirty minutes after an injection of [3H]thymidine, presumptive precursor ceils were found labeled within, or at the edges of, receptor cell lobules. At later survival times, label was seen over cells that were identified as receptors. The mean position of the labeled cells within the Iayer Of receptor cells became progres- sively more superficial with increasing survival times, indicating an upward migration of newly differentiated cells. The labeling index in the snail is ca. 0.7c/c, compared to 0.9% in the mouse. The turnover time is about 45 days, compared to 30-45 days in the mouse.

INTRODUCTION

The occurrence of receptor cell renewal in the ol-

factory mucosa of vertebrate animals is well estab-

lished 7's'ls-l'2. Although neurogenesis is otherwise

rare within the nervous system of adult vertebrates.

especially mammals, the dual processes of receptor

cell death and replacement are a constant feature of

the olfactory epithelium in every vertebrate species

which has been examined. The precursors of the re-

ceptors are the basal cells which lie beneath the re- ceptors at the base of the olfactory epithelium 71s21 .

The basal cells divide by mitosis, and the daughter

cells then migrate upward through the epithelium as

they continue to differentiate and mature as sensory neurons s-~5.~6.

An analogous phenomenon of renewal has not yet

been described for any invertebrate chemosensory

system. We tested its generality by examining the ol-

factory organ of a snail, which is situated at the tip of

the snaiFs tentacles, rather than in a nose 6. Despite

the difference in gross structure, and in phylogeny.

the histological appearance of the snail olfactory epi-

thelium is remarkably similar to that of vertebrates.

Like tts vertebrate counterpart, the olfactory epithe-

lium in the snail is pseudostratified. It contains a pop-

ulation of receptor cells, which are primary sensory

neurons, and a unique population of gland celts s. The

anatomical placement of the snail receptors, and

their morphology, is very similar to that of the verte-

brate receptors 4A-22. In Achatina fulica, the species used in this study, each posterior tentacle contains

ca. 100,000 receptor cells 3. Recently, it has been

found that some of the receptor axons terminate in

synaptic glomeruli 21. as in the vertebrate olfactory

bulb. The applicability of 2-deoxyglucose autoradio-

graphic methods has also been demonstrated 3. These

experimental results, together with the flatness of the

olfactory epithelium, its ready accessibility and the

morphological similarities to vertebrate systems. make the snail's olfactory system an attractive alter-

native model for the study of olfaction.

A mechanism for the renewal of olfactory receptor

cells in the snail tentacle was suggested because these

neurons, like their vertebrate counterparts, have

dendrites which penetrate through a layer of epithe-

Correspondence: R. Chase. Department of Biology, McGill University, 1205 Av. Docteur Penfield. Montr6at. Qud. H3A IB1. Ca- nada.

233

lial cells to an exposed surface. Although the snail's tentacle can be retracted inside the body for defense, or during rest, when it is operating as an olfactory sensor the receptor endings are vulnerable to irrita- tion from environmental sources, microbial infection and desiccation. The capability to generate new re- ceptor cells could provide for the continuity of olfac- tory function despite the loss of cells.

This study used the method of [3H]thymidine auto- radiography to establish cell proliferation and to identify the precursor population 11. Thymidine is in- corporated into newly synthesized DNA just prior to cell division. The interpretation of cell labeling with [3H]thymidine is greatly facilitated if it can be dem- onstrated that the [3H]thymidine is available for up- take during only a short interval following an injec- tion. A part of this report, therefore, concerns a de- termination of the [3H]thymidine availability time in the snail.

MATERIALS AND METHODS

Snails, Achatina fulica, were obtained from a labo- ratory culture 17. The animals weighed 25-35 g. Their sexual maturity was indicated by the reproductive ac- tivity of siblings. [3H]thymidine (spec. act. 60-90 Ci/mM) was injected into the haemocoel at the dorsal portion of the foot directly beneath the shell. A dos- age of 4/~Ci/g was mixed with ca. 1 ml of snail Ring- er's solution and a drop of blue food coloring. The food coloring allowed observations to determine the distribution of the injected solution and to check for leakage once the needle had been withdrawn. All in- jections were done in the morning in order to control for circadian variables.

The radioactive haemolymph content was ana- lyzed by means of samples drawn from a cannula that was inserted into one of the tentacles ~. The cannula consisted of a length of polyethylene tubing (PE 90), ca. 70 mm long, which was inserted into the tentacle following an injection of 0.5% succinylcholine chlor- ide to immobilize the animal. The cannula was tied in place with surgical silk and further secured with a drop of cyanoacrylate glue. The free end was sealed with wax; this end was cut off to collect samples and then subsequently resealed.

Samples of the haemolymph were placed in a boil- ing water bath for 3 min to precipitate proteins and to

inactivate enzymes. They were then centrifuged for 15 min at 13,000 rpm and placed on ice. A 5~tl sample of the supernatant was analyzed by thin layer chro- matography to determine the thymidine and thymine content. The solvent system was the upper phase of ethyl acetate:water:formic acid in the ratio 65:35:10. Standards were located using ultraviolet light, and corresponding areas of the experimental samples were scraped off. Radioactivity was eluted from the silica gel by adding ca. 1 ml of water, mixing thor- oughly, and centrifuging the suspension for 1 rain at 13,000 rpm. The procedure was twice repeated, re- sulting in a total volume of 3 ml for scintillation counting. A 25 gl aliquot of haemolymph superna- tant was also counted to determine the total tritium content. The counts were corrected for efficiency by internal standardization.

For autoradiography, the animals were sacrificed at various times following the injection of [3H]thymi- dine. The tentacles were excised, washed in 3 changes of snail Ringer's solution for 5 min each, then pinned flat in a solution of 1% glutaraldehyde and 1% paraformaldehyde for 1 h. They were em- bedded in Araldite resin and cut transversely as 1-/~m thick sections at intervals of 8 ~m. In pilot experi- ments, the entire tentacle tip was examined, but for the analytical work sections were taken only from the middle region of the olfactory epithelium, The slides were coated with NTB2 emulsion (Kodak) that was diluted 2:1 with distilled water at 40 C. Devel- opment was done with D-19 (Kodak) for 5 min at 16 C. The slides were counterstained with 0.5% to- luidine blue (pH 11).

RESULTS

Determination of the FH]thymidine availability time In order to establish the levels of radioactive com-

ponents at various times after an injection, the theo- retical distribution of tritium at 'zero time' was esti- mated assuming complete and instantaneous mixing in the haemolymph. This assumption is supported by observations using the blue dye that was part of the injected solution. Within seconds of an injection, the entire snail, including the tentacles, acquired a bluish tint, indicating a rapid dispersion of the injected ma- terial. Since the isotope was injected in a dosage of 4 uCi (8.88 1() ~ dpm) per gram, and since there is ca.

234

100 - -- Tota l t r i t ium

E ~% ~% Thymine T Bo

0 70 i

N60 i

U , - . 40 1

n t . . .30 - Ti ~

;20 i 10 L ~ I I

i i i i ~ ) ) i - , i i

1 12 3 4 5 10 20 30 60 120

Minutes a f te r In iec t ion

Fig. 1. Levels of radioactive substances in the haemolymph at various times after a single injection of [3H]thymidine. The points are means _+ S.D. The sample size is 5 animals at all times except at 60 min and 120 min, where the sample sizes are 4 animals and 3 animals, respectively.

0.40 ml of haemolymph per gram in Achatinafutcia ~=. the theoretical total radioactivity circulating at zero time is 2.22 x 107 dpm/ml.

The levels of total tritium, thymidine and thymine in the circulation at various times after an injection are shown in Fig. 1, relative to the amount of total tri- tium at zero time. It is assumed that all initial activity is present as [3H]thymidine, with little or no decom- position or product impurity. The thymidine concen- tration falls to 66.2% of its initial value after 30 s. 3.2% after 5 min and 1.6% after I h. From these data, a conservative estimate of the pulse duration for a single injection of [3H]thymidine is l h. It is un- likely that the small amount of [3H]thymidine still re- maining in the circulation at this time would make a significant contribution to the number of labeled cells '~.

Epithelium Muscle Receptor cell Iobules

Digits (neuropil) Gland cells 100/Jm

Fig. 2. The olfactory epithelium of Achatina fulica. Transverse histological section through the tip of a posterior tentacle. The olfacto- ry receptor cells are aggregated in lobules beneath the outlying epithelial and muscle layers. Protein secreting glands lie between the receptors and the digits, which are branched extensions of the tentacle ganglion.

235

~ ~ i~ fill ~ ~i ~ ~ ~i!~ii ~#~ ~i i~ i~ ~

~!!~ii~ ~, i~i ii!!!!~il ~ ~ ~

Fig. 3. Examples of cells labeled with [3H]thymidine (arrowheads) following a single pulse injection. A: 30-rain survival: the line marks the border between the muscle layer and the lobules containing receptor cell bodies (see Fig. 2). B: 30-rain survival; the labeled cells lie bctween thc receptor cells above and the protein-secreting gland cells (PG) below. C: l-day survival; the labeled cell lies su- perficially in a receptor cell lobule, D: 14-day survival; the labeled cell lies among receptor cells near the middle of a lobule.

236

Apart from [3H]thymidine, the radioactive com- pounds in the circulation after zero time consist of thymine and other catabolic products 2J9. None of

these substances is taken up by the cell nuclei, and therefore they do not affect the pulse duration. After about 5 min, most of the tritium activity is probably contained in tritiated water.

Autoradiography When the snail olfactory organ is viewed in cross-

section (Fig. 2), several types of cells are evident, each having a characteristic morphology and loca- tion. The cellular composition of the tentacular olfac- tory organ is thoroughly described elsewhere 4-~'1~'22. The autoradiographs that were examined in this inves- tigation contained labels over several types of cells, namely, epithelial cells, glial cells, protein glands and receptor cells ~s. Interneurons were never labeled. Cells belonging to each of the aforementioned types could be positively identified and unambiguously dis- tinguished from cells of the other types. There were, in addition, other labeled ceils whose appearance conformed to none of the previously described cell types in the snail tentacle. These latter cells are thought to be the undifferentiated precursors of the receptor cells. Only the receptors and their precur- sors are considered in this report. A cell was re- garded as labeled if there were 6 or more silver grains

overlying the nucleus. Labeled cells were seen at all survival times (Figs.

3 and 4). They were frequently observed as pairs

5

03

~3

d 2

"3

E 1

Z

g

I , \

i 1

12 24 5 10 15 20 Hours Days

T ime a f te r In jec t ion

060 r'-

o-

045 ~-

E 030 ,

Fig. 4. Time-course of cell labeling following a single injection of [3H]thymidine. One tentacle was examined from each ani- mal sacrificed at the times shown. The points are means +_ SD. for 2 animals (25 sections each) at 3 days and at 14 days; 3 ani- mals (25 sections each) at all other times.

(e.g. Fig. 3B). At 30 min, the majority of the labeled cells were situated at or near the borders of the lo- bules containing receptor cell bodies (Fig. 3A and

B). Usually, these cells had a size and a shape that was different from any of the previously described cell types in the snail olfactory epithelium (Fig. 3B), but other cells were similar in appearance to receptor cells (Fig. 3A).

At survival times after 30 min, all the labeled cells were situated within the receptor cell lobules (Fig. 3C and D). The receptor cells in Achatina are gener- ally ovoid in shape and measure approximately 6 8 l~m. The nucleus is highly chromatic and can vary in appearance from light to dark (Fig. 3). The darker cells seemed to be preferentially labeled at early sur- vival times and the lighter cells at longer survival times. However, this impression could not be quanti- fied because of the obscuring effect of the silver grains and the non-categorical variation in nuclear density.

The labeling index was estimated to be 0.7% at 30 min and it remained at about the same level until day 10, after which it fell to 0.3% on day 30 (Fig. 4). The gross distribution of the labeled cells appeared to be uniform in all regions of the olfactory organ at all times.

The labeled cells shown in Fig. 3 illustrate the gen- eral finding that cells were heavily labeled at short survival times, but more lightly labeled at long sur-

I It 35

~r

.$

"3

z

30 min 12 hrs 24 hrs 3 days days t0 days 14 days 30 (lays

T ime a f te r In jec t ion

Fig. 5. The number of silver grains over individual cells as a function of time after an injection of [3H]thymidine. Each bar represents a single tentacle, with each tentacle coming from a different snail. Standard deviations are shown. The line is the least squares regression; P < 0XX)I.

vival times (compare Fig. 3A and B with Fig. 3D).

The densities of autoradiographic labels were

measured by counting the number of silver grains over each labeled cell (Fig. 5). At 30 min the mean

number of grains was ca. 22. By day 30, the mean

number of grains had fallen to ca. i2. The coefficient

for the least squares regression for the data shown in

Fig. 5 is -0.23; P < 0.001. A measure of labeling that

is more sensitive to the dynamics of a dividing cell

population is the number of cells labeled at either ex-

treme of the grain density range. The cells were

therefore sorted into a lightly labeled class (6-12

grains) and a heavily labeled class (>20 grains). The

number of lightly labeled cells and the number of

heavily labeled cells are shown as a percentage of all

labeled cells in Fig. 6. It can be seen that the number of heavily labeled cells decreases with survival time,

whereas the number of lightly labeled cells increases.

The two curves cross at 16 h, which represents the

theoretical mean time for the division of those cells which incorporated [3H]thymidine at, or near, zero

time.

Although labeled cells were seen at all positions

within the lobules at all survival times, the mean

depth of the labeled cells, as measured from the epi-

thelial surface, progressively declined with increas-

ing survival times (Fig. 7). At 30 min, the mean depth

was 145 Hm, which corresponds to a position deep in

the Iobules. By day 30, the mean depth was 108 #m,

which is superficial in the lobules. The coefficient for

7o l co L ight ly labe led ce l l s

60

~ 50

3o

t t"-"----I c~ i l l / ~o .~ /_ c

Heav i ly labe led ce l l s

12 24 5 10 15 20 25 3Q

Hours Days

T ime a f te r In jec t ion

Fig. 6. Time-dcpendent changcs in the number of lightly la- beled cells (6 12 grains: solid line) and heavily labeled cells (>20 grains: dashed line). The points are means _+ S.D.

237

3

E

~z

_ LU CZ , . o

o

2t] T, 20o

30 min 12 hrs 24 hrs 3 days 7 days 10 days 14days 30 days

T ime a f te r In jec t ion

Fig. 7. The location of labeled cells at various times following an injection of [3H]thymidine. Each bar represents a single ten- tacle, with each tentacle coming from a different snail. Stan- dard deviations are shown. The line is the least squares regres- sion; P < 0.006.

the least squares regression for the data shown in Fig. 7 is -0.88; P < 0.006.

DISCUSSION

It is generally assumed that when [3H]thymidine is

delivered as a pulse, the cells labeled at short survival

times are those that are in the process of duplicat- ing DNA prior to mitosis ~1. At longer survival times

(ca. 20 h or more), a labeled cell is understood to be

the product of division by a cell which initially incor-

porated the isotope. The application of these as-

sumptions to the observations of cell labels in the

present experiments (Fig. 3) leads to the conclusion

that proliferation occurs within the receptor cell pop-

ulation of the snail olfactory epithelium. Further evi-

dence for cell replication is contained in the finding

that the density of the labels over individual cells de-

creases with time (Figs. 5 and 6), as would be ex-

pected if the radioactive DNA were distributed to

daughter cells. The replication of receptor cells ap-

pears to be part of a renewal process within the re-

ceptor population, rather than an expansion of the

population, because the number of labeled cells

eventually decreases (Fig. 4), indicating that cell rep- lication is accompanied by cell death.

In contrast to the situation in the vertebrate olfac- tory epithelium 7J5,2, the snail tentacle does not seem

to contain a readily identifiable and anatomically seg-

regated population of precursors for the receptor cell population. Our results indicate that the precursors

238

are scattered among the mature receptors within and around the iobules. In vertebrates, the precursors are generally believed to be the basal cells which lie beneath the receptors, but it should be noted that 20 min after an injection in a mouse, about 10% of the labeled cells are found above the region of basal cells 15. In the snail, there is an upward migration of newly formed receptors (Fig. 7) similar to that de- scribed in vertebrates s'~5"~6. The difference between the segregated arrangement for the precursors in

vertebrates and their distribution among the recep- tors in Achatina may be related to the size of the area occupied by the receptors. In vertebrates, the recep- tors form a layer ca. 20-50 f~m thick. Since the basal cells lie directly beneath the receptor layer, their pro- geny need only migrate a maximum of 50/~m. In Achatina, the lobules may be as thick as 160 urn. Were it not for the fact that the precursor cells are vertically distributed within the lobules, the required migration distance would be much greater than it is in vertebrates.

In two species of slugs, both closely related to Achatina, the tentacular receptor cell population can be differentiated into two types, using ultrastructural criteria t'22. The alpha neuron is described as smaller than the beta neuron; it has a slightly irregular and chromatic nucleus with a small nucleolus. The beta neuron has a larger, more spherical nucleus than the alpha cell; the nucleus is also less chromatic, and the nucleolus is larger. This distinction in receptor cell morphology is relevant to the present results because the alpha receptor type is found predominately near the base of the lobules, whilst the beta receptors are found at superficial positions 1'22. It is possible, therefore, that the alpha cells are immature recep- tors and the beta cells are mature receptors that have migrated upwards. A confirmation of this hypothesis will require electron microscopic autoradiography. An intriguing finding that relates to the proposed vertical migration is the fact that only superficial re- ceptor cells become labeled with [14C]2-deoxyglu- cose after exposure to odors 3, suggesting that matu- ration is accompanied by an increase in metabolism. or an increase in sensitivity to stimuli, or both.

The number of labeled cells decreases with time af- ter an injection, suggesting that cells die and disap- pear, as would be expected in a population which is continuously renewed, tf one extrapolates the de-

cline in the number of labeled cells from day 10 (Fig. 4), a turnover time of about 45 days can be estimated. This compares with estimates of ca. 30-45 days for the turnover time in mice ~>14.

It might be expected that the number of labeled cells would increase during the initial period after an injection, when the labeled DNA is presumably be- ing distributed to the progeny of dividing cells. Cu- riously, this result was obtained neither in the present work nor in the investigations of the mouse olfactory mucosa carried out by Moulton and co-workers is The absence of an initial increase in the labeling index may be explained, in part. by the assumption of a non-synchronous division of cellsl ,,)wing to variable times of [3H]thymidine uptake in relation to the dura- tion of the S-phase of the cell cycle A second factor contributing to the absence of a net increase in the la- beling index is the occurrence of cell death concur- rently with cell proliferation. Whereas the influence of cell death is only apparent in "d~e labeling index from day 10 onward, the death rate prior to that time may be just sufficient to offset gains in the population due to proliferation. It is interesting to note that in the mouse 15, as in the snail, the decline in the labeling index also begins at about day 1().

Because the precursor cell population cannot be readily identified, and therefore quantified, the la- beling index in this study is expressed as a percentage of the number of receptor cells (Fig. 4). During the first 24 h, the labeling index is ca. (1.7%. This value can be compared with a labeling index of 0.9% in the mouse, where the percentage is based on the total number of cells of all types in the olfactory epitheli- um j''. Because the labeling index reflects both the probability of DNA synthesis in a given cell at a given time and the availability time of [~Hlthymidine, the interpretation of the similarity between snail and mouse depends on an appreciation of the pulse dura- tion times in the two species. We have no information on the pulse duration in mice, but remarkably, the time course of [3H]thymidine availability m the rat corresponds closely to that seen in the snail, when similar methods are used for its determination. The comparison is shown in Table 1. If one assumes that the clearance rate in mice is close to that in rats, then evidently the proliferation of receptor cells proceeds at about the same rate in mice as it does in snails,

Taken together, the results ~f ~his investigation

239

TABLE I

Comparison of [~ H]thymidine clearance times in ,snail and rat folio wing a single injection

The data t;or rat are taken from ref. 2.

Time after injection Percentage of initial FH]thymidine remaining in the circulation

Rat Snail

5 rain 3.6 3.2 30 min 2.4 2.0 60 min I).4 1.6

demonst ra te a process of renewal in the o l factory ep-

i thel ium of a snail which is s imi lar to that which exists

in ver tebrates . P resumably , the common basis for the

renewal in both cases is the exposure of the receptors

to a harmfu l externa l env i ronment . Neurob io log is ts

now have avai lable two a l ternat ive mode l systems in

which to study the processes of death and renewal in

a nerve cell popu lat ion , and the prob lem of how the

new receptor cells are able to establ ish correct ana-

tomical connect ions w i thout d isrupt ing sensory func- t ion 13.

ACKNOWLEDGEMENTS

We thank Joan Marsden and Rober t Lev ine for

advice and crit icism. This work was suppor ted f inan-

cially by a grant f rom the Natura l Sciences and Engi-

neer ing Research Counci l of Canada.

REFERENCES

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