Choline acetyltransferase (CHAT) activity was determined in layers of optic tectum in control goldfish and in goldfish 4-20 days fol- lowing unilateral enucleation. Significant changes in activity were found in the periventricular (PV) and superficial gray and white (SGW) layers. Within 4 days, ChAT activity in the PV layer on the lesioned side was about 75% of that on the control side. By 20 days, ChAT specific activity in the SGW layer on the lesioned side was about 150-160% of that on the control side. This increase in specific activity in the SGW layer was accounted for by the decrease in volume and in density of the layer after enudeation, so that the total amount of activity in the layer did not change significantly, indicating that the optic terminals contain little to no ChAT activity. ChAT activity in the optic tract was very low and did not decrease after enucleation. These data strongly indicate that the retinotectal path- way in goldfish is not cholinergic and, therefore, that the ChAT activity in the SGW layer is related to sources other than retinal gangli- on cells. It is suggested that one such source might be neurons with somata in the PV layer.
INTRODUCTION
Acetylcholine has been repeatedly proposed to be
the transmitter of the retinotectal projection in gold- fish (Carassius auratus)22,27. One important piece of
evidence for this suggestion involves the binding and
action of a-bungarotoxin and other nicotinic cholin-
ergic antagonists. A high density of a-bungarotoxin binding sites (equated with presumed postsynaptic
nicotinic receptors) was localized to the tectal layers
receiving optic projection; this density significantly decreased following enucleation 23. In addition, a-
bungarotoxin and reversible nicotinic antagonists,
such as curare, were reported to reduce the postsyn- aptic response to optic nerve stimulation7, z7. Finally,
the high choline acetyltransferase (CHAT) activity
found in optic rectum was reported to decrease by about 10-15% following enucleation 5. These data were interpreted as being consistent with a choliner-
gic retinotectal projection in goldfish. However, while high activities of CHAT, the enzyme synthesiz- ing acetylcholine, do occur in cholinergic axons and terminals, it is well known that the interruption of a
cholinergic projection produces a rapid and drastic
loss of enzyme activity in the axons distal to the lesion and in the axonal terminals 14. The small magnitude of
the decrease in tectal C h A T activity following optic
nerve cut 5 could mean that the optic nerve terminals
contain little to no C h A T activity, an interpretation consistent with the extremely low C h A T activity in
the vertebrate optic nervet3, 25. However, it could
also be argued that if this small decrease in C h A T ac-
tivity, found in homogenates of the entire tectum, were actually concentrated within discrete layers,
such as those to which the optic axons project, the ac-
tivity loss in these specific layers might be much more
significant. The method of quantitative histochemis- try 17,18, providing both quantitation and high histo-
logical resolution, was used to address this question. Samples of individual tectal layers were cut from freeze-dried sections of goldfish optic tectum and as-
sayed for C h A T activity; activities in layers from
right and left tecta were compared at different times following enucleation of the right eye. Two questions were addressed: first, how does the distribution of ChAT activity among tectal layers compare to that of
Correspondence: C.D. Ross, Department of Physiology, Oral Roberts University, Tulsa, OK 74171, U.S.A.
optic nerve terminals and, second, what is the effect
of optic terminal degeneration on this enzyme activ- ity? Of particular relevance to the question of a cho- linergic retinotectal projection is the effect of enu- cleation on ChAT activity in the superficial gray and white (SGW) layer, which receives the highest densi- ty of optic terminals26.es.29, 32. If the optic projection
provided the only or major cholinergic input to the SGW layer, a drastic drop in both total and specific ChAT activity would be expected in that layer fol- lowing enucleation. If only some of the optic projec- tion were cholinergic, and much cholinergic input from other sources also terminated in the layer, then it is possible that cutting the optic nerve would reduce the total ChAT activity in the SGW layer, but might produce little change in the specific activity if the amount of enzyme loss was proportional to the reduc- tion in volume accompanying the degeneration of the optic terminals. If none of the optic projection were cholinergic, enucleation would produce essentially no change in total ChAT activity (except for possible transneuronal effects), but an increase in specific ac- tivity because of the reduced volume of the layer. The data to be presented from this study strongly fa- vor this last interpretation, indicating that the retino- tectal projection in goldfish is not cholinergic.
MATERIALS AND METHODS
Tissue handling Common goldfish (Carassius auratus) were used in
the study. Most fish, including all in the enucleated
group, were 10-13 cm in length and were obtained through a local supplier. Two additional goldfish, 7-8 cm in length, were obtained from Carolina Bio- logicals and were included in the control group. Ex- perimental fish were anesthetized in 0.1% ethyl m-aminobenzoate methanesulfonate (MS-222). The fight eye was removed surgically in 2-3 min, after which time the fish was returned to water. All fish survived without obvious difficulties until killed 4, 7, 10 or 20 days following enucleation. Brains were dis- sected out and frozen within about 10 min of death either on Freon cooled to -130 °C with liquid nitro- gen or on dry ice. Frozen tissue was mounted onto wooden dowels with brain paste and sectioned at 20-25 /~m thicknesses, at -20 °C. Frozen sections were saved in aluminum racks for freeze-drying at
-40 °C (ref. 18) or thawed onto microscope slides for staining either with thionin (Fig. 1), for acetylcholin- esterase (ACHE) activity 2.L~ or for ChAT-like immu- noreactivityL Freeze-dried sections were stored in tubes under vacuum at -20 °C.
Outlines of and boundaries within freeze-dried sections were mapped using a Wild dissecting micro- scope with drawing tube attachmentS. Boundaries and cell layers visualized in the thionin-stained sec- tions (Fig. 1) were traced onto the same map to aid in the identification of tectal layers. Samples were dis- sected from the rectum and the exact locations of sample boundaries were recorded on the map. Mapped sample boundaries were entered into a Hewlett Packard 9845B computer using a Hewlett
Packard 9874A digitizer, for computation of individ- ual sample areas and/or for printing, using a Hew-
lett Packard x - y plotter (Fig. 2). Dry weights of sam- ples were determined using quartz fiber balances 18 and ranged from about 0.05 to 0.6/~g, with the aver- age size being about 0.2/~g. Samples were loaded into 400/~l-capacity microcentrifuge tubes for assay of ChAT activity.
ChAT assay The determination of ChAT activity was based on
the radiometric method of McCaman and Hunt 19, using the sodium tetraphenylboron procedure of Fonnum3 for the extraction of [1-14C]acetylcholine. Samples were incubated for 30min at 37 °C. Further details of the assay have been published previously 10.
Data analysis Differences between mean values of data were ex-
amined for statistical significance using a t-test, with t expressed as the difference between the means divid- ed by the standard error of the difference and de- grees of freedom as combined number of values in the data sets minus two.
Materials [1-14C]acetyl-coenzyme A (58 mCi/mmol), sub-
strate for the ChAT assay, was obtained from New England Nuclear, Boston, Mass. [1-14C]acetylcho- line chloride (12 mCi/mmol), used as recovery stand- ard in the ChAT assay, was obtained from Amersham-Searle, Arlington Heights, IL. Tetraiso-
propyl pyrophosphoramide (ACHE assay and stain),
SGW
| ~
S G W
51
! I I
Fig. 1. Thionin-stained 25 am-thick section through the optic tectum of a goldfish with right eye removed 10 days prior to death. Reac- tive gliosis is evident in the superficial gray and white (SGW) layer and in the optic tract (OTr) in the left (L) tectum. Magnification bar, 1 mm, applies to the whole section; insets of left and right tecta are magnified an additional 2.5 times. The periventricular layer appears darkly stained.
I I M M I I
"~ :.-7, ~ ..
\ Z ! k ~ - - i i / . /
~ \ )t~',, -I - - ~ i - ~
i " r l l ~
I MM
Fig. 2. Map of sample boundaries, indicated by solid lines, as dissected from the left and right tecta in the freeze-dried section adjacent (and therefore comparable) to the thionin-stained section in Fig. 1. Cell layers within the tectum and outlines of structures outside the tectum are indicated by dashed lines. Dissected samples were weighed and assayed for ChAT activity. Maps such as these provide per- manent records of the locations of the samples for which enzyme activities have been measured.
52
acetylthiocholine iodide (ACHE stain), and ethyl m-aminobenzoate (anesthetic) were obtained from
Sigma, St. Louis, MO. 'Freeze-It ' was obtained from Curt in-Matheson Scientific, 400 ~l-capacity micro-
centrifuge tubes from Bio-Rad. A rat monoclonal
anti-ChAT antibody was generously supplied by Dr.
Felix Eckenstein.
RESULTS
Fig. 3 shows sections through the optic tract just
caudal to the chiasm in a fish enucleated 10 days be-
fore sacrifice. The tract on the left side, contralateral
to the removed eye, shows marked gliosis related to
I i
I m m ! 1
Fig. 3. ChAT activity in the optic tracts of one goldfish 10 days after enucleation of the right eye. (Upper) thionin-stained sec- tion, showing outlines of the optic tracts with gliosis on the left side. (Lower) map of dissection of the optic tracts in the freeze- dried section adjacent to thionin-stained section above. Large dashed lines represent boundaries of the tract on the left and right sides; small dashed lines outline other morphological fea- tures. Sample boundaries are indicated by solid lines con- taining the ChAT activity as determined by assay. The average of the sample activities in the left optic tract was 11 + 1 and in the right optic tract, 10 + 2/~mol/kg dry wt/min. The density (weight per volume) of samples from the left optic tract was al- most 20% less than the density of samples from the righL On a volume basis, therefore, the activity on the left side would be about 10% lower than that on the right side.
>- PV DW CG SCG SOW 0 M
_~,oo
60-
40- .o
~: 20- ~ o" o =o
o~ 00 Ioo 200 300 400 MICROMETERS
Fig. 4. Distribution of ChAT activity across tectal layers in 5 control fish. Each symbol represents the activity in one sample, and each symbol type represents one animal. The tine was drawn by eye to best fit the data. Data were normalized to 100% of maximum to compensate for inter-animal differences. Average enzyme activities for each layer are given in Table I. The abscissa plots the distance from beginning of the PV layer to the center of the sample. Locations of tectal layers are indi- cated by abbreviations: PV (periventricular), DW (deep wtxite), CG (central gray), SCG (superficial part of CG), SGW (superficial gray and white), O (optic), and M (marginal).
the degenerating optic fibers (Fig. 3, top). C h A T ac-
tivity was very low in the optic tract on both control
and lesion sides, about one hundredth or less of that
in the SGW layer, and the activity on the left side was
not reduced by cutting the optic nerve (Fig. 3, bot-
tom). ChAT activity was not uniformly distributed
among the tectal layers (Fig. 4). The lowest activities
were found in the marginal layer (the outermost
zone, M) and in the optic layer (O). The activity was highest in the superficial gray and white (SGW)
layer and gradually fell in magnitude through the central gray to a minimum in the deep white (DW)
layer. Activity in the periventricular (PV) layer was
about twice that in the deep white layer and about
two-thirds that in the SGW layer. Since the highest ChAT activity was in the SGW layer, the region hav- ing the greatest density of optic nerve termi- trials 26,28,29,32, the obvious question was" whether or
not this activity would be altered by loss of these ter-
minals. Enucleation significantly affected C h A T activity
only in the SGW and PV layers (Table I, Fig. 5). ChAT activity in the SGW layer was unchanged for 7 days following enucleation, after which time the spe- cific activity increased on the lesioned side to 130% of the control side by 10 days and 150-160% by 20
days. The difference between lesion and control sides at 20 days was statistically significant (P < 0.001,
53
TABLE I
ChAT activity (Izmol/kg dry wt/min) in goldfish optic tectal layers in control animals and in animals 4, 7, 10, and 20 days following enu- cleation of the right eye: average +_ S.E.M. (no. of samples)
Difference between left and fight side values significant at +P < 0.05, *P < 0.001. Abbreviations, see Fig. 4.
n = no. of samples, Table I; P < 0.05, n = no. of ani-
mals, Fig. 5). In order to compare the total C h A T activity in the
SGW layer on the left and right sides, it was nec- essary to convert the enzyme specific activities to a volume basis, then multiply by the total volume of the
SGW layer on each side. Da ta were converted to a
c E "~ ~ 0 0 ~ / / / / I .If
; / / ~ / SGW 3 0 0 0 - . /
E 2OOO-
* PV I . -
> i o o o - I-.-
<
0 - ' o 5
"? I'0 2'0 DAYS AFTER ENUCLEATION
Fig. 5. ChAT activity in superficial gray and white (SGW) and periventricular (PV) tectal layers in control animals (0 time) and in animals 4-20 days following enucleation of the fight eye. Activity values are averages + S.E.M. with n = no. of animals (n = 5 at 0 days and 3 each at 4-20 days after enucleation). At 4-20 days after enucleation, filled symbols are averages of the right (control) tectum and open symbols are averages of the left (lesion-affected) tectum. *, significant difference between left and right tecta (P < 0.05).
volume basis by multiplying enzyme activities per dry weight times estimated density (dry weight per vol- ume) of SGW on each side. The density of the SGW layer was calculated by dividing sample weights by their volumes determined from computer analysis of digitized sample boundaries. The density of the SGW layer on the left side decreased to 98 + 1% of that on the right side by 10 days and 87 + 2% by 20 days, pos-
sibly related to glial infiltration and replacement of myelin with glial cell protein. The difference in the volume of the SGW layer between the left and right sides was estimated using thionin-stained sections by comparing the depth of the layer in the superficial- deep orientation, the dorsa l -vent ra l extent of the
tectum, and the ros t ra l -caudal extent of the tectum. The volume of the SGW layer on the left side, ex- pressed as a percentage of the right side, was 103 + 3% in 3 controls, 98 + 4% at 4 days after enuclea- tion, 96 + 4% at 7 days, 76 _+ 4% at 10 days and 66 _+ 3% at 20 days. The estimate of total C h A T activity per SGW layer on each side demonstra ted a very slight lesion-side/control-side decrease (about 2% by 10 days and 7% by 20 days) which was not statistical- ly significant (Fig. 6).
ChAT activity in the PV layer was reduced to about 75% of the control value by 4 days, the earliest time examined after enucleation, after which time
54
O3 W
• "r- SGW
,oo: ........ 4 N N
I-- 5 0 - o - - - - o SPECIFIC ACTIVITY
I-- e e TOTAL ACTIVITY u
• ~ 0 - O 4 7 I0 20
DAYS A F T E R E N U C L E A T I O N
Fig. 6. Comparison of the effect of enucleation on specific and total ChAT activity in SGW layer. The activity in the SGW lay- er in the left tectum is expressed as a percentage of that in right (control) tectum. The plot for total activity in the SGW layer in the left tectum was obtained for each animal by multiplying the specific activity by the left/right volume and density ratios (n = number of animals). The average of the 20 day values, 2411 + 360, was not significantly different from the average of the right side activities, 2595 + 155/~mol/kg dry wt/min. The difference between the specific and the total activity reflects the reduced volume and density in the SGW layer on the left side from the loss of optic nerve fibers and terminals.
there was no additional significant change. The dif-
ference between lesion and control sides at 4 days was statistically significant (P < 0.001, n = no. of
samples, Table I; P < 0.05, n = no. of animals, Fig.
5). The only layer to show a significant loss of C h A T
activity after enucleation was the PV layer. Consid-
ering that the PV layer accounts for about 10% or
less of the total tectal volume, the 25% decrease in
ChAT activity in that layer after enucleation, togeth-
er with the small decrease in the SGW layer, would prorate to an estimated decrease in total tectal ChAT
activity of less than 5%.
Staining patterns for A C h E activity and for CHAT- like immunoreactivity were consistent with the distri-
bution of C h A T activity in the tectum. The most in-
tense staining was in the SGW layer, with moderate staining in the PV layer, in both control and lesioned
sides.
DISCUSSION
The distribution of ChAT activity among goldfish tectal layers correlates only partly with the distribu-
tion of optic terminals. Although the C h A T activity and the density of optic terminals are both highest in
the SGW layer, relatively high ChAT activity also oc-
curs in the PV layer, which is composed of cell bodies which have not been reported to receive direct optic projection 32.
It is well known that cutting a cholinergic pathway
produces within a few days a significant loss of C h A T
activity in axons and terminals distal to the le- sion 4,14J6. In specific examples, cutting the olivo-
cochlear bundle, a centrifugal cholinergic pathway to
the cochlea, produced a 74-99% drop in ChAT ac-
tivity in the cochlea within 2 days and complete loss of activity within 7 days 9, while cutting the centrifug-
al cholinergic pathways to the olfactory bulb pro-
duced an almost complete loss of C h A T activity in the bulb within 7 days 11. Degeneration of optic termi-
nals in the SGW layer is well under way by 4 days af- ter enucleation 2°. Therefore, if the retinal ganglion
cells were cholinergic and were supplying C h A T to
terminals in the SGW layer through the optic axons, cutting these axons should have produced some de-
crease in ChAT activity in that layer by 4 days and a substantial decrease by 7 days. If the optic terminals
were the only cholinergic endings in the SGW layer,
then virtually all the ChAT activity should have been lost within the 20-day time frame of this experiment.
However, since the total ChAT activity in the SGW layer did not change significantly following optic
nerve cut, activity was not lost by the degeneration of
the optic terminals. The results found in this study fit
only the last possibility presented in the introduction,
that the optic terminals contain essentially no C h A T activity. The increase in ChAT specific activity in the
SGW layer is best correlated with an increase in con-
centration of the cholinergic terminals that remain after the loss of the volume of the non-cholinergic op-
tic terminals. These are the conditions that would be expected if the optic projection contained no C h A T
activity 6. Axons of cholinergic neurons have been shown to
contain high C h A T activity 9.12-14. In contrast, the ChAT activity in vertebrate optic nerve is extremely low13,25 (Fig. 3). Cutting a cholinergic nerve produc-
es a significant drop in ChAT activity in the nerve dis- tal to the lesion 9,u.t4. However, cutting the optic nerve in goldfish caused no reduction in the very low
ChAT activity in the optic tract, even at 20 days post- enucleation. It is difficult t o know exactly the func- tion of this low ChAT activity in the optic tract, but if
it were related to even a few cholinergic optic axons it should have been reduced after cutting the optic
nerve. The conclusion that the optic projection contains
at most only insignificant amounts of ChAT activity is consistent with immunocytochemical staining studies reporting no decrease in ChAT-like immunoreactive staining in the tectum after optic nerve cut and no fi- ber staining in the optic nerve 3°,31.
The relatively high ChAT activity in the PV layer is consistent with results showing somata in that layer stained for ChAT-like immunoreactivity 31. It is possi- ble to estimate what proportion of the neurons in the PV layer might be cholinergic based on the ChAT ac- tivity in the layer, given an estimate of the ChAT spe- cific activity in a goldfish cholinergic neuron. In order to estimate the amount of ChAT activity in a goldfish cholinergic neuron, several samples were dissected from goldfish brain that contained groups of large motoneuron-like cells that, in an adjacent section, showed positive ChAT-like immunoreactive stain- ing. A sample coinciding with an area containing sev- eral stained cells in the adjacent section had ChAT activity of 8925; a sample coinciding with an area containing slightly fewer such cells had activity of 6450; and a sample with borders adjoining these first two, but with no obviously stained cells in the corre- sponding area in the stained section, had an activity of 147 #mol/kg dry wt/min. From calculations of the proportion of each sample volume occupied by stained neurons, it was estimated that a pure sample of cholinergic somata could have ChAT activity as high as 50,000-60,000 k~mol/kg dry wt/min. From the average ChAT activity of the PV layer (Table I), it can then be estimated that about 5% or less of the volume in the PV layer would be occupied by cholin- ergic neurons. (This estimation assumes that the ChAT activity in cholinergic cells in the PV layer and in large motoneuron-like cells is about the same).
The simplest interpretation of the results presen- ted is that the ChAT activity in the SGW layer is not in the optic projection, but related to non-optic ter- minals. Although it might be argued that the small re- duction in total ChAT activity in the SGW layer could represent a minor population of cholinergic retinal afferents, it can be countered that, if this were the case, the decrease in activity should have become apparent before 7 days following enucleation. Non-
55
optic cholinergic terminals could derive from outside
the tectum, such as from the nucleus isthmi 24, and/or from neurons intrinsic to the rectum. Based on immu- nocytochemical staining studies30, 31, together with the present results, it seems likely that at least some of the high ChAT activity in the SGW layer is related to cholinergic neurons with somata in the PV layer that send processes into the SGW layer.
The rapid loss of ChAT activity in the PV layer af- ter enucleation is difficult to explain with the present information. The rapid decline of ChAT activity could have been taken to support a cholinergic reti- nal projection to this layer if it were not for the lack of evidence supporting a retinal projection to the PV layer 32. The timing of the decrease in ChAT activity could be consistent with a retrograde effect of axoto- my. Axons other than optic fibers, such as in a possi- ble centrifugal projection to the retina, would have also been interrupted by enucleation. However, if such a projection contained cholinergic fibers, they must be a very small proportion of the total number of axons in the optic tract since the ChAT activity in the tract is so low. Further, no evidence has been found for a centrifugal cholinergic projection to the retina originating from cells in the PV layer27, 32. An- other possible explanation for the reduction of ChAT activity in the PV layer is some unusually rapidly oc- curring effect in the cell bodies in that layer second- ary to the functional loss of optic input to their proc- esses in the SGW layer. It would not be unreasonable to associate the decrease in ChAT activity, observed at 4 days, with the possible slight decrease in ChAT total activity in the SGW layer at 10 and 20 days. A reduction in activity in a neuronal cell body would be expected to be followed by a reduction in the proces- ses, since there would be less enzyme to be trans- ported to the processes.
It is concluded from this study that cholinergic mechanisms are important in the tectal processing of retinal input and that retinal input is important in maintaining cholinergic mechanisms within the tec- rum, but that the retinotectal projection itself is not cholinergic.
ACKNOWLEDGEMENTS
The authors express their appreciation to Janis Kardatzke for dissecting freeze-dried sections, to
56
Ka t r ina Be ranek , L u a n n Juenge l and Judy Parli for
per forming C h A T assays, to the O R U Photography
D e p a r t m e n t and Pam Darne l l for help in p roduc ing
the manuscr ip t and to Bruce Wi n eg a r for writ ing the
compute r digitizing program. This work was suppor t -
ed by N I H G r a n t EY-03838 and by O R U in t r amura l
funds.
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