Neurochcmi.stry International. Vol. 5, No. 1, pp. 95 to 99, 1993. 0197-0186/83/010095-05503.00/0 Prinled in Greal Britain. © 1983 Pergamon Press Ltd.

COLLAGEN-TAILED AND GLOBULAR FORMS OF ACETYLCHOLINESTERASE IN THE DEVELOPING

CHICK VISUAL SYSTEM

ANA BARAT, ELENA ESCUDERO, CORONA RODRIGtJEZ-BORRAJO and GALO RAMiREZ*

Centro de Biologia Molecular, CSIC-UAM, Universidad Aut6noma, Canto Blanco. Madrid-M, Spain

(Receiced 16 May 1982; accepted 12 July 1982)

Abstract--We have carried out a comparative study of the developmental profiles of the enzyme acetylcholine- sterase, and of its collagen-tailed and globular structural forms, solubilized in the presence of 1 M NaCI, 174, (w/v) sodium cholate and 2 mM EDTA, in the chick retina and optic lobes. The overall acetylcholinesterase activities, both per mg protein and per embryo or chick, are substantially higher in tectum than in retina, from embryonic day 16. The A12 collagen-tailed form of the enzyme is present in similar amounts in the embryonic retina and optic tectum; however, while the A12 activity increases significantly in retina after birth, both by percentage and in absolute terms, the tectal tailed enzyme follows a declining developmental profile, reaching a minimum after 6 months of life. On the other hand, the globular G4 species shows developmental profiles, both in retina and tectum, rather similar to those obtained for the overall enzyme activity, while the G2 and G~ forms are present in comparable concentrations in both tissues. Besides, G 4 is the predominant globular form in the chick optic lobe after hatching, G2 and G1 being enriched in the embryonic tectum. In the case of retina, however, all the globular forms contribute more evenly to the total acetylcholinesterase activity, along the developmental period considered.

The potential significance of some of the postnatal developmental profiles is discussed in terms of the progressive adjustment of retina and tectum to the requirements of visual function.

A recent report from our laboratory described the developmental profiles of different molecular forms of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7; ACHE), characterized by velocity sedimenta- tion in sucrose gradients, in the chick retina and optic tectum (Villafruela, Barat, Manrique, Villa and Rami- rez, 1981). However, at the time that this work was done we did not have a suitable method to solubilize intact collagen-tailed AChE molecules from chick central nervous system tissues, their existence being merely inferred, and their relative abundance roughly estimated, by collagenase-digestion of detergent- extracted tissue samples (Villafruela, Barat, Villa and Ramirez, 1980). We have, in the meantime, developed procedures to solubilize intact collagen-tailed AChE structural forms, both in chick visual structures and in other vertebrate central nervous system tissues (Barat, Escudero, G6mez-Barriocanal and Ramirez, 1980a, b; G6mez-Barriocanal, Barat, Escudero, Rodriguez-Borrajo and Ramirez, 1981; Rodriguez- Borrajo, Barat and Ramirez, 1982), that are both effi-

* To whom correspondence should be addressed.

95

cient and reproducible. We have therefore used this new experimental approach to carefully estimate the relative concentration of collagen-tailed AChE forms in chick retinal and tectal tissues during embryonic and postnatal developmental. Furthermore, we have taken advantage of the associated improvements in AChE solubilization to re-evaluate the overall enzyme developmental profiles. Our previous discussion on the possible significance of AChE developmental pro- files in the broader context of the structural and func- tional maturation of the chick visual system has been revised to accommodate the new data.

EXPERIMENTAL PROCEDURES

We have tested the four different extraction procedures described by G6mez-Barriocanal et al. (1981) in retinal and tectal samples, at different developmental ages, both embryonic and postnatal, and we have finally adopted for this study the one using l0 mM Tris-HC1, pH 7; 1 M NaC1; 1% (w/v) sodium cholate and 2 mM EDTA, taking into account both the efficiency of solubilization and the con- sistency of the pattern of distribution of the total AChE activity in different molecular forms, for a given age and tissue. Extraction in the absence of detergent results in the

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E M B R Y O N I C AND P O S T - H A T C H I N G AGE

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Fig. I. Developmental protiles of AChE activity in chick retina and tectum, per mg of protein (left) and per pair of retinas or optic lobes (right), from embryonic day 8 to 6 mouths after birth. Points and bars represent mean + S.D. for 4 determinations. The arrows in the time scale indicates hatching. (@ @),

retina: { 0 0 } , rectum.

solubilization of only about one third of the total tissue ACHE. while if Triton X-100 is used, instead of sodium cholate, more cxtraction/centrifugation cycles are required to achieve similar levels of enzyme solubilization.

Retinas and tectal lobes were dissected over ice and homogenized in 5 10ml of the above extraction solution (volume varying according to age) and, alter 10 min at 4 C. the homogenates were centrifuged at l l0,000,q for 30 rain, at 2 (7. The supernatants were put aside, and the pellets were re-extracted as in the initial homogenization, and the new extracts centrifuged under the same conditions. Both supernatants were pooled and aliquots were taken for sedi- mentation analysis in sucrose gradients, and for the deter- ruination of the solubilized AChE activity. The final resi- dual pellet contained less than I",, of the solubilized ac- tivity; in turn, the activity of the pooled supernatants was equal or slightly higher than the initial homogenate ac- tivity. It was. therefore, considered that R)r all practical purposes this extraction procedure resulted in a total solu- bilization of the enzyme.

The methods used for scdimentation analyses, AChE and marker enzymes assays, and AChE peak localization and quantitative evaluation have been described in detail m a previous publication {G6mez-Barriocanal et ul., 1981). Some other experimental details are given in the figure legends.

R E S U L T S

Figure 1 shows the developmenta l profiles of AChE in retina and opt ic tectum, both in te rms of specific activity per mg of tissue prote in (left) and of total activity per pair of ret inas or opt ic lobes (right). Taking into account these results, and the sedimenta- t ion profiles ob ta ined for the different age points [-not shown, but similar to those in Villafruela et al. (1981) except for the presence of a 20S peak (19.8 _+ 0.2), as

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EMBRYONIC AND POST-HATCHING AGE ((Joys)

Fig. 2, Developmental profiles of the A~2 collagen-tailed structural form of AChE in chick retina and tectum. Per cent activities (upper panell have been calculated from the sedimentation profiles at the different age points (see text), as previously described {Gomez-Barriocanal et al., 1981). Specific (per nag of protein) and total (per pair of retinas or optic lobes) A~2 activities (mid and lower panels, respect- ively} have been obtained from the curves in Fig. 1, and the per cent values in the upper panel. Other details as in

Fig. 1.

Development of molecular forms of AChE 97

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details as in Figs I and 2.

seen in Gdmez-Barriocanal et al. (1981), Fig. 1, panel 3b], as described in 'Experimental Procedures', we have calculated the per cent distribution, and the specific and total activities of the different molecular forms of the enzyme, namely A12 (three tetramers of catalytic subunits linked to a three-stranded tail; nominal sedimentation coefficient, 20S), G4 (a tetramer of catalytic subunits; 1 IS) and G2 + G1 (the dimer and the monomer, respectively; 7S, 5S). These profiles are illustrated in Figs 2-4, respectively. Actual sedimentation coefficients in the presence of sodium cholate are statistically similar in retina and tectum and can be found in a previous paper (G6mez-Barrio- canal et al., 1981).

DISCUSSION

It was expected that a change in the tissue hom- ogenization solution would entail more or less signifi- cant modifications in the measured AChE activity in

the homogenate. A re-evaluation of total AChE ac- tivities was, therefore, in order. Furthermore, the new developmental profiles shown in Fig. 1 include two additional age points not considered previously (Vil- lafruela et al., 1981), namely day 1, to assess the acute effects of eye-opening, and day 180 (6 months), taken as a measurement of the adult, steady-state levels of AChE activity. Apart from the sharp and transient rise in specific activity during the first day of life, both in retina and tectum (Fig. 1, left), the most important departure from our previous results (Villafruela et al., 1981) can be seen in the postnatal points of both tec- tal curves, where the AChE specific activity reaches a maximum of 816 (nmol acetate/min/mg protein), at day 6 after hatching, vs 490 in our former report. Tectal AChE specific activity declines consistently from day 6 onwards, to a value of 538, after 6 months of life.

The potentially most interesting set of new data in the present report refers, however, to the development of collagen-tailed AChE species, more specifically the

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so-called A~2 form (Massoulie, 1980), that in the chick central nervous system tissues has an apparent sedi- mentation coefficient of about 20S (Barat et al..

1980a, b: Gomez-Barriocanal et at., 1981: Rodriguez- Borrajo et al.. 1982). Interestingly, the relative propor- tion o f the A~2 AChE follows a very difl'erent pattern of development in retina and tectum (Fig. 2, upper paneli. Thus, while prior to hatching A~: represents 2 3",, of the total AChE activity in both tissues, the postnatal percentage of A~_~ increases continuously in retina with age, up to about 10",, after {~, months, but decreases in rectum to practically disappear in the adult chick. The other specific [per mg of protein) and total A~2 activity curves (F'ig. 2. mid and lower panels) reflect the same phenomenon, from a different standpoint.

It is worth noting that. in spite of the much higher overall AChE activity in the optic rectum, the ab- solute activity of Ale in retina is consistently higher than that of tectt, m after the onset of visual function. with the exception of the first day of life. This, again. illustrates the danger of attempting a correlation of the total levels of AChE activity with some particular aspects of cholinergic function (Villafiuela et al..

1981). The relatively low activity of the collagen-tailed

forms of AChE in the postnatal chick optic lobe, which led us in a previous paper to question the use- fulness of EDTA as a solubilizing agent in this area of the brain (Barat et al.. 1980b}, does not lend itself to a straightforward interpretation. However. the pro-

gressive disappearance of the collagen-tailed forms of AChE has already been described in the case of a slow tonic muscle in the chick {Lyles and Barnard. 1980). All these contrasting developmental patterns do not rule out the possible (although not necessarily exclusivel synaptic role postulated by different authors (see Massoulie, 1980, for a review) for the asymmetric collagen-tailed forms of AChE since the molecular structure and functional mechanisms of cholinergic synapses, both in muscle and in the ner- vous system, are themselves variable. Thus. different synapses may require different populations of AChE molecules for proper function, and the necessary adjustments to reach this optimum configuration might as well take place after birth, at the time when function is being established.

On the other hand, the developmental profiles of the globular forms of AChE {Figs 3 and 4) show only minor differences with those previously published by us (Villafruela et al., 1981) except for the eye-opening peaks, in all cases, and the 6-day peaks in specific activity in the Ga curves, that simply reflect the

changes in total tissue activities commented at thc beginning of this Discussion. These curves do fltrther- more confirm our previous conclusion {Villafruela et al.. 1981) that in the chick tectum the postnatal m- crease in AChE activity reflects mainly an accumu- lation of the G4 form, while in retina all globular l\)rms [and the A12 form, as discussed above) contrib- ute more evenly to the overall tissue activity.

The possible relationship between AChE appear- ancc and accumulation, and synaptogenesis and onset of visual flmction was thoroughly discussed in our previous paper (Villafruela t't al., 1981). While the present results do not allow us to modify substantially our former tentative statements, we could nevertheless suggest, in view of the Aj2 developmental changes depicted in Fig. 2 and discussed above, that ftmction- dependent synaptic validation and stabilization, even more than synaptogenesis, could in some way bc related to the postnatal developmental profiles of col- lagen-tailed AChE in different neural structures (and perhaps, different types of musclesl, if we assume that these profiles, and the resulting adult levels of the

A-R)rms, reflect the progressive adjustment of this set of molecules to the varying functional requirements of each structure.

In any case, the role and localization of the differ- enl forms of AChE should be further clarified before a meaningful interpretation of all these developmental data can be successfully attempted.

.4ckmm'h, dgemems This work was supported by grants l'rom the Comision Asesora de lnvestigaci6n Cientifica y Tccnica, the Plan Nacional de Prevenci6n de la Subnorma- lidad and the Fondo de hwestigaciones Sanitarias. C.R.-B. is a fellow of the Caja de Ahorros y Monte de Picdad dc Madrid.

REFERENCES

Barat, A.. Escudero, E., G6mez-Barriocanal, J. and Rami- rez, G. {1980a). Solubilization of 20S acetylcholinesterase from the chick central nervous system. Neurosci. Lett. 20, 205 210.

Barat. A., Escudero, E., Gomez-Barriocanal , J. and Rami- lez, G. 11980b). Solubilization of 20S acetylcholinesterasc from chick retina, Biochem. hiophy.~. Res. Commun. 96, 1421 1426.

Gomez-Barriocanal, J., Barat, A., Escudero, E., Rodriguez- Borrajo, C. and Ramirez. G. (19811. Solubilization of col- lagen-tailed acetylcholinesterase from chick retina: Effect of different extraction procedures. J. Neurochem. 37, 1239 1249.

Lyles, J. M. and Barnard, E. A. (1980). Disappearance of the "endplate" form of acetylcholinesterase from a slow tonic muscle. FEBS Lett. 109, 9 12.

Development of molecular forms of AChE 99

Massouli6, J, (1980). The polymorphism of cholinesterases and its physiological significance. Trends Biochem. Sci. 5, 16(~164.

Rodriguez-Borrajo, C., Barat, A. and Ramirez, G. (1982). Solubilization of collagen-tailed molecular forms of acetylcholinesterase from several brain areas in different vertebrate species. Neurochem. Int. 4, 563 568.

Villafruela, M. J., Barat, A., Villa. S. and Ramirez. G. (1980). Molecular forms of acetylcholinesterase in the chick visual system. FEBS Lett. !10, 91 95.

Villafruela, M. J., Barat, A.. Manrique, E., Villa, S. and Ramirez, G. (1981}. Molecular forms of acetylcholine- sterase in the developing chick visual system. Develop. Neurosci. 4, 25 36.