Neurochem. Int. Vol. 11, No. 4, pp. 425-431, 1987 0197-0186/87 $3.00 + 0.00 Printed in Great Britain. All fights reserved © 1987 Pergamon Journals Ltd

THE GABA A RECEPTOR COMPLEX IN THE DEVELOPING CHICK OPTIC TECTUM:

ONTOGENY OF [3H]MUSCIMOL, [3H]FLUNITRAZEPAM A N D [35S]TBPS BINDING SITES

ALIC1A BATUECAS, ANA CUBERO, ANA BARAT and GALO RAMJREZ* Centro de Biologia Molecular (CSIC-UAM), Universidad Aut6noma, Canto Blanco,

28049 Madrid, Spain

(Received 5 May 1987; accepted 2 July 1987)

Abstract--The developmental profiles of the neurotransmitter recognition site, labeled by [ 3H]muscimol, and of the two main modulatory sites, labeled by [3H]flunitrazepam and [35S]t-butylbicyclophospho- rothionate, respectively, within the GABAA receptor complex, have been determined in chick tectal lobes between embryonic day 8 and postnatal day 20. The consonance among the rates of appearance and accumulation of the three receptor sites in tectal membranes suggests a coordinated expression and assembly of the protein subunits involved in the spatial configuration of the complex and its three binding sites, although the existence of isolated muscimol binding subunits during early embryogenesis cannot be excluded at the present time. Furthermore, the total number of binding sites of each kind, per pair of lobes, is compatible with a 1/1/1 stoichiometry. The GABA A receptor complex reaches a maximum of expression, relative to total membrane protein, immediately after hatching, suggesting that the tectal GABAergic system may be instrumental in damping the effects of sudden exposure to light of the chick visual system upon eye opening.

Our recent work on the GABAA receptor complex in the developing chick optic tectum has focused on the general properties of the transmitter recognition site (Batuecas et al., 1987) and of the two modulatory sites specifically recognized by benzodiazepines and picrotoxinin, respectively (Cubero et al., 1987), the latter site being associated to the chloride channel. We are now in a position to undertake a comparative ontogenetic study of the three main sites of the complex, looking for temporal correlation or div- ergence of the three developmental profiles, before and after the critical hatching time. The degree of maturat ion of the three sites at birth, both in absolute terms and in comparison with the known devel- opmental profiles of excitatory amino acid receptors (Ramirez et al., 1981; G6mez-Barriocanal et aL, 1982; Ramirez et aL, 1983) may suggest a role for this tectal GABAergic system in damping the effects of sudden exposure to light upon eye opening.

*To whom correspondence should be addressed. Abbreviations: BZ, benzodiazepine; BZ-site, benzo-

diazepine binding site; FNZ, flunitrazepam; PTX-site, picrotoxinin recognition site; TBPS, tert-butyl- bicyclophosphorothionate; En, embryonic day n; Pn, postnatal day n.

EXPERIMENTAL PROCEDURES

All the information relative to egg incubation, hatching and general care of young chicks has been given in sufficient detail in previous papers of this series (Batuecas et al., 1987; Cubero et al., 1987). Membrane preparations to be used for [3H]muscimol, [3H]flunitrazepam (FNZ) and [35S]tert- butylbicyclophosphorothionate (TBPS) binding assays have also been described in the same publications. The standard radioligand concentrations used to determine the devel- opmental profiles were: [3H]muscimol, 40nM for regular membranes, and 4nM for Triton X-100-extracted mem- branes; [3H]FNZ, 10nM, and ['S]TBPS, 2 nM. Binding conditions were the same as the reference conditions estab- lished in the above papers, and non-specific binding was measured by displacement with an excess (104 times) of non-radioactive muscimol, flunitrazepam or picrotoxinin, respectively. To be able to establish meaningful comparisons among the different developmental profiles we have always measured the optimized basal binding, thus avoiding stimu- lation by ions or allosteric effectors (e.g. CI- and GABA, respectively, when measuring [3H]FNZ binding); in the case of [35S]TBPS however, the presence of chloride ions is a necessary requisite for specific binding. The effect of de- tergent on [3H] muscimol binding has been analyzed at different developmental ages by use of independent onto- genetic profiles for membrane preparations extracted or not with Triton X-100, as previously described (Batuecas et al., 1987). The procedures used for the three radioligands are summarized in Table 1.

A dedicated preparation was used for protein deter- 425

426 ALICIA BATUECAS et al.

Table I. Procedures used for membrane preparation and binding assays in the analysis of three receptor sites of the GABA A receptor complex

I Detergent extraction

(0.05% Triton X-100)

I 2 washing cycles

I Incubation

4 nM [3H] muscimol, pH 7, 4°C, 30rain

Homogenization

Centrifugation (72,000g × 30 min)

Pellet

I Osmotic shock

!

Centrifugation

Pellet: keep frozen, for at least 24 h

Thaw

Supernatant (discard)

Supernatant (discard)

2 washing cycles I washing cycle 3 washing cycles

I l I Incubation: Incubation: Incubation:

40 nM 13H]muscimol, 10 nM [3H]FNZ, 2 nM ['S]TBPS, pH 7, 4°C, 30 rain pH 7, 4°C, 30 min pH 7.4, 23°C, 2 h

The procedures are described in detail in previous papers (see Experimental Procedures). All eentrifugation steps (detergent extraction, washing cycles and membrane recovery after incubation) were done at 72,000g, 4°C, for 30 rain. No significant binding of any of the radioligands was found in the 72,000g supernatant, either by equilibrium dialysis or by polyetliyleneglyeol precipitation in the presence of carrier ~,-giobulin. The incubation buffer was, in all cases, 10 mM potassium phosphate, at the pHs shown above, including 200 mM KCI when measuring [3sS]'I'BPS binding.

mination in both the initial homogenates and the membrane fractions assayed for radioligand binding, for the different age points. The material to be used for protein measurement was dissolved in 0.5 M NaOH and further processed as described (Villafruela et aL, 1981).

Twelve embryos or 6 birds were pooled in the starting homogenates for any receptor binding or protein deter- mination, so to minimize the influence of a possibly abnor- mal avian specimen. Besides, all developmental profiles are based on three independent sets o f measurements, with duplicate samples in binding assays and sextuplicate sam- pies (3 aliquots of increasing size, in duplicate) in protein measurements. In this way, the standard deviation values were kept well below 10% of the mean value.

The developmental period considered here (embryonic day 8 to postnatal day 20) encompasses the visual mat- uration of the chick; adult values are given in some cases for comparative purposes.

Two different types of developmental profiles are given for each binding site within the GABA^ receptor complex.

The binding per mg of membrane protein is a measure of the preferential expression of each site, relative to the bulk of membrane protein, whereas the binding per pair of tectal lobes represents the accumulation of sites in the developing organ. While the first type of profile is useful to learn about the rates of synthesis/post-translational processing/ externalization of the receptor protein, the second type reflects the global functional capacity of the tectal GAB- Aergic system at any given time during development.

RESULTS

Developmental changes in the membrane preparations used for the different binding assays

Figure 1 d isplays the deve l opmen t a l var ia t ions in the p ro t e in con t en t s o f the m e m b r a n e f rac t ions used for the different b ind ing assays. T h e u p p e r pane l gives actual p ro t e in values (in mg) per pa i r o f tectal lobes

GABA^ receptor complex in chick optic rectum 427

0 - 12

~) )o E

~ 8 hi F--

Z 4

al ~ 2 hi

o

i I l i v I I //'---'Z'~ /

/

,/z /~.//

~ ' - "~ ' , - , ,~, - , , / / ,

8 12 "16 20 2 6 10 20

used in Figs 2-4 to calculate the total number of binding sites per pair of tectal lobes.

Developmental profiles of [3H]muscimol, [3H]FNZ and [3SS]TBPS recognition sites in the GAB.4 A recep- tor complex

Figures 2-4 display the developmental profiles of the three receptor sites considered in this paper. The upper curves in the three figures show an increasing density of binding sites (pmol or fmol per mg protein) in tectal membranes during embryonic life, with a maximum at 1-2 days after hatching, and a slow postnatal decline down to adult values, which are very similar to those measured in 20-day chicks (for

z 50 ,T, I--

~ 40

~ 3o I-- 0 F-

2O (4. 0

N 10

i I i i i i i / /

,. ,-"._,C~:" : ~o . . . . . . o - - - - /~ - - o ~ I P " °. .o ' ~p-~ . . .o "°

o- . . . . . °~ °

hatching

8 12 16 20 2 6 10 20

EMBRYONIC AND POSTNATAL AGE (days)

Fig. I. Protein contents of the different chick tectal mem- brane preparations, used to measure binding of [3H]- muscimol, [3H]FNZ and [3SS]TBPS, as a function of em- bryonic and postnatal development. Membrane fractions were prepared in all cases as summarized in Table 1. Upper panel: absolute amounts of protein in rag/pair lobes. Lower panel: percent of total protein in the initial homogenate. The points in the different curves are the mean of 3 independent sets of determinations. The standard deviation bars have been omitted to avoid confusion (but see Experimental Procedures). (O O), membrane preparations for [3H]muscimoi binding (no detergent); (O- - - -O) , mem- brane preparations for [3H]muscimol binding (Triton- X-100-extracted); (&----&), membrane preparations for [3H]FNZ binding; (A- - ' - -A) , membrane preparations

for [35S]TBPS binding.

(i.e. per chick), whereas the lower panel expresses the same data in percentual terms relative to the total tectal protein measured in the initial homogenate. The differences seen in the actual protein values (upper panel) reflect the different treatments under- gone by each membrane preparation according to the receptor site analyzed (Table 1): protein is lost upon consecutive washing cycles, and notably upon de- tergent extraction. These protein values have been

1.2

= 1 . 0

o 0.8

~ 0 . 6

c ~ 0.4 Z o.

o 0 .2 m . 0 0 :E 7

u~ 6

"-~ 4

"6 3

2

i w ~ i i i l / / i

hatchln ' /T g

/ e--..-m - - f ~ - •

/ ° '°" "~ ' °"q /P"°

1 ~ hatching

0 - - . - 7 ~ , t , , , / / i 8 12 16 20 2 6 10 20

EMBRYONIC AND POSTNATAL AGE (days)

Fig. 2. Developmental appearance and accumulation of specific [3H]muscimol binding sites in chick tectal mem- branes. The upper curves show the changes in binding sites/rag of membrane protein, while the lower curves display total binding sites per pair of tectal lobes (i.e. per chick), calculated from the upper curves values and the protein data in Fig. I. Values given in the upper curves are mean + SD for 3 independent developmental experiments (see Experimental Procedures) (0 O), binding of 40nM [3H]muscimol to a washed membrane preparation. ( 0 - - - - 0 ) , binding of 4 nM [3H]muscimol to a detergent- extracted/washed membrane preparation (see Experim©ntal Procedures and Table 1). The radioligand concentrations used for the two membrane preparations (helf the respective Kds ) are in agreement with the observed differences in K0s

(Batuecas et al., 1987).

428 ALICIA BATUECAS et al.

12

._.E I.O o

08

r , ~ 0.6

~ 0.2 a .

w 0 N

n-"

I ..... I I I I I f ~ / ~

4 ¢ , , , ~ hoichin~li

I I I I / I I I / / - - - +

F-

E_ 8

2

0 I L__// 8 12 16 20 2 6 I0 2 0

E M B R Y O N I C A N D P O S T N A T A L AGE ( d a y s )

Fig. 3. Developmental appearance and accumulation of specific [3H]FNZ binding sites in chick tectal membranes.

See Fig. 2 and Table 1 for more details.

140

120

"~ I 0 0

~ 8o

"- 60 a ~ Z ~ 4 0

O m 20

a. 0 ,-n v- 1.2 ~g

o

0.8

g o.6 -6

0.4

0.2

I I I - - I i I I / ~ I

,o,°.,.,, -t I I i - I T I t I / / I

Aj,~I~ ~/fA

/ x t

,

B 12 16 20 2 6 10 20

EMBRYONIC AND POSTNATAL AGE (days)

Fig. 4. Developmental appearance and accumulation of specific [3SS]TBPS binding sites in chick tectal membranes.

See Fig. 2 and Table l for more details.

example, in the case of muscimol, we have found receptor densities of 0.64 and 0.62 pmoi/mg after 3 and 6 months, respectively, of postnatal life).

The two curves in Fig. 2, upper panel, confirm our previous assumption on the effect of Triton X-100 extraction on [3H]muscimol binding to tectal mem- branes: the 10-fold increase in affinity (Batuecas e t al. , 1987) due to the detergent treatment is consistently seen all throughout tectal development, with nearly identical binding values when using low radioligand concentrations proportional to the respective dis- sociation constants (half the respective Kds, in this case).

The lower profiles in the three figures show the amount of receptor sites accumulated during onto- genesis and are probably proportional to the func- tional capacity of the tectum within the chick visual system. In agreement with the upper profiles, there is a sharp decrease in the rate of accumulation of receptor sites in the membranes, in all three cases, immediately after hatching.

The developmental profiles of Figs 2-4 demon- strate a stricklingly close coordination in the onto- genetic expression of the three sites involved in the

optimal function of the GABAA receptor complex, as one would expect for a macromolecular oligomeric structure with an unitary and well-defined phys- iological role, and suggest that the complex modu- latory phenomena described in the GABAA complex (Olsen et al. 1984) may be operative at very early stages in tectal development. Nevertheless, to have a more detailed picture of this developmental syn- chronization among the three sites we have calculated some arbitrary m a t u r a t i o n ra te s expressing all bind- ing data (per mg of protein) as % of P2 values. These maturation rates for the [3H]FNZ, [sSS]TBPS and [3H]muscimol (detergent-extracted membranes) have in turn been expressed as % of the corresponding [3H]muscimol (washed membranes) values, and plot- ted as such against developmental age (Fig. 5). We can detect a certain lag in the relative maturation of the BZ- and PTX-sites of the complex, which disap- pears late in the embryonic period. The correspon- dence in maturation rates for the three sites is how- ever quite good from E20 onwards. The possible significance of this finding will be taken up in the Discussion.

To stress the embryonic timing of the GABAA

GABA A receptor complex in chick optic tectum 429

w 14o /

.~ 120 LIJ 0 z 100 fie W It. ,,, 80 ,.y

la. 0

6O

40 i 8

I I I I I I , / / I

...dr ' /~

",,,. ....

o-

/

h a t c h i n ¢ t

I I I~ i I i / / t 12 16 20 2 6 10 20

EMBRYONIC AND POSTNATAL AGE (days)

Fig. 5. Comparison of maturation rates for the transmitter and modulatory recognition sites in the chick tectal GABA^ receptor complex. [3H]FNZ and [35S]TBPS binding data, and also [3H]muscimol data in detergent-extracted mem- branes (per mg of protein), expressed as % of values at P2, were divided by the corresponding [3H]muscimol binding values (no detergent extraction), equally expressed as % of P2, and the ratios were plotted against developmental age. (A-- - -A) , [3H]FNZ/[3H] muscimol. (A-- . - -A) , ['S]- TBPS/[3H]muscimol (©-- - -©) , [3H]muscimol (detergent- extracted membranes)/[ 3H]muscimol (reference membranes).

receptor complex maturation we have calculated the E20/P20 ratio for the different binding sites: in Table 2, these ratios are compared with the corresponding values for tectal [3H]kainate receptors, which appear mainly after hatching (G6mez-Barriocanal et al., 1982; Ramirez et al., 1981, 1983).

Stoichiometry o f the transmitter and modulatory sites in the G A B A A receptor complex

The values of Bmax for the three binding sites considered in this study have been given in the foregoing paper (Cubero et al., 1987), both at El6 and P10. Taking into account the protein contents of the respective membrane preparations at the corre- sponding age points (Fig. 1, upper panel) we have estimated the absolute number of sites of each type

Table 2. E20/P20 ratios for different binding sites

pmol/mg E20/' Radioligand pmol/mg P20

[3H]muscimol (washed membranes) 1.39 [3H]muscimol (detergent-extracted 1.36

membranes) [ 3H]FNZ 1.13 [3sS] TBPS 0.93 [ 3H]kainic acid 0.18

The E20 and P20 (pmol/mg) values have been taken from curves in Figs 2-4. Data for [3H]kainic acid are from G6mez-Barriocanal et aL (1982).

Table 3. Stoichiometry of the transmitter and modulatory sites in the chick tectal GABAA receptor complex

B~x x mg protein (pair of lobes)

Radioligand E 16 P 10 [3H]muscimol (washed membranes) 3.96 21.2 [3H]muscimol (detergent-extracted 2.86 16.0

membranes) [ 3H]FNZ 2.49 18.2 [ 3sSITBPS 2.02 17.6

B~a x for the different radioligands are from Batuecas et al. (1987), and Cubero et al. (1987). Protein data have been taken from Fig. 1.

for a pair of tectal lobes to detect possible changes in the stoichiometry of the complex from embryonic to postnatal ages. Table 3 gives the results of such calculations. While embryonic values (El6) suggest a discrete predominance of the transmitter over the modulatory sites (perhaps in consonance with the curves in Fig. 5), postnatal figures are substantially similar and compatible with a 1 / 1 / 1 stoichiometry for the three recognition sites.

DISCUSSION

Unlike mammals, chicks are born with a mature visual system so that locomotion, feeding, and other visually-controlled behavioral tasks are possible vir- tually right after hatching. This implies, among other requirements, that the tectal circuitry has to be ready to handle the sudden influx of light-derived electrical signals at eye opening (Peters et al., 1958; Sedlacek, 1969; Rager, 1976). Some years ago we advanced the hypothesis that a developmental coordination be- tween excitatory, or permissive, and inhibitory, or signal-blocking, transmitter systems could be a rea- sonably efficient means to ensure the electrical sta- bility of the chick visual centers, and especially the optic tectum, at the critical time of hatching (Ramirez et al., 1981; G6mez-Barriocanal et al., 1982; Ramirez et al., 1983). Preliminary experiments on the relative timing of the appearance and accumulation of recep- tors for excitatory and inhibitory amino acids trans- mitters.in the chick optic tectum lent support to our views on this matter (Ramirez et al., 1983).

The present paper confirms that not only [3H]muscimoi receptor sites, but the whole GABAA receptor complex system is ready in the tectum at birth to help control the flow of information. Indeed, the sharp decrease after birth of the rates of accumu- lation of transmitter and modulatory recognition sites in chick tectal membranes suggests that the first

430 ALICIA BATUECAS et al.

exposure to light might be a signal to a slow down, or even shut off, the genomic program responsible for the a c c e l e r a t e d synthesis, processing, assembly and externalization of GABAA receptor complexes, es- pecially operative in the latest embryonic stages. This precocious maturation of the GABA A receptor- dependent GABAergic system stands in sharp con- trast with the gradual, postnatal ontogenesis of the excitatory system mediated by tectal kainate recep- tors (G6mez-Barriocanal e t al., 1982); the relative importance of the embryonic and postnatal times for the maturation of these amino acid- mediated excitatory and inhibitory systems is illustrated in Table 2.

Although the similarity in the maturation profiles of the three receptor sites recognizing [3H]muscimol, [3H]FNZ and [35S]TBPS, respectively, is rather re- markable (Figs 2-4), a more detailed quantitative analysis of the individual maturation rates reveals a certain discrepancy in the early embryonic period, with a lag in the development of the two modulatory sites as compared with the transmitter site (Fig. 5). Among the possible explanations for this finding, those based on a relative inefficiency of the radio- ligands to label the embryonic BZ- and PTX-sites, or on a higher sensitivity to inactivation of the receptors themselves, are not supported by the nearly identical kinetic behavior of the embryonic and postnatal receptors (Cubero et al. , 1987). Alternatively, the early transient excess of [3H]muscimol binding sites would support the existence of isolated receptor subunits containing only the transmitter recognition site, which might possibly be involved in the alleged trophic effects of GABA on neurogenesis (Wolff et al. , 1978; Madtes and Redburn, 1983; Meier et aL,

1985). Another important point of discussion concerns

the stoichiometry of the three receptor sites within the GABAA complex. Recent work by Nielsen and Brae- strup (1983) in rat cortex, and by Chang and Barnard (1982), Martini et al. (1983), Sigel and Barnard (1984) and Casalotti et aL (1986), using solubilized and purified receptor preparations, suggest the existence of different polypeptide subunits for each receptor site, in a l/l/1 ratio. Our paper is the first one to provide developmental evidence supporting this ten- tative receptor configuration (Table 3). Other reports, however, favor transmitter/benzodiazepine site ratios of about 2 to l: Asano and Ogasawara, 1981; Steph- enson et al. 1982; Korneyev e t al. , 1984; Kirkness and Turner, 1986 (see Sigel and Barnard, 1984, and Schwartz e t al. , 1985, for additional comments on this topic).

Systematic comparative analyses of the onto- genesis of the three binding sites in the GABAA receptor complex are almost non-existent. A recent report by Tehrani and Barnes (1986) claims a coordi- nated expression of the three receptors in chick cerebral hemispheres, which is, however, not quite borne out by the developmental profiles shown, perhaps due to the heterogeneity of the structure used. Values at hatching were in this case just over 50% of adult values. Although the possible stoichi- ometry of the receptor is not discussed by the authors, the biphasic Scatchard plots for [35S]TBPS binding make comparative estimations very difficult (Cubero e t al., 1987). Reports on the ontogenetic behavior of either the transmitter site (labeled by [3H] muscimol or [3H]GABA) or the BZ-site (labeled by [3H]FNZ or [3H]diazepam) are nevertheless more plentiful. The use of [3H]GABA to label the trans- mitter site in the chick optic tectum (Flores et al. , 1986) has a few problems of its own that we will discuss in a later paper. In the case of chick cere- bellum, Fiszer de Plazas (1982) was also able to show a precocious maturation of the transmitter site, using [3H]GABA as the radioligand. This, and our own results stress the marked differences in developmental patterns between chicks, relatively mature and self- sufficient after hatching, and mammals, mother- dependent and functionally blind at birth. Thus, studies on brain cortex and cerebellum of rats and/or mice show that it usually takes from 10 to 20 days of postnatal development to reach the 50%- point in the profiles of receptor density (sites/mg protein) for either [3H]muscimol/[3H]GABA (Coyle and Enna, 1976; Palacios et al. , 1979; East and Dutton, 1981) or [3H]FNZ/[3H]diazepam (Braestrup and Nielsen, 1978; Regan et al. , 1980; Aldinio et al., 1981). The same is true, for instance, in the case of rabbit retina (Madtes and Basher-Elahi, 1986).

Within this context, the results described in this paper confirm our previous findings on the close relationship between receptor development and onset and maintenance of function (vision, in our case; G6mez-Barriocanal et al., 1982; Ramirez et al., 1983). Our present efforts are directed to understand how the increasing functional demands during retinotectal development are conveyed to the genome to modify the expression of selected genes involved in GABA- ergic and glutamatergic neurotransmission.

Acknowledgemen t s - -Th i s work was supported by Grants from the Comisi6n Asesora de Investigaci6n Cientifica y Trcnica, and the Fondo de Investigaciones Sanitarias.

GABA A receptor complex in chick optic tectum 431

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