Brain Research, 166 (1979) 245-257 © Elsevier]North-Holland Biomedical Press
C H O L I N E R G I C B I N D I N G SITES IN RAT H I P P O C A M P A L F O R M A T I O N : PROPERTIES A N D ONTOGENESIS
JACOB BEN-BARAK and YADIN DUDAI Department of Neurobiology, The WeizmannInstitute of Science, Rehovot (Israel)
(Accepted August 17th, 1978)
SUMMARY The hippocampal formation of the rat contains two types of membrane-bound cholinergic binding sites, as revealed by specific binding of [3H]quinuclidinyl benzilate (QNB) or of a-[lzSI]bungarotoxin (a-Btx). The sites differ in pharmacological profile, sensitivity to detergents and ontogenesis. The major binding site (about 17 pmol per adult hippocampus) is of a muscarinic nature, and binds [3H]QNB with an on-rate of 2 × 106 M -1 sec -1 and an apparent KD of 0.4 nM. This binding is displaced by low concentrations of muscarinic ligands but not of nicotinic ligands. The earliest increase in binding level is detected at about day 4 postnatal and a sharp increase in total binding takes place between days 10 and 15. Total binding continues to increase gradually about 3-fold until an age of about 7 weeks, at a rate resembling that of acetylcholinesterase, a-Btx-binding sites (about 0.6 pmol per adult hippocampus) display a nicotinic profile with an on-rate constant for a-[l~I]Btx of 6 × 104 M -1 see -1 and an apparent KD of 2 nM. Ontogenesis of these sites clearly differs from that of muscarinic sites and acetylcholinesterase. Absolute binding reaches mature levels at an age of 12-14 days postnatal, and binding per tissue protein is higher during the first postnatal days than at maturity. It appears that the level of toxin-binding sites attains mature values before the major synaptogenetic events in the area are completed.
INTRODUCTION The hippocampal formation of rat brain can serve as a suitable model for studying the ontogenesis of cholinergic receptors in the central nervous system and the possible role of these receptors in developmental processes such as axonal invasion and synaptogenesis. The region is one of the richest cholinergic structures in the brain 2a. Detailed information on its development and anatomy is available 1-a,11,25,27.
246 Only one extrinsic cholinergic afferent, the septohippocampal tract, which provides up to 90 ~ of the cholinergic innervation to the region, has been identified ~2,23,a3. In recent years powerful biochemical tools have been developed which make it possible to directly study the pharmacological and biochemical properties of cholinergic receptors. The potent muscarinic antagonist, quinuclidinyl benzilate (QNB) 36, binds to hippocampal sections 20 and homogenates 37. The nicotinic antagonist, abungarotoxin 10, also binds to hippocampal sections16,29,3z and homogenates3L Although it is not yet clear whether a-toxins label functional acetylcholine receptors in ganglia and in the central nervous systemT,8,1a,zS, as they do in peripheral systems4,9, lO,19, toxin-binding sites may be regarded as putative cholinergic receptors. QNBbinding sites and toxin-binding sites differ in their localization within the hippocampal formation~O, 32. In the following, we present studies on the properties and ontogenesis of both muscarinic and nicotinic cholinergic binding sites in rat hippocampal formation. The data is correlated with previous studies, reported by other laboratories, on the development of cholinergic innervation in the hippocampal formation of the rat25, 27.
MATERIALS AND METHODS Animals Wistar rats (from the Weizmann Institute Breeding Center) were used. They were killed by cervical dislocation, the brain immediately removed and the hippocampal formation (including the hippocampus, the dentate gyrus and the subiculum) was dissected on ice. Tissue was homogenized (100 mg/ml) in 0.32 M sucrose in a glass-Teflon homogenizer driven by a Heidolph motor at half-maximal speed. Chemicals a-Bungarotoxin (a-Btx) was purified from crude Bungarus multicintus venom (Miami Serpentarium, Miami) 10. Purified toxin displayed a single band on SDS-polyacrylamide gel electrophoresis. Toxin was iodinated with 12zI by the chloramine-T method 4, to a specific activity of 30-60 Ci/mmol. Molarity of toxin was determined by isotopic dilution experiments performed on purified Torpedo nicotinic receptor. The latter was kindly provided by Dr. Sara Fuchs. [3H]Quinuclidinyl benzilate ([3H]QNB), 29.4 Ci/mmol, was from New England Nuclear (Boston, Mass.). [3H]Acetylcholine chloride (100-500 mCi/mmol) was from the Radiochemical Centre (Amersham). Acetylcholine chloride, O,L-muscarine chloride, scopolamine hydrochloride, D-tubocurarine chloride and procaine hydrochloride were from Sigma (St. Louis, Mo.). Atropine sulfate and oxotremorine were from Fluka A.G. (Buchs, Switzerland), gallamine triethiodide and decamethonium bromide were from ICN-K and K (Plainview, N.Y.), and eserine sulfate was from Calbiochem (Los Angeles, Calif.). Dihydro-fl-erythroidine hydrobromide was a generous gift of Merck, Sharp and Dohme (Quebec, Canada) and dexetimide hydrochloride was a generous gift of Janssen Pharmaceutica (Beerse, Belgium). Diethylfluorophosphate was
247 kindly provided by Dr. Gavriel Amitai. All other chemicals were of analytical grade.
a-[leSjBtx binding assays Binding of a-[125I]Btx to hippocampal homogenates was assayed by two methods. (a) Aliquots of homogenate (containing up to about 200 #g protein) were incubated in 0.12 M NaC1, 2 mg/ml BSA, 0.05 M Tris.HC1, pH 7.4, in total volume 50/~1. Reaction was started by addition of a-[125I]Btx (final concentration 12-15 nM) and was carried on for 120 rain at 25 °C. Reaction was terminated by diluting with 2 ml of incubation buffer, followed immediately by vacuum filtration over a wet Millipore EGWP 02500 filter, as described by Vogel and Nirenberg 35. The filter was then washed three times with 2 ml portions of the buffer, and counted in a Packard AutoGamma Spectrometer. This assay method, which determines the amount of labelled aBtx bound to particulate fractions, was employed for the kinetic and pharmacological studies and for two complete sets of developmental studies. The drawback of this assay is the relatively limited amount of protein which could be used without deviation in linearity (usually up to about 200 #g). In addition, the Millipore assay may be expected a priori not to reveal soluble binding sites, which might exist during various developmental stages, or generated in subcellular fractionation experiments. (b) An alternative assay method was used for subcellular fractionation and solubilization experiments, and to repeat one complete set of developmental studies. In this assay, aliquots (containing up to about 800 #g protein in final volume 150 #1) were incubated as above, but reaction was terminated by addition of 2 ml of 30 ~o saturated ammonium sulfate, pH 7.0, containing 2 mg/ml BSA. The reaction mixture was then filtered over a wet glass-fiber filter (GF/C, Tamar, Israel), and the filter was then washed three times with 2 ml portions of the same ammonium sulfate solution and counted as above. This assay enables determinations of toxin-binding in samples containing up to about 1.0 mg protein. In addition, it is known that 30 ~ saturated ammonium sulfate precipitate soluble peripheral nicotinic receptors 26. It was reported that solubilized a-Btx-binding sites from brain resemble peripheral receptors in size31, and therefore we assumed that soluble binding sites might be revealed by an ammonium sulfate assay. We found that when the assays were performed on the same aliquots of particulate fractions, results obtained by the two assay methods mentioned above closely agreed.
[3HI Q NB binding assay Aliquots of homogenate (containing up to about 200 #g protein) were incubated in 0.06 M NaC1, 0.025 M Tris.C1, pH 7.4, in total volume 150 or 300 #1. Reaction was started by addition of [3H]QNB (final concentration 5 nM) and was carried on for 60 min at 25 °C. Reaction was terminated by diluting with 2 ml of incubation buffer, followed immediately by vacuum filtration over a wet glass-fiber filter (GF/C, Tamar, Israel). The filter was then washed three times with 2 ml portions of buffer, dried and placed in vials containing 4 ml 33 ~ (v/v) Triton X-100, 0.8 700 2,5-diphenyloxazole (PPO) and 0.01 ~ 1,4-bis [2-(5 phenyloxazolyl)]benzene(POPOP) in toluene. Vials were maintained for 12-24 h at 25 °C and counted by liquid scintillation spectrometry.
Acetylcholinesterase was determined as described by Johnson and RusselP s, employing [~H]acetylcholine (3.3 mM) as substrate. Protein was determined according to Lowry et al. 24, using BSA as standard. RESULTS
(A) Characterization of cholinergic binding sites Rat hippocampal formation is known to contain cholinergic receptors as revealed by electrophysiologicalS, ~4 and, more recently, by biochemical 2°,32,36,37 methods. The powerful muscarinic antagonist, QNB, has been used to characterize rat brain muscarinic receptors6, 36, and was shown to bind to hippocampal sections z0 and homogenates 37. The pharmacology of QNB-binding sites in the hippocampal formation has only been briefly described 37. It has also been reported that muscarinic binding sites in hippocampus, which react with QNB, display nicotinic characteristics 5. a-Btx, a potent nicotinic antagonist in peripheral systems 4,9,1°,19, specifically binds to hippocampal sections16,29, 32 and homogenates 3z. The pharmacology of abungarotoxin-binding sites in the hippocampal formation has also been only briefly described 32. As a first step in our work, basic properties of both QNB- and a-Btx-binding sites in the hippocampal formation have been studied. Specific binding of [3H]QNB, defined as total binding minus binding in the presence of 10 -6 M atropine, was saturable, with saturation being reached at [3H]QNB concentrations of 1-2 nM (Fig. 1A). The apparent KD, calculated from the binding
i m z o i
2(? I B
I I I I I I0 Labelled ligand, nM
Fig. l. Specificbinding ot [3H]QNB (A) and a-[t251]Btx(B) to homogenate of rat hippocampal formation, as a function of labeled ligand concentration. Aliquots of 20/~1 of homogenate (100 mg/ml in 0.32 M sucrose) were incubated with the appropriate concentration of labeled ligand at 25 °C for 1 h (A) or 2 h (B), and the amount of radioactivity bound was determined by a filtration assay as described under Methods. Specificbinding of [3H]QNBwas defined as total binding minus binding in the presence of 10-6 atropine. Specific binding of a-[125I]Btxwas defined as total binding minus binding in the presence of 10-4 M nicotine.
249 isotherms, was found to be 0.4 =k 0.1 nM. In comparing receptor levels at various developmental stages, it is necessary to work under conditions where saturation of sites is achieved. A concentration of 5 nM [3H]QNB was therefore chosen for routine work. Under these conditions, non-specific binding was about 5 ~ of total binding, and binding reached equilibrium in about 30 min. Assuming a simple, bimolecular reaction, the on-rate constant,//1, was found to be 2 × 106 M -1 sec -1. Less than 20 of binding was released from saturated binding sites 3 h after adding 10.4 M atropine to the aliquot. Specific binding of a-[125I]Btx, defined as total binding minus binding is the presence of 10-4 M nicotine, was also saturable, with saturation being reached at a[125I]Btx concentrations higher than 10 nM (Fig. 1B). The apparent K9, calculated from the binding isotherms, was found to be 2 ~ 1 nM. A concentration of 12-15 nM was routinely employed to achieve saturation. Under these conditions, non-specific binding was about 40 ~ of total, and was not decreased by 1 mM nicotine, 1 mM Dtubocurarine, or even by 10.5 M non-labeled a-Btx. Binding reached equilibrium in about 2 h. Again, assuming a simple, bimolecular reaction, the on-rate constant, K1, was calculated to be 6 × 10a M -~ sec -1. Less than 20 ~ of binding was released from saturated binding sites 2 h after adding 10.5 M non-labeled a-Btx to the aliquot. The pharmacological profiles of [3H]QNB-binding sites and of a-[125I]Btxbinding sites are presented in Table I. Because of the low binding levels obtained with a-[12aI]Btx, it was impractical to perform initial rate studies and hence determine I50 values. Inhibition constants of various ligands for both toxin- and QNB-binding sites were therefore determined under similar saturation conditions, as follows. Concentrations of various ligands that displace 50 ~ of the binding of either [3H]QNB or a[~zSI]Btx at saturation (EDs0 values) were determined from competition experiments. K, the apparent dissociation constant of each ligand, was then estimated from the relation: K=
where K9 is the apparent dissociation constant of either [3H]QNB or a-[125I]Btx obtained from direct binding studies (see above), and [L] is their concentration under the assay conditions 21. From the data presented in Table I it is seen that QNB-binding sites in the hippocampal formation display a muscarinic profile; dihydro-fl-erythroidine, which was reported to effectively block a muscarinic receptor in the hippocampus 5, had no effect on [aH]QNB-binding at relatively high concentrations. On the other hand, gallamine, which is of a nicotinic nature, did display a considerable affinity for the muscarinic site, but was several orders of magnitude less potent than typical muscarinic antagonists. Of the ligands tested, dexetimide, scopolamine and atropine were the most potent in protecting [3H]QNB-binding sites. a-[125I]Btx-binding sites in the hippocampal formation display a nicotinic
250 TABLE I
Inhibition of [3H] Q NB binding and a-f12SI]Btx binding to hippocampal formation homogenate by various ligands Aliquots of homogenate were preincubated for 30 min with the appropriate concentrations of ligands. Reaction was started by addition of either [3H]QNB (final concentration 5 nM) or a-l125I]Btx (final concentration 15 riM) and was carried out for 60 rain (QNB binding) or 120 min (bungarotoxin binding) at 25 °C. EDs0 values were determined from plots of relative specific binding vs. ligand concentration and apparent dissociation constants (K values) were calculated as described in the text. DEFP, diethylfluorophosphate, a potent cholinesterase inhibitor.
K, [3H]QNBbinding sites (M)
K, a-[lZSl]Btx-binding sites (M)
Atropine Scopolamine Dexetimide D,L-Muscarine Oxotremorine Acetylcholine** Gallamine Eserine Nicotine D-Tubocurarine a-Bungarotoxin Dihydro-fl-erythroidine Decamethonium Procaine DEFP
1 5 4 2 1 1 6 5
> 3 x > 3 x 1 x > 3 x > 3 x 1 x 3 x 2 x 1 x 2 x 4 x 1 × 2 x > 3 x >
x x x x X x x x > 4 x > > 6 x 1 x >
10 -9 10 -lo 10 10 10 -5 10-6 10 s 10-6 10-5 10 4, 10-5 10 -6* 10-4* 10-5 10-5 10 -5*
10 -4* 10 4, 10 -5 10-4* 10 4, 10-5 10 -5 10 4 10 -6 10 -° 10-1° 10 5 10 4 10-4* 10 s*
* Little or no effect at this concentration. ** In the presence of 10-5 M DEFP, which has no significant effect on binding activity.
profile, w i t h n i c o t i n e a n d t u b o c u r a r i n e b e i n g t h e m o s t p o t e n t in p r o t e c t i n g t o x i n b i n d i n g sites. A t r o p i n e , s c o p o l a m i n e a n d o x o t r e m o r i n e h a d n o significant effect o n b i n d i n g e v e n at h i g h c o n c e n t r a t i o n s . D e x e t i m i d e d i s p l a y e d s u b s t a n t i a l affinity f o r t o x i n - b i n d i n g sites, b u t still 4 - 5 o r d e r s o f m a g n i t u d e less t h a n the affinity o f t h e s a m e d r u g f o r Q N B - b i n d i n g sites. D e c a m e t h o n i u m , w h i c h is e x t r e m e l y p o t e n t in c o m p e t i n g f o r t o x i n - b i n d i n g sites o f p e r i p h e r a l n i c o t i n i c r e c e p t o r s 9, h a d o n l y a s m a l l effect o n t o x i n - b i n d i n g sites in the h i p p o c a m p u s , as is t h e case f o r o t h e r b r a i n r e g i o n s 31. C h o l i n e r g i c r e c e p t o r s are e x p e c t e d to be m e m b r a n e - b o u n d . I n d e e d , s u b c e l l u l a r f r a c t i o n a t i o n studies r e v e a l e d t h a t b o t h m u s c a r i n i c a n d n i c o t i n i c b i n d i n g sites in t h e h i p p o c a m p a l f o r m a t i o n a r e l o c a l i z e d m a i n l y in t h e ' c r u d e m i t o c h o n d r i a l f r a c t i o n ' , i.e. t h e p a r t i c u l a t e f r a c t i o n s e d i m e n t i n g b e t w e e n 1000 a n d 20,000 x g ( T a b l e II). T h e s a m e is t r u e f o r a c e t y l c h o l i n e s t e r a s e . O n l y 10-15 ~ o f t h e t o t a l m u s c a r i n i c sites, n i c o t i n i c sites a n d esterase sites are l o c a t e d at t h e m i c r o s o m a l f r a c t i o n (i.e., f r a c t i o n s e d i m e n t i n g b e t w e e n 20,000 a n d 100,000
x g), a n d essentially n o a c t i v i t y was f o u n d in t h e
s u p e r n a t a n t o f c e n t r i f u g a t i o n at 100,000 x g f o r 1 h. H o w e v e r , a l t h o u g h similar in t h e i r s u b c e l l u l a r d i s t r i b u t i o n in a n i s o t o n i c h o m o g e n a t e , m u s c a r i n i c sites, n i c o t i n i c sites a n d a c e t y l c h o l i n e s t e r a s e differed in t h e i r sensitivity t o s o l u b i l i z a t i o n p r o c e d u r e s .
251 TABLE II
Subcellular distribution of muscarinic binding sites, nicotinic binding sites and acetylcholinesterase in rat hippocampal formation Hippocampi from adult rats were pooled (total of 770 mg wet weight) and homogenized in 7.5 ml of 0.32 M sucrose. The homogenate was than centrifuged at 1000 x g for 10 min, the pellet (P, 1000 x g ) dispersed in 0.32 M sucrose and the supernatant (S, 1000 x g ) further centrifuged at 20,000 × g for 30 rain. Again the pellet was dispersed (P, 20,000 x g) and the supernatant (S, 20,000 x g) further centrifuged at 100,000 x g for 1 h. The latter centrifugation yielded S, 100,000 x g and P, 100,000 x g. Activities were determined as described under Methods, and are presented both as activity in each fraction and as percentage of the appropriate total activity in the complete homogenate.
Homogenate S, 1000 x g P, 1000 x g S, 20,000 x g P, 20,000 x g S, 100,000 x g P, 100,000 x g
[3H]QNB specific binding
a-[12Sl]Btx specific Acetylcholinesbinding terase activity
pmol in each fraction
/o°/ total activity
pmol in each fraction
~ total activity
nmol/min % total in each activity fraction
mg in each fraction
~ total protein
56.4 44.6 4.8 5.9 37.1 0.04 7.9
100 79 9 10 66 0.1 14
1.35 1.36 0.15 0.26 1.23 0 0.18
100 101 11 19 91 0 13
4182 3451 259 846 2277 127 490
66.3 47.4 9.0 14.9 32.4 13.7 5.0
100 71 14 22 49 21 8
100 83 6 20 54 3 12
Effect of high ionic strength and a detergent on the subcellular distribution of muscarinic binding sites, nicotinic binding sites and acetylcholinesterase in rat hippocarnpal formation Homogenates (100 mg/ml) were prepared in 0.025 M Tris, pH 7.4 (-- Buffer), Buffer ÷ 2 M NaCI, or Buffer ÷ 1 ~ Triton X- 100, and were stirred for 2 h at 4 °C. Activities were then determined in aliquots of the homogenate and of the supernatant of 100,000 x g centrifugation for 1 h. For a-[125I]Btx binding, the ammonium sulfate precipitation assay was used (see under Methods). For [ZH]QNB binding, preliminary experiments were performed to test whether precipitation with ammonium sulfate (in the range 20-70 ~ saturation), or filtration on Millipore E G W P filter, yield different subcellular distribution profiles. Results did not significantly vary with various assay procedures and the data presented are for a Millipore filter assay. The experiment was repeated twice and values are percentage activity relative to activity in buffer homogenate. Values in parentheses are total activities normalized per 1.4 ml homogenate in each case.
Buffer Homogenate %
Buffer + NaCI
Buffer + Triton
100,000 x g Homogenate 100,000 x g Homogenate 100,000 x g supernatant % supernatant % supernatant
[SH]QNB binding 100 5 (14.2 pmol) a-[12aI]Btx 100 0 binding (0.36 pmol) Acetylcholin100 4 esterase (632 nmol/min)
,o an Z O 3~ o
- ;;',, I
' , ' , ; ; I ','.',', I ' , ' , ' , ; I ' , ' , ' , ; I I',.,s/ I
I,,,,I,,,, i,,,l ,,,I, . 20 25 30 35 40 " A
Age(day s )
Fig. 2. Development of specific [3H]QNB binding in rat hippocampal formation. Data are presented as total binding per hippocampus (A) and as binding per tissue protein (B). Each point represents one experiment,in which binding was determined in an homogenate prepared from 1-3 pooled hippocampi. Values for adult animals (older than 3 months) are mean ± S.E.M. for 8 experiments.
High ionic strength did not solubilize any of the sites, and was inhibitory for toxinbinding. On the other hand, Triton X-100 completely destroyed muscarinic binding activity, but was very efficient in releasing both toxin-binding sites and esterase into the supernatant of a 100,000 × g, centrifugation (Table Ill). On the whole, we found that the hippocampal formation contains about 30-40fold more muscarinic binding sites than toxin-binding sites.
(B) Developmentof cholinergic binding sites Very little binding of [aH]QNB was detected until day 3 after birth. The earliest increase in muscarinic receptors starts at about day 4, and an especially sharp increase in total binding takes place between days 10 and 15 (Fig. 2A). Thus between days 10 and 15, absolute binding increases about 2.5-fold. After the end of the second postnatal week, total activity increases gradually and reaches mature levels at an age of about 7 weeks. On the other hand, specific activity of [3H]QNB-binding sites already attains mature values at an age of about 15 days (Fig. 2B). Thus the absolute level of muscarinic receptors increases about 3-fold between day 15 postnatal and maturity (Fig. 2A), but protein content of the hippocampal formation increases at a similar rate (Fig. 4).
253 E ~
. _o. ,oo
"" • • :;-
2o ,I . . . . -5
I .... 0
I .... 5
I .... I0
I .... I .... I .... I .... t .... I,,~ 15 20 25 30 35 40 Age(days)
Fig. 3. Development of specific a-[zzSI]Btx binding in rat hippocampal formation. Data are presented as total binding per hippocampus (A) and as binding per tissue protein (B). Each point represents an experiment in which binding was determined by the Millipore filter assay in an homogenate prepared from 1-3 pooled hippocampi. Values for adult animals (older than 3 months) are mean ::k S.E.M. for 8 experiments. The study was repeated with another set of animals, employing the ammonium sulfate precipitation assay (see under Methods), and results obtained were essentially the same.
~. t5 E tn
- 300 0
, ~1, , , _ . ~ _ _ . . . . 0
Fig. 4. Development of acetylcholinesterase activity ( O ©) and protein content (O Q) in rat hippocampal formation. Each point represents a single determination performed on an homogenate prepared from 1-3 pooled hippocampi, and values for adult animals are mean i S.E.M. for 8 determinations.
.20 0 I00
~ 8o x ii1 ~
l I iO'm Ligond
Fig. 5. Displacement of specific [aH]QNB binding by various concentrations of atropine (A) and of specific a-[Z25I]Btx binding by various concentrations of nicotine (B), in hippocampal formation homogenates of newborn and adult rats. For experimental details, see Table I. (3 ©, 5 days postnatal; ~ ~, 10 days postnatal; • • , adult.
Development of a-Btx-binding sites in the hippocampal formation markedly differs from that of muscarinic binding sites. Absolute binding increases about 12-fold from birth until days 12-14, when it reaches mature levels (Fig. 3A). Specific activity of the nicotinic binding sites is higher in the first postnatal days than at maturity (Fig. 3B). A similar developmental profile was obtained both when the assay was performed by the Millipore method or by the ammonium sulfate precipitation method (see Methods). When compared to the development of another cholinergic component of the hippocampal formation, acetylcholinesterase, it could be seen that the ontogenesis of muscarinic sites is essentially similar to that of the esterase, whereas that of the nicotinic sites is strikingly different (Fig. 4). We did not detect significant differences between the apparent dissociation constants of the cholinergic binding sites from the hippocampal formation of newborn rats and the same sites from adult rats. Moreover, no significant age-dependent differences were found in protection of labeled-ligand binding by competitive drugs such as atropine or nicotine (Fig. 5).
DISCUSSION The hippocampal formation contains at least two distinct types of cholinergic binding sites, aside from the enzymes acetylcholinesterase and choline acetylase. The major site, as revealed by binding of [SH]QNB, is of a muscarinic nature. It has been reported, on the basis of iontophoretic studies, that the hippocampus contains a cholinergic receptor with a mixed muscarinic-nicotinic nature, that is blocked by
255 muscarinic antagonists, e.g. QNB, as well as by some nicotinic antagonists, e.g. dihydro-fl-erythroidine5. Our direct binding studies in vitro show that dihydro-flerythroidine does not protect QNB-binding sites even at high concentrations. QNBbinding sites did reveal considerable affinity for the nicotinic antagonist gallamine. However, gallamine is a flexible triquaternary compound, and a reasonable affinity for a cholinergic muscarinic site may be expected, o-Tubocurarine, which is a more potent nicotinic antagonist than gallamine, but which is a bisquaternary compound, showed less affinity for the muscarinic binding site. Potent nicotinic ligands, such as nicotine or a-Btx, did not affect the muscarinic sites even at high concentrations. Thus our results do not support the suggestion that the hippocampus cholinergic receptor is of a mixed nicotinic-muscarinic type~. The rate of development of muscarinic receptors in the hippocampus resembles that of acetylcholinesterase. Cholinergic receptors and esterase are also concomitantly expressed in other developing cholinergic systems, e.g. muscle~°. However, in muscle both receptor and esterase are synthesized by the same cell, whereas, in the hippocampus, the receptor is postsynaptic87, but up to 90 ~ of the enzyme is ofa presynaptic origin 22,33. This may imply that the expression of both these components of the cholinergic system is coordinated in the hippocampus by some intercellular signal. A similar situation was reported by Enna et al. la for the GABA system in chick brain; no appreciable time difference was detected between the development of GAD activity (which is presynaptic) and the GABA receptor (which is postsynaptic). It is of interest to correlate the ontogenesis of cholinergic binding-sites in the hippocampal formation with innervation and synaptogenesis in the region, processes which were previously described by other laboratoriesll,25,2L In the hippocampal formation synaptic density, as revealed by ultrastructural studies, reaches adult values at about 25 days postnatally~1. Septohippocampal axons start to invade the hippocampus at about 4 days postnatally, as shown by esterase staining, and proceed toward the temporal end of the region, until, about 11 days postnatally, all parts of the hippocampal formation are innervated by cholinergic afferents2~. Major synaptogenetic events take place in the hippocampal formation between 11 and 21 days after birth zT. It appears therefore that toxin-binding nicotinic sites reach mature levels before the major synaptogenetic events in the hippocampal formation are completed. However, one has to consider the possibility that the binding in vitro does not reflect maturation of a physiological function, as was recently suggested for the muscarinic acetylcholine receptor in developing chick heart 15. Muscarinic receptors in the hippocampal formation attain mature binding levels after the gross synaptogenetic events are completed. But processes such as increase in density of synaptic connections as revealed by ultrastructural studies ~1 or arrival of presynaptic terminals as demonstrated by histochemical techniques25, 27 do not necessarily imply maturation of synaptic function. Thus whereas the number of synapses in the dentate gyrus of the rat approximates adult values by 25 days postnatal, complex spine formation is completed only latedL Synaptin, a synaptosomal antigen, also reaches mature values in the mouse after major synaptic events are completed~7. It is therefore plausible to assume that the rate of development of acetylcholinesterase and muscarinic binding-sites in
256 the hippocampal f o r m a t i o n reflects the time course of functional maturation o f synapses. The differential distribution o f a-toxin-binding nicotinic sites 82, and their rate o f development (Fig. 3), imply that they fulfil a different physiological function f r o m muscarinic sites. W h a t is that function ? Are a-Btx-binding sites functional acetylcholine receptors? Several studies have demonstrated non-equivalence of toxinbinding sites and acetylcholine-binding receptor sites in ganglia 7,8,28, and as yet there is no p r o o f that a-toxins indeed label the acetylcholine site o f a nicotinic receptor in the brain. It is possible that in ganglia and brain a-toxins bind to a cholinergic receptor at a site which is distinct f r o m the acetylcholine site. A n o t h e r possibility is that the toxins label macromolecules which are not receptors involved in neurotransmission. Nevertheless, these components seem to be associated with synapses. Thus it was recently demonstrated by a u t o r a d i o g r a p h y that a-Btx-binding sites in rat hippocampus are located in synaptic complexes 16. In addition we have shown by fornix lesion studies that toxin-binding sites in the h i p p o c a m p u s are not located on septal afferants 12. In conclusion, although their physiological function is not yet known, it is o f interest to note that the ontogenesis of toxin-binding sites does not resemble the ontogenesis of other components of the cholinergic system in the hippocampal formation. ACKNOWLEDGEMENTS The skilled technical assistance o f Shoshana N a h u m is gratefully acknowledged. This work was supported by a grant f r o m the United States-Israel Binational Science Foundation, Jerusalem, Y.D. is incumbent o f the Barecha F o u n d a t i o n Career Development Chair. REFERENCES 1 Altman, J. and Das, G. D., Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats, J. comp. Neurol., 124 (1965) 319-335. 2 Angevine, J. B., Time of neuron origin in the hippocampal region, Exp. Neurol., 2 Suppl. (1965) 1-70. ~3 Bayer, S. A. and Altman, J., Hippocampal development in the rat. Cytogenesis and morphogenesis examined with autoradiography and low-level X-irradiation, J. eomp. Neurol., 158 (1974) 55-80. 4 Berg, D. K., Kelly, R. B., Sargent, P. B., Williamson, P. and Hall, Z. W., Binding of a-bungarotoxin to acetylcholine receptors in mammalian muscle, Proe. nat. Acad. Sci. (Wash.), 69 (1972) 147-151. 5 Bird, S. J. and Aghajanian, G. K., The cholinergic pharmacology of hippocampal pyramidal cells: A microiontophoretic study, Neuropharmacology, 15 (1976) 273-282. 6 Birdsall, N. J. M. and Hulme, E. C., Biochemical studies on muscarinic acetylcholine receptors, J. Neurochem., 27 (1976) 7-16. 7 Brown, D. H. and Fumagalli, L., Dissociation of a-bungarotoxin binding and receptor block in the rat superior cervical ganglion, Brain Research, 129 (1977) 165 168. 8 Carbonetto, S. T., Fambrough, D. M. and Muller, K. J., Nonequivalence of a-bungarotoxin recertors and acetylcholine receptors in chick sympathetic neurons, Proe. nat. Aead. Sci. (Wash.), 75 (1978) 1016-1020. 9 Changeux, J. P., The cholinergic receptor protein from fish electric organ. In L. L. Iverson, S. P. Iverson and S. H. Snyder (Eds.), Handbook ofPsychopharmaeology, 6 (1975) 235-301. 10 Changeux, J. P., Kasai, M. and Lee, C. Y., Use of a snake venom toxin to characterize the cholinergic rezeptor protein, Proe. nat. Acad. Sci. (Wash.), 67 (1970) 1241-1247.
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