Brain Research, 163 (1979) 263-275 © Elsevier/North-HollandBiomedicalPress
263
DEVELOPMENT OF HIGH AFFINITY CHOLINE UPTAKE AND ASSOCIATED ACETYLCHOLINE SYNTHESIS IN THE RAT FASCIA DENTATA
DAVID L. SHELTON,J. VICTOR NADLER* and CARL W. COTMAN Department of Psychobiology, University of California, lrvilie, Calif. 92717 (U.S.A.)
(Accepted July 13th, 1978)
SUMMARY The ontogenic development of hemicholinium-sensitive, high affinity choline uptake and the synthesis of acetylcholine from exogenous choline have been studied in particulate preparations of the rat fascia dentata. Between 6 days of age and adulthood the rate of high affinity choline uptake increases 3-fold, when expressed with respect to protein, and 125-fold, when expressed independently of protein. This process develops most rapidly during the period around 16-17 days of age, similar to the ontogenesis of choline acetyltransferase activity. This observation supports the idea that cholinergic septohippocampal boutons develop mainly at this time. Unlike choline acetyltransferase activity, the velocity of high affinity choline uptake increases to as much as 161 ~ of the adult value at about 30 days of age. It is suggested that at 25-31 days of age a relatively high endogenous septohippocampal firing rate increases the rate of choline uptake. At 6 days of age we detected no synthesis of acetylcholine from the accumulated choline. Uptake-synthesis coupling develops mainly between 6 and 13 days of age, earlier than any other presynaptic cholinergic property. Acetylcholine synthesis from exogenous choline develops in parallel with high affinity choline uptake, but developmental increases in uptake velocity result in comparable increases in synthesis rate only after a delay of several days. Some limiting factor other than choline acetyltransferase activity appears to link the accumulation of exogenous choline to acetylcholine synthesis during development.
INTRODUCTION The rat fascia dentata is particularly suitable for detailed developmental studies * To whomall correspondenceshouldbe addressed. Presentaddress: Departmentof Pharmacology, Duke UniversityMedicalCenter, Durham, N.C. 27710 U.S.A.
264 of cholinergic innervation. This part of the hippocampal formation is comprised of a single homogeneous layer of granule cells overlain with a molecular layer, in which the dendrites of the granule cells ramify and afferent fibers establish their connections in a laminar fashionS, 10. There are no intrinsic cholinergic neurons, the entire cholinergic innervation of the fascia dentata originating in the septum. All presynaptic cholinergic properties have been related to the septohippocampal tract. These include choline acetyltransferase (CAT) activity29,31, '50, acetylcholinesterase (ACHE)activity ":~.~.~, AChE-dependent histochemical staining31,33,37,49, 50, high affinity choline uptake 29, acetylcholine (ACh) contentZg, 42 and ACh release12,13,3s,zL Thus one can relate developmental events to a single morphological entity. The growth of septohippocampal fibers into the rat fascia dentata has been described 36, as well as the development of CAT and AChE activities within these fibers 4°. In this report we describe the development of high affinity choline uptake and its coupling to acetylation of the accumulated choline. High affinity choline uptake serves as a marker for presynaptic cholinergic membrane2S,Zg,sL It is dependent upon extracellular Na + and C I - (refs. 21, 23, 25, 45, 53), requires a source of cellular energy45, 53 and is exceedingly sensitive to drugs of the hemicholinium series 2l 25, 46,53. This transport system provides nearly all the choline required for ACh synthesis3,23,3°,41,53 and in sucrose homogenates is localized mainly in nerve-ending particles25, 53. High affinity choline uptake should thus be a sensitive indicator of the development of cholinergic axon terminals. We have therefore related the development of hemicholinium-sensitive, high affinity choline uptake and the associated ACh synthesis to the ontogenic growth of cholinergic fibers and development of AChrelated enzyme activities. METHODS
Preparation of particulate fractions Female Sprague-Dawley rats were purchased from Simonsen Laboratories (Gilroy, Calif.) during pregnancy. The day of birth was taken as day 0. At ages ranging from 6 to 105 days, the pups were decapitated, and the brain was quickly removed and immersed in ice-cold 0.32 M phosphate-buffered sucrose (pH 7.1-7.3 with 50 #M sodium phosphate). This operation took less than 1 min. After a few minutes in cold sucrose, the hippocampal formation was isolated from one side of the brain and cut into transverse slices of 500 #m thickness with a Mcllwain tissue chopper (Brinkmann Instruments, Westbury, N.Y.). The tissue was maintained in ice-cold sucrose during the cutting and subsequent dissection procedures. When animals 16 days of age or older were used, the fascia dentata was divided into molecular and granular layers. With the aid of a dissecting microscope and sharp dissecting knifO °, the fascia dentata of each slice was first divided parallel to the granule cell layer about one-fourth to onethird the distance from the outer edge of the latter to the outer edge of the molecular layer (Fig. 1). The molecular layer was then isolated by cutting along the hippocampal fissure, and the granular layer was removed by cutting immediately beneath it, where a natural break in the tissue occurs. The 'molecular layer' thus actually included
265
Fig. 1. Dissection of layers of the fascia dentata shown on a section (from a postfixed transverse hippocampal slice) stained for AChE activity. This histochemical staining localizes the septohippocampal fibers al,aT,4a. M, molecular layer; G, granular layer. Layers were isolated by cutting along the dashed lines, as described in the text. Scale bar = 0.5 mm.
approximately the outer 70 ~ of this layer, that is, the perforant path terminal zone35, 4s. The 'granular layer' included not only the granule cell bodies themselves, but also a somewhat variable amount of inner molecular layer. Since it was not possible to divide the layers accurately in animals younger than 16 days, the fascia dentata was isolated whole at these ages. Choline uptake was determined on particulate fractions which contained synaptosomes. Corresponding layers from all slices were pooled and homogenized in 0.5 ml of 0.32 M phosphate-buffered sucrose with a Teflon-glass homogenizer. The homogenate was diluted with an additional 7 ml of sucrose and centrifuged at 42,000 × g and 4 °C for 20 min. Preliminary experiments showed that this procedure sedimented all particles that accumulated choline under our incubation conditions. The resulting supernatant was discarded and the pellet resuspended in 0.5 ml of sucrose. This particulate preparation was immediately assayed for choline uptake activity after removal of a small portion for determination of protein 32.
Choline uptake For determination of choline uptake activity, 0.1 ml of particulate suspension was added to 0.9 ml of Elliott's artificial CSF 15 (122 m M NaC1, 3.1 m M KCI, 1.2 m M
266 MgSO4, 0.4 mM KHzPO4, 1.3 mM CaClz, 25 mM NaHCO3, 10 mM D-glucose, pH 7.37.5 with 95 % compressed air/5 % COz). This mixture was preincubated at 38 °C under an atmosphere of 95 o/,, compressed air/5 % CO2. After 10 min, 10 #1 of [methyl-ZH]choline chloride (10.1 Ci/mmol, Amersham-Searle Corp., Des Plains, I11.) was added to yield a final choline concentration of 0. l/~M. The incubation was continued for an additional 4 min. At the end of this period the incubation mixture was diluted with 2 ml of Elliott's medium at room temperature and filtered by gentle suction through a glass fiber filter (Whatman GF/A, 2.5 cm diameter) underlain with a membrane filter (GE Nuclepore, 2.5 cm diameter, 0.4/~m pore size). The tissue trapped on the filter was then washed with an additional eight 2 ml portions of medium. Filtration and washing were accomplished in less than 25 sec. Preliminary experiments showed that this procedure removed essentially all the unbound [3H]choline from the filter. When only choline uptake was measured, the filters were immersed in 2 ml of 1 ~ (w/v) sodium dodecylsulfate, 20 mM EDTA (pH 8.0 with NaOH) overnight to solubilize the tissue. The solubilized tissue was then counted with use of Tritoso116. Parallel control incubation mixtures contained 0.8/~M hemicholinium-3 (HC-3) (Aldrich Chemical Co., Milwaukee, Wisc.) to inhibit high affinity choline uptake selectively3. At this concentration HC-3 inhibits virtually all the high affinity uptake, but has little effect on low affinity uptake and should not affect presynaptic cholinergic function in any other way. High affinity choline uptake was calculated by subtracting the radioactivity on the filter after incubation in the presence of HC-3 from that remaining on the filter after incubation in its absence. After subtraction of this blank value, choline uptake was found to be linear with time of incubation for 5-6 min, and the rate of uptake was proportional to the quantity of tissue incubated up to at least 100/zg of protein. We found little evidence of a low affinity uptake process under our incubation conditions, in agreement with those who have used Na -~-free controls 44. Preliminary experiments without tissue and with varying amounts of tissue showed that most of the apparent HC-3-resistant uptake of [3H]choline could be accounted for by binding to the filters.
Extraction and separation of choline and A Ch When both choline uptake and ACh synthesis were measured, the washed tissue was treated with 1.2 ml of a 3-heptanone solution containing 5 mg/ml sodium tetraphenylboron (TPB) to extract choline and ACh, 20/~M diisopropyl fluorophosphate to inhibit extracellular cholinesterase (intracellular cholinesterase is said to be absent from cortical tissue 6) and 1 nCi of [methyl-a4C]choline chloride to determine the efficiency of the separation procedure. Choline and ACh were extracted from the organic phase with 0.2 ml of 0.4 N HCI. A 50 /zl portion of the acid extract was counted for determination of choline uptake activity and 100 /zl was dried under vacuum and stored at --22 °C prior to separation of choline and ACh. Values of high affinity choline uptake from these studies equalled those from studies in which the tissue was dissolved in sodium dodecylsulfate, suggesting that negligible amounts of [ZH]phosphorylcholine and other metabolites not extracted by TPB were formed. ACh and choline were separated by the enzymatic method of Goldberg and
267 McCaman 19. The dried residue was dissolved in 10 #1 of an ice-cold solution consisting of 0.15 unit/ml choline kinase (Sigma Chemical Co., St. Louis, Mo.), 1 mM ATP, 5 mM MgC12 and 50 mM sodium phosphate buffer, pH 8.0. This mixture was incubated for 15 min at 38 °C to phosphorylate the choline. The samples were then placed on ice, and ACh was extracted with 200 #1 of 75 mg/ml TPB in 3-heptanone. After addition of 90 #1 of ice-cold 1-I20, the phases were separated by centrifugation and portions of each counted. Greater than 95 700 of the choline was recovered (as phosphorylcholine) in the aqueous phase, as determined from the [14C]choline internal standards. External standards of [14C]ACh, with or without unlabeled choline, showed that at least 93 70 of the ACh in the sample was extracted by TPB. The rate of ACh synthesis was calculated by subtracting the amount of [aH]ACh synthesized in 4 min in the presence of HC-3 from the amount synthesized in the absence of HC-3. Less than 10 70 of accumulated choline was converted to ACh when 0.8 #M HC-3 was present during incubation. RESULTS HC-3-sensitive, high affinity choline uptake is demonstrable in the rat fascia dentata at 6 days of age, the earliest developmental stage examined in this study. From this point to adulthood, the initial velocity increases 125-fold, when expressed independently of protein (Fig. 2B), and about 3-fold, when expressed relative to protein (Fig. 2A). The most rapid phase of development occurs during the period around 16-17 days of age, similar to the ontogenesis of CAT activity (Fig. 2A and ref. 40), a better established presynaptic cholinergic marker. After this age, however, the velocity of choline uptake increases much faster than CAT activity. At 29-31 days of age particulate fractions of the fascia dentata already transport choline at the adult rate, when expressed independently of protein (Fig. 2B). If the values are expressed relative to protein (Fig. 2A), uptake velocities at 25 and 29-31 days of age exceed those of adult fascia dentata by 38 ~ and 61 70, respectively (P < 0.01 at 25 days and P < 0.001 at 29-31 days, two-tailed Student's t-test). In contrast, the fascia dentata acquires CAT activity more gradually, and the specific activity of CAT never exceeds the adult value at any age studied. The development of high affinity choline uptake differs even more markedly from that of AChE activity, although both are considered to be associated mainly with the presynaptic cholinergic membrane29,4a, 49. Values of the HC-3 blanks remained approximately constant at all ages (0.03-0.1 pmol/4 min/sample). Hence they decreased considerably during development, when expressed relative to protein. As discussed in Methods, we have attributed most of the apparent HC-3-resistant choline uptake in this preparation to non-specific filter binding. At 6 days of age we detected no conversion of the accumulated choline to ACh (Fig. 2). Conversion of as little as 10 70 could have been detected. From 11 days of age onward, however, the development of ACh synthesis from exogenous choline parallels that of high affinity choline uptake, suggesting that the availability of recently accumulated choline regulates ACh synthesis during most of the developmental
268
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Fig. 2. Development ofcholinergic markers in the rat fascia dentata. Symbols: filled triangles, HC-3sensitive, high affinity choline uptake; open triangles, ACh synthesis from exogenous choline; filled circles, CAT activity; open circles, AChE activity. Bars indicate S.E.M. Data for development of CAT and AChE activities were recalculated from ref. 40 (hilus layer was excluded). For number of experiments represented by each mean see ref. 40 and Figs. 3 and 4. A: velocities calculated with respect to protein. Adult values were: choline uptake, 13.0 4- 0.6 pmol/4 min/mg protein; ACh synthesis, 8.13 ± 0.38 pmol/4 rain/rag protein; CAT activity, 13.0 ± 0.7/~mol/30 min/g protein; AChE activity, 1600 ± 70/~mol/30 min/g protein. B: velocities calculated independently of protein. Adult values were: choline uptake, 5.80 ± 0.45 pmol/4 min/fascia dentata of one side of brain; ACh synthesis, 3.64 i 0.28 pmol/4 min/fascia dentata of one side of brain; CAT activity, 2.60 ± 0.19 tLmol/30 min/1.2 mm length of fascia dentata; AChE activity, 322 ~ 28/~mol/30 min/l.2 mm length of fascia dentata.
period, as it does in adult rats2,26,89,41,44. Surprisingly, however, the developmental time course of ACh synthesis does not coincide with that of choline uptake. Rather, changes in the overall rate of synthesis follow those in uptake velocity only after a delay of several days. When animals 16 days of age or older were examined, we analyzed high affinity choline uptake and ACh synthesis in separated granular and molecular layers to determine whether developmental changes are uniformly distributed in the fascia dentata or restricted to a particular lamina. In adult rats choline uptake velocity expressed relative to protein is about 20 ~ greater in the granular layer than in the molecular layer (Fig 3), a distribution very similar to that of CAT activity 4°. The difference between the two layers first becomes evident between 21 and 25 days of age. The rate of high affinity choline uptake appears to increase slightly earlier in the
269
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Fig. 3. Development of HC-3-sensitive, high affinity choline uptake in layers of the fascia dentata. Values are means ± S.E.M. for the number of animals given in parentheses. Results on whole fascia dentata of animals 6, 11, and 13 days of age are given for comparison.
molecular layer than in the granular layer between 16 and 31 days of age, but the magnitude of increase is much greater in the granular layer. In adult rats, particulate fractions of the granular layer synthesize about 28 more [3H]ACh from exogenous [SH]choline than particulate fractions of the molecular layer (Fig. 4), in accordance with the distribution of high affinity uptake. The difference between the layers first becomes evident at 29-31 days of age. Between 16 and 31 days of age changes in ACh synthesis follow a similar time course in the two layers, in each case lagging behind the increases in high affinity choline uptake.
20' Molecular Layer
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Fig. 4. Development of ACh synthesis from exogenous choline in layers of the fascia dentata. See Fig. 3 for other details.
270 8O Molecul0r Layer
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Fig. 5. Developmentof coupling between high affinitycholine uptake and ACh synthesisin layers of the fascia dentata. Values are percentages of accumulated [all]cholineconverted to [aH]ACh.See Fig. 3 for other details.
Particulate fractions of both layers from adult rats convert 60-65 ~ of the accumulated choline to ACh (Fig. 5), values similar to those obtained from studies in which 0 °C or Na÷-free blanks were used and cholinesterase activity was suppressed44,z3. The percentage conversion of transported precursor to intraparticulate product serves as a measure of the coupling of uptake to transmitter synthesis. This coupling develops mainly between 6 and 13 days of age, during which period the conversion of [all]choline to [aH]ACh increases from undetectable to 40-50 ~. The most rapid development of uptake-synthesis coupling thus precedes that of any other presynaptic cholinergic property. An additional increase occurs between 29 and 31 days of age and adulthood. Preparations of granular and molecular layer at all ages convert approximately equal percentages of accumulated choline to ACh. DISCUSSION Within the rat hippocampal formation high affinity choline uptake is a property of the septohippocampal presynaptic membrane 29. Previous workers have suggested a predominant localization for this transport process in cholinergic synaptic boutons14, 50 and synaptosomes zS,z3, and our finding that high affinity choline uptake and CAT activity are similarly distributed between layers of the fascia dentata further supports this view. Thus one would expect measurements of high affinity choline uptake velocity during development to indicate the time course of cholinergic synaptogenesis. However, some caution is required on two counts. First, the velocity of high affinity choline uptake varies not only with the number of cholinergic boutons (or, more properly, the amount of cholinergic presynaptic membrane), but also, at least in adult rats, with the electrophysiological activity of the cholinergic fibers 1,2,44. Studies of CAT activity should, however, distinguish growth-induced from activity-
271 induced alterations of the choline uptake rate, since CAT activity would be expected to vary with the number of cholinergic boutons, but not with short-term changes in impulse flow44. Second, changes in uptake velocity should be referable to an altered number of uptake sites and not a change in the affinity of the carrier for choline. Sorimachi and Kataoka have shown no change in the Km for Na+-dependent, high affinity uptake of choline during the developmental period covered by our study 47. Unfortunately, we were unable to perform comparable kinetic studies on dentate particulate preparations, because choline uptake saturated during a 4 min incubation at concentrations of 0.2 #M or above, considerably below the reported Km at 38 °C44. Damage to the synaptosomes during preparation might account for this result. This explanation appears unlikely, however, since the adult choline uptake rate obtained in the present study actually exceeds that which may be calculated from Michaelis-Menten kinetic parameters computed by others who incubated hippocampal preparations at 38 °C44. Possibly our preparations contained a higher level of endogenous choline than those used by other workers, thus limiting the quantity of exogenous precursor which could be accommodated within the synaptosome. We cannot therefore exclude the possibility that a change in the affinity of the transport carrier for choline might account for developmental changes in choline uptake, although previous evidence suggests that this is unlikely. The velocity of high affinity choline uptake by particulate preparations of fascia dentata changes little between 6 and 13 days of age, when calculated with respect to protein. Neither does the CAT activity of the hippocampal formation as a whole4°. These results suggest little formation of cholinergic boutons during this period. Whatever increases may occur can probably be attributed to continued growth of the septohippocampal fibers, which are present in the septal two-thirds of the hippocampal formation at 6 days of age and invade the temporal extreme between 6 and 11 days a6. Enzyme and choline transport carrier may be located mainly in the axonal growth cones at this time. Both uptake and CAT activities increase most rapidly during the period around 16-17 days of age, a time at which large numbers of synapses are forming in the dentate molecular layer 11. Indeed both parameters attain adult values (per unit protein) at this age. In accordance with developmental studies in other systems4,7-9,34, the changes in CAT activity have been interpreted to signify a rapid formation of cholinergic boutons in the period around 16-17 days of age 4°. Results of the present study support this interpretation. They do not, however, agree with the report of Sorimachi and Kataoka, who found that Na+-dependent, high affinity choline uptake develops somewhat more rapidly in the hippocampal formation than CAT activity between 7 and 17 days of age 47. Our studies and theirs show a similar development of CAT activity, but we find a more delayed development of high affinity choline uptake than they do. This discrepancy may be attributable to our measuring CAT and choline uptake activities in the fascia dentata alone (whereas Sorimachi and Kataoka assayed them in the whole hippocampal formation), to a difference in the strain of rats studied or, least likely, to a difference in the type of blank used. After 17 days of age high affinity choline uptake develops very differently from CAT activity. Uptake activity expressed relative to protein increases to 161% of the adult
272 value at 29-31 days of age, whereas no other presynaptic cholinergic measure exceeds the adult value at any time point studied. This excess uptake activity was not reported in the previously cited developmental study of the hippocampal formation as a whole 47, but the 25-31-day period was not investigated. On a protein-independent basis, high affinity choline uptake activity reaches, but does not exceed, adult rates around 25 days of age. One could interpret these results to imply that cholinergic presynaptic membrane expands much more rapidly than other tissue elements between 16 and 31 days of age, completing its development around the end of this period. Neither CAT activity nor AChE activity, another property of cholinergic membrane, shows any comparable change, however. Alternatively, an increase in the endogenous septohippocampal firing rate could conceivably account for the specific augmentation of choline uptake activity. This possibility is supported by results of a preliminary study in which pentylenetetrazol (PTZ) failed to increase the velocity of high affinity choline uptake in dentate preparations from two 30-day-old rats, but was effective in adults, as others have reportedZ, 44. PTZ is thought to act by increasing the septohippocampal firing rate. Around 30 days of age these fibers may be activated to such a a degree normally that PTZ cannot significantly augment their firing. High affinity choline uptake is thought to regulate ACh synthesis in adult rats, since the choline accumulated by the high affinity carrier is extensively and preferentially acetylated 2°,2~,4~,51,53 and treatments that alter the velocity of uptake affect the rate of ACh synthesis to the same degree 44. This tight coupling between uptake and synthesis has led to the concept that the membrane-associated high affinity carrier may be physically joined to the cytoplasmic CAT 3. On the other hand, treatments which reduce the availability of acetyl-CoA reduce ACh synthesis by a similar amount ~7, ~s,z7 without significantly affecting choline uptake 27. It would thus appear that uptake and acetylation are stoichiometrically coupled only when the intracellular acetyl-CoA concentration is saturating. Indeed the synthesis of acetyl-CoA may be as closely coupled to ACh synthesis as choline uptake. Results of the present study suggest that high affinity choline uptake and acetylation of the accumulated choline are not inherently coupled, but that regulation of ACh synthesis by the uptake process develops over an extended period. At 6 days of age less than 10~ of the accumulated choline is converted to ACh. Lack of CAT activity probably cannot account for this result, since the hippocampal formation as a whole already shows one-fourth to one-third the adult activity (per unit protein) at this age 40 and cholinergic fibers have entered the fascia dentata 36. Uptake becomes coupled to acetylation between 6 and 13 days of age, just before the period during which both processes develop rapidly. Possibly either the independently synthesized and transported carrier and enzyme first become associated at this time or else acetylCoA becomes increasingly available to the enzyme. Between 31 days of age and adulthood the percentage conversion of accumulated choline to ACh again increases. This tightening of uptake-acetylation coupling may have a similar explanation. That acetylation of choline is, in fact, regulated in part by high affinity choline uptake during the developmental period is indicated by the remarkable parallelism in their rates of development (Fig. 2). In contrast to adult hippocampal formatiom in
273 which an altered u p t a k e rate i m m e d i a t e l y translates into an a l t e r e d rate o f A C h synthesis 44, d e v e l o p m e n t a l increases in the rate o f A C h synthesis f r o m exogenous choline lag b e h i n d increases in choline u p t a k e b y 5-8 days. Deficient C A T activity c a n n o t a c c o u n t for this delay. This result f u r t h e r suggests the existence in d e v e l o p m e n t o f some limiting factor (possibly a c e t y l - C o A ) necessary to couple high affinity choline u p t a k e to acetylation. Finally, o u r l a m i n a r d a t a suggest a distinction b e t w e e n s e p t o h i p p o c a m p a l projections to the d e n t a t e g r a n u l a r a n d m o l e c u l a r layers. The f u r t h e r rise in high affinity choline u p t a k e which occurs after 16-17 days o f age a p p e a r s slightly earlier in the m o l e c u l a r layer, b u t the m a g n i t u d e o f increase is m u c h greater in the g r a n u l a r layer. Possibly different s e p t o h i p p o c a m p a l fibers innervate these two lamina. ACKNOWLEDGEMENT This study was s u p p o r t e d by N S F G r a n t BNS76-09973.
REFERENCES 1 Atweh, S. F. and Kuhar, M. J., Effects of anesthetics and septal lesions and stimulation on aH-acetylcholine formation in rat hippocampus, Europ. J. Pharmacol., 37 (1976) 311-319. 2 Atweh, S. F., Simon, J. R. and Kuhar, M. J., Utilization of sodium-dependent high affinity choline uptake in vitro as a measure of the activity of cholinergic neurons in vivo, Life Sci., 17 (1975) 1535-1544. 3 Barker, L. A. and Mittag, T. W., Comparative studies of substrates and inhibitors of choline transport and choline acetyltransferase, J. PharmacoL exp. Ther., 192 (1975) 86-94. 4 Black, I. B., Hendry, I. A. and Iversen, L. L., Trans-synaptic regulation of growth and development of adrenergic neurones in a mouse sympathetic ganglion, Brain Research, 34 (1971) 229-240. 5 Blackstad, T. W., Cortical grey matter: a correlation of light and electron microscopic data. In H. Hyden (Ed.), The Neuron, Elsevier, Amsterdam, 1967, pp. 49-118. 6 Bourdois, P. S. and Szerb, J. C., The absence of 'surplus' acetylcholine formation in prisms prepared from rat cerebral cortex, J. Neurochem., 19 (1972) 1189-1193. 7 Burt, A. M., Acetylcholinesterase and choline acetyltransferase activity in the developing chick spinal cord, J. exp. ZooL, 169 (1968) 107-112. 8 Burt, A. M., Choline acetyltransferase and acetylcholinesterase in the developing rat spinal cord, Exp. Neurol., 47 (1975) 173-180. 9 Chiappinelli, V., Giacobini, E., Pilar, G. and Uchimura, H., Induction of cholinergic enzymes in chick ciliary ganglion and iris muscle cells during synapse formation, J. Physiol. (Lond.), 257 (1976) 749-766. 10 Cotman, C. W. and Nadler, J. V., Reactive synaptogenesis in the hippocampus. In C. W. Cotman (Ed.), Neuronal Plasticity, Raven, New York, 1978, pp. 227-271. 11 Crain, B., Cotman, C. W. Taylor, D. and Lynch, G., A quantitative electron microscopic study of synaptogenesis in the dentate gyrus of the rat, Brain Research, 63 (1973) 195-204. 12 Dudar, J. D., The effect of septal nuclei stimulation on the release of acetylcholine from the rabbit hippocampus, Brain Research, 83 (1975) 123-133. 13 Dudar, J. D., The role of the septal nuclei in the release of acetylcholine from the rabbit cerebral cortex and dorsal hippocampus and the effect of atropine, Brain Research, 129 (1977) 237-246. 14 Eisenstadt, M. L., Treistman, S. N. and Schwartz, J. H., Metabolism of acetylcholine in the nervous system of Aplysia californica. I1. Regional localization and characterization of choline uptake, J. gen. PhysioL, 65 (1975) 275-291. 15 Elliott, K. A. C., The use of brain slices. In A. Lajtha (Ed.), Hahdbook ofNeurochemistry, Vol. 2, Plenum, New York, 1969; pp. 103-114. 16 Fricke, U., Tritosol" a new scintillation cocktail based on Triton X-100, Analyt. Biochem., 63 (1975) 555-558.
274 17 Gibson, G. E. and Blass, J. P., Impaired synthesis of acetylcholine in brain accompanying mild hypoxia and hypoglycemia, J. Neurochem., 27 (1976) 37-42. 18 Gibson, G. E., Jope, R. and Blass, J. P., Decreased synthesis of acetylcholine accompanying impaired oxidation of pyruvic acid in rat brain minces, Biochem. J., 148 (1975) 17-23. 19 Goldberg, A. M. and McCaman, R. E., An enzymatic method for the determination of picomole amounts of choline and acetylcholine. In I. Hanin (Ed.), Choline and Acetylcholine: Handbook of Chemical Assay Methods, Raven, New York, 1974, pp. 47-61. 20 Guyenet, P. G., Lefresne, P., Beaujouan, J. C. and Glowinski, J., The role of newly taken up choline in the synthesis of acetylcholine in rat striatal synaptosomes. In P. G. Waser (Ed.), Cholinergic Mechanisms, Raven, New York, 1975, pp. 137-144. 21 Guyenet, P., Lefresne, P., Rossier, J., Beaujouan, J. C. and Glowinski, J., Effect of sodium, hemicholiniumo3 and antiparkinson drugs on [14C]acetylcboline synthesis and [~H]choline uptake in rat striatal synaptosomes, Brain Research, 62 (1973) 523-529. 22 Guyenet, P., Lefresne, P., Rossier, J., Beaujouan, J. C. and Glowinski, J., Inhibition by hemicholinium-3 of [14C]acetylcholine synthesis and [3H]choline high-affinity uptake in rat striatal synaptosomes, Molec. Pharmacol., 9 (1973) 630-639. 23 Haga, T. and Noda, H., Choline uptake systems of rat brain synaptosomes, Biochim. biophys. Acta (Amst.), 291 ~1973) 564-575. 24 Holden, J. T., Rossier, J., Beaujouan, J. C., Guyenet, P. and Glowinski, J., Inhibition of highaffinity choline transport in rat striatal synaptosomes by alkyl bisquaternary ammonium compounds, Molec. Pharmacol., II (1975) 19-27. 25 Hudick, J. P., Wajda, 1. J. and Lajtha, A., Isotopic labeling of synaptosomes that accumulate choline and the effect of narcotic drugs, Neurochem. Res., 1 (1976) 609-625. 26 Jenden, D. J., Jope, R. S. and Weiler, M. H., Regulation of acetylcholine synthesis: does cytoplasmic acetylcholine control high affinity choline uptake? Science, 194 (1976) 635-637. 27 Jope, R. S. and Jenden, D. J., Synaptosomal transport and acetylation of choline, Life Sci., 20 (1977) 1389-1392. 28 Kuhar, M. J., DeHaven, R. N., Yamamura, H. l., Rommelspacher, H. and Simon, J. R., Further evidence for cholinergic habenulo-interpeduncular neurons: pharmacologic and functional characteristics, Brain Research, 97 (1975) 265 275. 29 Kuhar, M. J., Sethy, V. H., Roth, R. H. and Aghajanian, G. K., Choline: selective accumulation by central cholinergic neurons, J. Neurochem., 20 (1973) 581-593. 30 Lefresne, P., Guyenet, P., Beaujouan, J. C. and Glowinski, J., The subcellular localization of ACh synthesis in rat striatal synaptosomes investigated with the use of Triton X-100, J. Neurochem., 25 (1975) 415-422. 31 Lewis, P. R., Shute, C. C. D. and Silver, A., Confirmation from choline acetylase analyses of a massive cholinergic innervation to the rat hippocampus, J. Physiol. (Lond.), 191 (1967) 215--224. 32 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 33 Lynch, G. S., Matthews, D. A., Mosko, S., Parks, T. and Cotman, C. W., Induced acetylcholinesterase-rich layer in rat dentate gyrus following entorhinal lesions, Brain Research, 42 (1972) 311-318. 34 Marchisio, P. C. and Consolo, S., Developmental changes of choline acetyltransferase (ChAc) activity in chick embryo spinal and sympathetic ganglia, J. Neurochem., 15 (1968) 759-764. 35 Matthews, D. A., Cotman, C. W. and Lynch, G. S., An electron microscopic study of lesioninduced synaptogenesis in the dentate gyrus of the adult rat. I. Magnitude and time course of degeneration, Brain Research, 115 (1976) 1-21. 36 Matthews, D. A., Nadler, J. V., Lynch, G. S. and Cotman, C. W., Development of cholinergic innervation in the hippocampal formation of the rat. I. Histocbemical demonstration of acetylcholinesterase activity, Develop. BioL, 36 (1974) 130-141. 37 Mellgren, S. I. and Srebro, B., Changes in acetylcholinesterase and distribution of degenerating fibres in the hippocampal region after septal lesions in the rat, Brain Research, 52 (1973) 19-36. 38 Mulder, A. H., Yamamura, H. I., Kuhar, M. J. and Snyder, S. H., Release of acetylcholine from hippocampal slices by potassium depolarization: dependence on high affinity choline uptake, Brain Research, 70 (1974) 372-376. 39 Murrin, L. C. and Kuhar, M. J., Activation of high-affinity choline uptake in vitro by depolarizing agents, Molec. Pharmacol., 12 (1976) 1082-1090. 40 Nadler, J. V., Mathews, D. A., Cotman, C. W. and Lynch, G. S., Development of cholinergic
275 innervation in the hippocampal formation of the rat. 11. Quantitative changes in choline acetyltransferase and acetylcholinesterase activities, Develop. Biol., 36 (1974) 142-154. 41 Polak, R. L., Molenaar, P. C. and Van Geider, M., Acetylcholine metabolism and choline uptake in cortical slices, J. Neurochem., 29 (1977) 477-485. 42 Sethy, V. H., Roth, R. H., Kuhar, M. J. and Van Woert, M. H., Choline and acetylcholine: regional distribution and effect of degeneration of cholinergic nerve terminals in the rat hippocampus, Neuropharmacology, 12 (1973) 819-823. 43 Shute, C. C. D. and Lewis, P. R., Electron microscopy of cholinergic terminals and acetylcholinesterase-containing neurons in the hippocampal formation of the rat, Z. Zellforsch., 69 (1966) 334-343. 44 Simon, J. R., Atweh, S. and Kuhar, M. J., Sodium-dependent high affinity choline uptake: a regulatory step in the synthesis of acetylcholine, J. Neurochem., 26 (1976) 909-922. 45 Simon, J. R. and Kuhar, M. J., High affinity choline uptake: ionic and energy requirements, J. Neurochem., 27 (1976) 93-99. 46 Simon, J. R., Mittag, T. W. and Kuhar, M. J., Inhibition of synaptosomal uptake of choline by various choline analogs, Biochem. Pharmacol., 24 (1975) 1139-1142. 47 Sorimachi, M. and Kataoka, K., High affinity choline uptake: an early index of cholinergic innervation in rat brain, Brain Research, 94 (1975) 325-336. 48 Steward, O., Topographic organization of the projections from the entorhinal area to the hippocampal formation of the rat, J. comp. Neurol., 167 (1976) 285-314. 49 Storm-Mathisen, J., Quantitative histochemistry of acetylcholinesterase in rat hippocampal region correlated to histochemical staining, J. Neurochem., 17 (1970) 739-750. 50 Storm-Mathisen, J., Glutamate decarboxylase in the rat hippocampal region after lesions of the afferent fibre systems. Evidence that the enzyme is localized in intrinsic neurons, Brain Research, 40 (1972) 215-235. 51 Suszkiw, J. B. and Pilar, G., Selective localization of a high affinity choline uptake system and its role in ACh formation in cholinergic nerve terminals, J. Neurochern., 26 (1976) 1133-1138. 52 Szerb, J. C., Hadh~izy, P. and Dudar, J. D., Release of [aH]aeetyicholine from rat hippocampal slices: effect ot septal lesion and of graded concentrations of muscarinic agonists and antagonists, Brain Research, 128 (1977) 285-291. 53 Yamamura, H. I. and Snyder, S. H., High affinity transport of choline into synaptosomes of rat brain, J. Neurochem., 21 (1973) 1355-1374.
263
DEVELOPMENT OF HIGH AFFINITY CHOLINE UPTAKE AND ASSOCIATED ACETYLCHOLINE SYNTHESIS IN THE RAT FASCIA DENTATA
DAVID L. SHELTON,J. VICTOR NADLER* and CARL W. COTMAN Department of Psychobiology, University of California, lrvilie, Calif. 92717 (U.S.A.)
(Accepted July 13th, 1978)
SUMMARY The ontogenic development of hemicholinium-sensitive, high affinity choline uptake and the synthesis of acetylcholine from exogenous choline have been studied in particulate preparations of the rat fascia dentata. Between 6 days of age and adulthood the rate of high affinity choline uptake increases 3-fold, when expressed with respect to protein, and 125-fold, when expressed independently of protein. This process develops most rapidly during the period around 16-17 days of age, similar to the ontogenesis of choline acetyltransferase activity. This observation supports the idea that cholinergic septohippocampal boutons develop mainly at this time. Unlike choline acetyltransferase activity, the velocity of high affinity choline uptake increases to as much as 161 ~ of the adult value at about 30 days of age. It is suggested that at 25-31 days of age a relatively high endogenous septohippocampal firing rate increases the rate of choline uptake. At 6 days of age we detected no synthesis of acetylcholine from the accumulated choline. Uptake-synthesis coupling develops mainly between 6 and 13 days of age, earlier than any other presynaptic cholinergic property. Acetylcholine synthesis from exogenous choline develops in parallel with high affinity choline uptake, but developmental increases in uptake velocity result in comparable increases in synthesis rate only after a delay of several days. Some limiting factor other than choline acetyltransferase activity appears to link the accumulation of exogenous choline to acetylcholine synthesis during development.
INTRODUCTION The rat fascia dentata is particularly suitable for detailed developmental studies * To whomall correspondenceshouldbe addressed. Presentaddress: Departmentof Pharmacology, Duke UniversityMedicalCenter, Durham, N.C. 27710 U.S.A.
264 of cholinergic innervation. This part of the hippocampal formation is comprised of a single homogeneous layer of granule cells overlain with a molecular layer, in which the dendrites of the granule cells ramify and afferent fibers establish their connections in a laminar fashionS, 10. There are no intrinsic cholinergic neurons, the entire cholinergic innervation of the fascia dentata originating in the septum. All presynaptic cholinergic properties have been related to the septohippocampal tract. These include choline acetyltransferase (CAT) activity29,31, '50, acetylcholinesterase (ACHE)activity ":~.~.~, AChE-dependent histochemical staining31,33,37,49, 50, high affinity choline uptake 29, acetylcholine (ACh) contentZg, 42 and ACh release12,13,3s,zL Thus one can relate developmental events to a single morphological entity. The growth of septohippocampal fibers into the rat fascia dentata has been described 36, as well as the development of CAT and AChE activities within these fibers 4°. In this report we describe the development of high affinity choline uptake and its coupling to acetylation of the accumulated choline. High affinity choline uptake serves as a marker for presynaptic cholinergic membrane2S,Zg,sL It is dependent upon extracellular Na + and C I - (refs. 21, 23, 25, 45, 53), requires a source of cellular energy45, 53 and is exceedingly sensitive to drugs of the hemicholinium series 2l 25, 46,53. This transport system provides nearly all the choline required for ACh synthesis3,23,3°,41,53 and in sucrose homogenates is localized mainly in nerve-ending particles25, 53. High affinity choline uptake should thus be a sensitive indicator of the development of cholinergic axon terminals. We have therefore related the development of hemicholinium-sensitive, high affinity choline uptake and the associated ACh synthesis to the ontogenic growth of cholinergic fibers and development of AChrelated enzyme activities. METHODS
Preparation of particulate fractions Female Sprague-Dawley rats were purchased from Simonsen Laboratories (Gilroy, Calif.) during pregnancy. The day of birth was taken as day 0. At ages ranging from 6 to 105 days, the pups were decapitated, and the brain was quickly removed and immersed in ice-cold 0.32 M phosphate-buffered sucrose (pH 7.1-7.3 with 50 #M sodium phosphate). This operation took less than 1 min. After a few minutes in cold sucrose, the hippocampal formation was isolated from one side of the brain and cut into transverse slices of 500 #m thickness with a Mcllwain tissue chopper (Brinkmann Instruments, Westbury, N.Y.). The tissue was maintained in ice-cold sucrose during the cutting and subsequent dissection procedures. When animals 16 days of age or older were used, the fascia dentata was divided into molecular and granular layers. With the aid of a dissecting microscope and sharp dissecting knifO °, the fascia dentata of each slice was first divided parallel to the granule cell layer about one-fourth to onethird the distance from the outer edge of the latter to the outer edge of the molecular layer (Fig. 1). The molecular layer was then isolated by cutting along the hippocampal fissure, and the granular layer was removed by cutting immediately beneath it, where a natural break in the tissue occurs. The 'molecular layer' thus actually included
265
Fig. 1. Dissection of layers of the fascia dentata shown on a section (from a postfixed transverse hippocampal slice) stained for AChE activity. This histochemical staining localizes the septohippocampal fibers al,aT,4a. M, molecular layer; G, granular layer. Layers were isolated by cutting along the dashed lines, as described in the text. Scale bar = 0.5 mm.
approximately the outer 70 ~ of this layer, that is, the perforant path terminal zone35, 4s. The 'granular layer' included not only the granule cell bodies themselves, but also a somewhat variable amount of inner molecular layer. Since it was not possible to divide the layers accurately in animals younger than 16 days, the fascia dentata was isolated whole at these ages. Choline uptake was determined on particulate fractions which contained synaptosomes. Corresponding layers from all slices were pooled and homogenized in 0.5 ml of 0.32 M phosphate-buffered sucrose with a Teflon-glass homogenizer. The homogenate was diluted with an additional 7 ml of sucrose and centrifuged at 42,000 × g and 4 °C for 20 min. Preliminary experiments showed that this procedure sedimented all particles that accumulated choline under our incubation conditions. The resulting supernatant was discarded and the pellet resuspended in 0.5 ml of sucrose. This particulate preparation was immediately assayed for choline uptake activity after removal of a small portion for determination of protein 32.
Choline uptake For determination of choline uptake activity, 0.1 ml of particulate suspension was added to 0.9 ml of Elliott's artificial CSF 15 (122 m M NaC1, 3.1 m M KCI, 1.2 m M
266 MgSO4, 0.4 mM KHzPO4, 1.3 mM CaClz, 25 mM NaHCO3, 10 mM D-glucose, pH 7.37.5 with 95 % compressed air/5 % COz). This mixture was preincubated at 38 °C under an atmosphere of 95 o/,, compressed air/5 % CO2. After 10 min, 10 #1 of [methyl-ZH]choline chloride (10.1 Ci/mmol, Amersham-Searle Corp., Des Plains, I11.) was added to yield a final choline concentration of 0. l/~M. The incubation was continued for an additional 4 min. At the end of this period the incubation mixture was diluted with 2 ml of Elliott's medium at room temperature and filtered by gentle suction through a glass fiber filter (Whatman GF/A, 2.5 cm diameter) underlain with a membrane filter (GE Nuclepore, 2.5 cm diameter, 0.4/~m pore size). The tissue trapped on the filter was then washed with an additional eight 2 ml portions of medium. Filtration and washing were accomplished in less than 25 sec. Preliminary experiments showed that this procedure removed essentially all the unbound [3H]choline from the filter. When only choline uptake was measured, the filters were immersed in 2 ml of 1 ~ (w/v) sodium dodecylsulfate, 20 mM EDTA (pH 8.0 with NaOH) overnight to solubilize the tissue. The solubilized tissue was then counted with use of Tritoso116. Parallel control incubation mixtures contained 0.8/~M hemicholinium-3 (HC-3) (Aldrich Chemical Co., Milwaukee, Wisc.) to inhibit high affinity choline uptake selectively3. At this concentration HC-3 inhibits virtually all the high affinity uptake, but has little effect on low affinity uptake and should not affect presynaptic cholinergic function in any other way. High affinity choline uptake was calculated by subtracting the radioactivity on the filter after incubation in the presence of HC-3 from that remaining on the filter after incubation in its absence. After subtraction of this blank value, choline uptake was found to be linear with time of incubation for 5-6 min, and the rate of uptake was proportional to the quantity of tissue incubated up to at least 100/zg of protein. We found little evidence of a low affinity uptake process under our incubation conditions, in agreement with those who have used Na -~-free controls 44. Preliminary experiments without tissue and with varying amounts of tissue showed that most of the apparent HC-3-resistant uptake of [3H]choline could be accounted for by binding to the filters.
Extraction and separation of choline and A Ch When both choline uptake and ACh synthesis were measured, the washed tissue was treated with 1.2 ml of a 3-heptanone solution containing 5 mg/ml sodium tetraphenylboron (TPB) to extract choline and ACh, 20/~M diisopropyl fluorophosphate to inhibit extracellular cholinesterase (intracellular cholinesterase is said to be absent from cortical tissue 6) and 1 nCi of [methyl-a4C]choline chloride to determine the efficiency of the separation procedure. Choline and ACh were extracted from the organic phase with 0.2 ml of 0.4 N HCI. A 50 /zl portion of the acid extract was counted for determination of choline uptake activity and 100 /zl was dried under vacuum and stored at --22 °C prior to separation of choline and ACh. Values of high affinity choline uptake from these studies equalled those from studies in which the tissue was dissolved in sodium dodecylsulfate, suggesting that negligible amounts of [ZH]phosphorylcholine and other metabolites not extracted by TPB were formed. ACh and choline were separated by the enzymatic method of Goldberg and
267 McCaman 19. The dried residue was dissolved in 10 #1 of an ice-cold solution consisting of 0.15 unit/ml choline kinase (Sigma Chemical Co., St. Louis, Mo.), 1 mM ATP, 5 mM MgC12 and 50 mM sodium phosphate buffer, pH 8.0. This mixture was incubated for 15 min at 38 °C to phosphorylate the choline. The samples were then placed on ice, and ACh was extracted with 200 #1 of 75 mg/ml TPB in 3-heptanone. After addition of 90 #1 of ice-cold 1-I20, the phases were separated by centrifugation and portions of each counted. Greater than 95 700 of the choline was recovered (as phosphorylcholine) in the aqueous phase, as determined from the [14C]choline internal standards. External standards of [14C]ACh, with or without unlabeled choline, showed that at least 93 70 of the ACh in the sample was extracted by TPB. The rate of ACh synthesis was calculated by subtracting the amount of [aH]ACh synthesized in 4 min in the presence of HC-3 from the amount synthesized in the absence of HC-3. Less than 10 70 of accumulated choline was converted to ACh when 0.8 #M HC-3 was present during incubation. RESULTS HC-3-sensitive, high affinity choline uptake is demonstrable in the rat fascia dentata at 6 days of age, the earliest developmental stage examined in this study. From this point to adulthood, the initial velocity increases 125-fold, when expressed independently of protein (Fig. 2B), and about 3-fold, when expressed relative to protein (Fig. 2A). The most rapid phase of development occurs during the period around 16-17 days of age, similar to the ontogenesis of CAT activity (Fig. 2A and ref. 40), a better established presynaptic cholinergic marker. After this age, however, the velocity of choline uptake increases much faster than CAT activity. At 29-31 days of age particulate fractions of the fascia dentata already transport choline at the adult rate, when expressed independently of protein (Fig. 2B). If the values are expressed relative to protein (Fig. 2A), uptake velocities at 25 and 29-31 days of age exceed those of adult fascia dentata by 38 ~ and 61 70, respectively (P < 0.01 at 25 days and P < 0.001 at 29-31 days, two-tailed Student's t-test). In contrast, the fascia dentata acquires CAT activity more gradually, and the specific activity of CAT never exceeds the adult value at any age studied. The development of high affinity choline uptake differs even more markedly from that of AChE activity, although both are considered to be associated mainly with the presynaptic cholinergic membrane29,4a, 49. Values of the HC-3 blanks remained approximately constant at all ages (0.03-0.1 pmol/4 min/sample). Hence they decreased considerably during development, when expressed relative to protein. As discussed in Methods, we have attributed most of the apparent HC-3-resistant choline uptake in this preparation to non-specific filter binding. At 6 days of age we detected no conversion of the accumulated choline to ACh (Fig. 2). Conversion of as little as 10 70 could have been detected. From 11 days of age onward, however, the development of ACh synthesis from exogenous choline parallels that of high affinity choline uptake, suggesting that the availability of recently accumulated choline regulates ACh synthesis during most of the developmental
268
io o
50/1
_ .
//-,--
= _ ~ o ~ o -i ~ B. U 50Jb
2b
3b
4o
'~io+
Age (days)
Fig. 2. Development ofcholinergic markers in the rat fascia dentata. Symbols: filled triangles, HC-3sensitive, high affinity choline uptake; open triangles, ACh synthesis from exogenous choline; filled circles, CAT activity; open circles, AChE activity. Bars indicate S.E.M. Data for development of CAT and AChE activities were recalculated from ref. 40 (hilus layer was excluded). For number of experiments represented by each mean see ref. 40 and Figs. 3 and 4. A: velocities calculated with respect to protein. Adult values were: choline uptake, 13.0 4- 0.6 pmol/4 min/mg protein; ACh synthesis, 8.13 ± 0.38 pmol/4 rain/rag protein; CAT activity, 13.0 ± 0.7/~mol/30 min/g protein; AChE activity, 1600 ± 70/~mol/30 min/g protein. B: velocities calculated independently of protein. Adult values were: choline uptake, 5.80 ± 0.45 pmol/4 min/fascia dentata of one side of brain; ACh synthesis, 3.64 i 0.28 pmol/4 min/fascia dentata of one side of brain; CAT activity, 2.60 ± 0.19 tLmol/30 min/1.2 mm length of fascia dentata; AChE activity, 322 ~ 28/~mol/30 min/l.2 mm length of fascia dentata.
period, as it does in adult rats2,26,89,41,44. Surprisingly, however, the developmental time course of ACh synthesis does not coincide with that of choline uptake. Rather, changes in the overall rate of synthesis follow those in uptake velocity only after a delay of several days. When animals 16 days of age or older were examined, we analyzed high affinity choline uptake and ACh synthesis in separated granular and molecular layers to determine whether developmental changes are uniformly distributed in the fascia dentata or restricted to a particular lamina. In adult rats choline uptake velocity expressed relative to protein is about 20 ~ greater in the granular layer than in the molecular layer (Fig 3), a distribution very similar to that of CAT activity 4°. The difference between the two layers first becomes evident between 21 and 25 days of age. The rate of high affinity choline uptake appears to increase slightly earlier in the
269
o'°ni oioi
20"
30
c E 20
° t
/
6
II
301
13
Gr~ulor Layer
16-17 21
25 29"31 70+
Age (clays)
Fig. 3. Development of HC-3-sensitive, high affinity choline uptake in layers of the fascia dentata. Values are means ± S.E.M. for the number of animals given in parentheses. Results on whole fascia dentata of animals 6, 11, and 13 days of age are given for comparison.
molecular layer than in the granular layer between 16 and 31 days of age, but the magnitude of increase is much greater in the granular layer. In adult rats, particulate fractions of the granular layer synthesize about 28 more [3H]ACh from exogenous [SH]choline than particulate fractions of the molecular layer (Fig. 4), in accordance with the distribution of high affinity uptake. The difference between the layers first becomes evident at 29-31 days of age. Between 16 and 31 days of age changes in ACh synthesis follow a similar time course in the two layers, in each case lagging behind the increases in high affinity choline uptake.
20' Molecular Layer
6 o. 20-
I0-
]n N
"E: o
I0
"p,
Granular Layer
.¢: I0
6
II
13
16t7 21
25
D
29"31 70+
Age (days)
Fig. 4. Development of ACh synthesis from exogenous choline in layers of the fascia dentata. See Fig. 3 for other details.
270 8O Molecul0r Layer
~,
80-
o
4080
GranularLayer
g
6
II
13
16-17'
21
25
29-31 70+
Age (days)
Fig. 5. Developmentof coupling between high affinitycholine uptake and ACh synthesisin layers of the fascia dentata. Values are percentages of accumulated [all]cholineconverted to [aH]ACh.See Fig. 3 for other details.
Particulate fractions of both layers from adult rats convert 60-65 ~ of the accumulated choline to ACh (Fig. 5), values similar to those obtained from studies in which 0 °C or Na÷-free blanks were used and cholinesterase activity was suppressed44,z3. The percentage conversion of transported precursor to intraparticulate product serves as a measure of the coupling of uptake to transmitter synthesis. This coupling develops mainly between 6 and 13 days of age, during which period the conversion of [all]choline to [aH]ACh increases from undetectable to 40-50 ~. The most rapid development of uptake-synthesis coupling thus precedes that of any other presynaptic cholinergic property. An additional increase occurs between 29 and 31 days of age and adulthood. Preparations of granular and molecular layer at all ages convert approximately equal percentages of accumulated choline to ACh. DISCUSSION Within the rat hippocampal formation high affinity choline uptake is a property of the septohippocampal presynaptic membrane 29. Previous workers have suggested a predominant localization for this transport process in cholinergic synaptic boutons14, 50 and synaptosomes zS,z3, and our finding that high affinity choline uptake and CAT activity are similarly distributed between layers of the fascia dentata further supports this view. Thus one would expect measurements of high affinity choline uptake velocity during development to indicate the time course of cholinergic synaptogenesis. However, some caution is required on two counts. First, the velocity of high affinity choline uptake varies not only with the number of cholinergic boutons (or, more properly, the amount of cholinergic presynaptic membrane), but also, at least in adult rats, with the electrophysiological activity of the cholinergic fibers 1,2,44. Studies of CAT activity should, however, distinguish growth-induced from activity-
271 induced alterations of the choline uptake rate, since CAT activity would be expected to vary with the number of cholinergic boutons, but not with short-term changes in impulse flow44. Second, changes in uptake velocity should be referable to an altered number of uptake sites and not a change in the affinity of the carrier for choline. Sorimachi and Kataoka have shown no change in the Km for Na+-dependent, high affinity uptake of choline during the developmental period covered by our study 47. Unfortunately, we were unable to perform comparable kinetic studies on dentate particulate preparations, because choline uptake saturated during a 4 min incubation at concentrations of 0.2 #M or above, considerably below the reported Km at 38 °C44. Damage to the synaptosomes during preparation might account for this result. This explanation appears unlikely, however, since the adult choline uptake rate obtained in the present study actually exceeds that which may be calculated from Michaelis-Menten kinetic parameters computed by others who incubated hippocampal preparations at 38 °C44. Possibly our preparations contained a higher level of endogenous choline than those used by other workers, thus limiting the quantity of exogenous precursor which could be accommodated within the synaptosome. We cannot therefore exclude the possibility that a change in the affinity of the transport carrier for choline might account for developmental changes in choline uptake, although previous evidence suggests that this is unlikely. The velocity of high affinity choline uptake by particulate preparations of fascia dentata changes little between 6 and 13 days of age, when calculated with respect to protein. Neither does the CAT activity of the hippocampal formation as a whole4°. These results suggest little formation of cholinergic boutons during this period. Whatever increases may occur can probably be attributed to continued growth of the septohippocampal fibers, which are present in the septal two-thirds of the hippocampal formation at 6 days of age and invade the temporal extreme between 6 and 11 days a6. Enzyme and choline transport carrier may be located mainly in the axonal growth cones at this time. Both uptake and CAT activities increase most rapidly during the period around 16-17 days of age, a time at which large numbers of synapses are forming in the dentate molecular layer 11. Indeed both parameters attain adult values (per unit protein) at this age. In accordance with developmental studies in other systems4,7-9,34, the changes in CAT activity have been interpreted to signify a rapid formation of cholinergic boutons in the period around 16-17 days of age 4°. Results of the present study support this interpretation. They do not, however, agree with the report of Sorimachi and Kataoka, who found that Na+-dependent, high affinity choline uptake develops somewhat more rapidly in the hippocampal formation than CAT activity between 7 and 17 days of age 47. Our studies and theirs show a similar development of CAT activity, but we find a more delayed development of high affinity choline uptake than they do. This discrepancy may be attributable to our measuring CAT and choline uptake activities in the fascia dentata alone (whereas Sorimachi and Kataoka assayed them in the whole hippocampal formation), to a difference in the strain of rats studied or, least likely, to a difference in the type of blank used. After 17 days of age high affinity choline uptake develops very differently from CAT activity. Uptake activity expressed relative to protein increases to 161% of the adult
272 value at 29-31 days of age, whereas no other presynaptic cholinergic measure exceeds the adult value at any time point studied. This excess uptake activity was not reported in the previously cited developmental study of the hippocampal formation as a whole 47, but the 25-31-day period was not investigated. On a protein-independent basis, high affinity choline uptake activity reaches, but does not exceed, adult rates around 25 days of age. One could interpret these results to imply that cholinergic presynaptic membrane expands much more rapidly than other tissue elements between 16 and 31 days of age, completing its development around the end of this period. Neither CAT activity nor AChE activity, another property of cholinergic membrane, shows any comparable change, however. Alternatively, an increase in the endogenous septohippocampal firing rate could conceivably account for the specific augmentation of choline uptake activity. This possibility is supported by results of a preliminary study in which pentylenetetrazol (PTZ) failed to increase the velocity of high affinity choline uptake in dentate preparations from two 30-day-old rats, but was effective in adults, as others have reportedZ, 44. PTZ is thought to act by increasing the septohippocampal firing rate. Around 30 days of age these fibers may be activated to such a a degree normally that PTZ cannot significantly augment their firing. High affinity choline uptake is thought to regulate ACh synthesis in adult rats, since the choline accumulated by the high affinity carrier is extensively and preferentially acetylated 2°,2~,4~,51,53 and treatments that alter the velocity of uptake affect the rate of ACh synthesis to the same degree 44. This tight coupling between uptake and synthesis has led to the concept that the membrane-associated high affinity carrier may be physically joined to the cytoplasmic CAT 3. On the other hand, treatments which reduce the availability of acetyl-CoA reduce ACh synthesis by a similar amount ~7, ~s,z7 without significantly affecting choline uptake 27. It would thus appear that uptake and acetylation are stoichiometrically coupled only when the intracellular acetyl-CoA concentration is saturating. Indeed the synthesis of acetyl-CoA may be as closely coupled to ACh synthesis as choline uptake. Results of the present study suggest that high affinity choline uptake and acetylation of the accumulated choline are not inherently coupled, but that regulation of ACh synthesis by the uptake process develops over an extended period. At 6 days of age less than 10~ of the accumulated choline is converted to ACh. Lack of CAT activity probably cannot account for this result, since the hippocampal formation as a whole already shows one-fourth to one-third the adult activity (per unit protein) at this age 40 and cholinergic fibers have entered the fascia dentata 36. Uptake becomes coupled to acetylation between 6 and 13 days of age, just before the period during which both processes develop rapidly. Possibly either the independently synthesized and transported carrier and enzyme first become associated at this time or else acetylCoA becomes increasingly available to the enzyme. Between 31 days of age and adulthood the percentage conversion of accumulated choline to ACh again increases. This tightening of uptake-acetylation coupling may have a similar explanation. That acetylation of choline is, in fact, regulated in part by high affinity choline uptake during the developmental period is indicated by the remarkable parallelism in their rates of development (Fig. 2). In contrast to adult hippocampal formatiom in
273 which an altered u p t a k e rate i m m e d i a t e l y translates into an a l t e r e d rate o f A C h synthesis 44, d e v e l o p m e n t a l increases in the rate o f A C h synthesis f r o m exogenous choline lag b e h i n d increases in choline u p t a k e b y 5-8 days. Deficient C A T activity c a n n o t a c c o u n t for this delay. This result f u r t h e r suggests the existence in d e v e l o p m e n t o f some limiting factor (possibly a c e t y l - C o A ) necessary to couple high affinity choline u p t a k e to acetylation. Finally, o u r l a m i n a r d a t a suggest a distinction b e t w e e n s e p t o h i p p o c a m p a l projections to the d e n t a t e g r a n u l a r a n d m o l e c u l a r layers. The f u r t h e r rise in high affinity choline u p t a k e which occurs after 16-17 days o f age a p p e a r s slightly earlier in the m o l e c u l a r layer, b u t the m a g n i t u d e o f increase is m u c h greater in the g r a n u l a r layer. Possibly different s e p t o h i p p o c a m p a l fibers innervate these two lamina. ACKNOWLEDGEMENT This study was s u p p o r t e d by N S F G r a n t BNS76-09973.
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