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(The FASEB Journal. 1998;12:349-357.)
© 1998 FASEB

RESEARCH COMMUNICATION

Prenatal availability of choline modifies development of the hippocampal cholinergic system

Jennifer Marie Cermaka, Thomas Hollera, Darrell A. Jacksona, and Jan Krzysztof Blusztajnb,1

a Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA
b Department of Psychiatry, Boston University School of Medicine, Boston, Massachusetts 02118, USA


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Choline supplementation during fetal development [embryonic days (E) 11–17] permanently enhances memory performance in rats. To characterize the neurochemical mechanisms that may mediate this effect, we investigated the development of indices of the cholinergic system in the hippocampus: choline acetyltransferase (ChAT), acetylcholinesterase (AChE), synthesis of acetylcholine (ACh) from choline transported by high-affinity choline uptake (HACU), and potassium-evoked ACh release. During E11–E17, Sprague-Dawley pregnant rats consumed 0 [choline-deficient (ChD)], 1.3 [control (ChC)], and 4.6 [choline-supplemented (ChS)] mmol/(kg·day) of choline, respectively. On postnatal days 17 and 27, hippocampi of the ChD animals had the highest AChE and ChAT activities, and increased synthesis of ACh from choline transported by HACU, concomitant with reductions of tissue ACh content relative to the ChC and ChS rats and an inability to sustain depolarization-evoked ACh release relative to the ChS animals. In contrast, AChE and ChAT activities, and ACh synthesized from choline transported by HACU, were lowest in ChS rats whereas depolarization-evoked ACh release was the highest. This pattern of changes suggests that the hippocampus of the ChD animals is characterized by fast ACh recycling and efficient choline reutilization for ACh synthesis, presumably to maintain adequate ACh release despite the decrease of the ACh pool, whereas in the ChS animals ACh turnover and choline recycling is slower while the evoked release of ACh is high. Together, the data show a complex adaptive response of the hippocampal cholinergic system to prenatal choline availability and provide a novel example of developmental plasticity in the nervous system governed by the supply of a single nutrient.—Cermak, J. M., Holler, T., Jackson, D. A., Blusztajn, J. K. Prenatal availability of choline modifies development of the hippocampal cholinergic system. FASEB J. 12, 349–357 (1998)


Key Words: acetylcholine • acetylcholinesterase • acetylcholine release • choline acetyltransferase • cortex • development • high-affinity choline uptake • hippocampus • memory • pregnancy • rat


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE NUTRITIONAL REQUIREMENTS of the developing nervous system are complex and remain inadequately understood. Protein malnutrition during pregnancy results in multiple changes of brain development in the offspring that manifest as inferior scores on a variety of behavioral and cognitive tests in both human subjects and experimental animals (for a review, see ref 1). Among the needs for individual nutrients, the requirement for essential fatty acids in nervous system development (2) and for folic acid during the periconceptual period in the prevention of neural tube defects (3) is generally acknowledged. The latter has led to a public policy aimed at women of child-bearing age to ensure that their diets contain adequate amounts of folate.

Choline is an essential nutrient necessary for the growth of mammalian cells (4); dietary choline deficiency in adult animals (5) and humans (6, 7) causes a variety of systemic abnormalities (for a review, see ref 8). Choline is the precursor of phosphatidylcholine, sphingomyelin, and plasmenylcholine, major membrane components of all cells including neurons and glia. Therefore, an adequate supply of choline during development of the nervous system is crucial as neuronal and glial progenitor cells divide, axons and dendrites grow, synapses form, and myelin is laid down. Choline is also necessary to establish a pool of acetylcholine (ACh),2 the neurotransmitter of cholinergic neurons (9). Little is known about the requirements of choline in pre- and early postnatal nutrition. In rats consuming a normal diet, pregnancy causes a dramatic depletion of choline pools (10), suggesting that choline requirements during pregnancy are increased and that the need for this nutrient by either the mother or the fetuses may exceed the amounts provided by normal rat chow. Thus, if choline is a limiting nutrient for the fetus and neonate, choline supplementation would be expected to influence brain development and behavior. Indeed, supplementation with choline during pre- and postnatal development in rats causes long-lasting improvements in spatial memory, as determined by a radial-arm maze (11, 12), or water maze (13) task, and of timing and temporal memory (14). Supplementation with choline during a 7-day period in the second half of fetal development [i.e., during embryonic days (E) 11–17] was found to be sufficient to improve memory performance in rats as old as 24–26 months of age (15). This memory facilitation correlated with altered distribution and morphology of septal neurons (16), suggesting that it involved organizational changes in the basal forebrain during development. The neurochemical mechanisms by which choline supplementation in utero leads to the improvement in memory are not known; however, they may include alterations of ACh synthesis and release (9), because basal forebrain cholinergic neurons are known to participate in memory processes (17), or they may be related to the function of choline-containing phospholipids in cell signaling events in the brain (8). Consistent with the latter possibility, we recently reported that choline supplementation in pregnant rats increases both basal- and metabotropic glutamate receptor-stimulated phospholipase D (PLD) activity in the hippocampus of the offspring (18). The purpose of this study was to determine whether cholinergic neurotransmission is altered by prenatal choline availability. We report that postnatal development of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities, synthesis of ACh from choline transported by sodium-dependent high-affinity choline-uptake (HACU), and potassium-evoked ACh release in the hippocampus are influenced by choline supply in utero.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Sprague-Dawley CD strain rats were obtained on the 10th day of pregnancy from Charles River Laboratories (Kingston, N.Y.). Modified rat AIN-76A diets, custom formulated to contain no choline, or the chloride salt of choline (7.9 mmol/kg) instead of the standard bitartrate were supplied by Dyets Inc. (Bethlehem, Pa.). [3H]Acetylcholine iodide and [3H-acetyl]coenzyme A were supplied by Du Pont/New England Nuclear (Boston, Mass.). [14C]Choline was purchased from ICN Radiochemicals (Irvine, Calif.). All other chemicals were from Sigma Chemical Company (St. Louis, Mo.) or Fisher (Pittsburgh, Pa.).

Subjects
Timed pregnant Sprague-Dawley CD strain rats were housed in individual cages with a 12 h light/dark cycle. Prenatal choline treatments were carried out from E days 11 through 17. The dams were divided into three groups: deficient, control and supplemented. The deficient group received an AIN-76A diet that contained water sweetened with 50 mM saccharine and no choline. Controls received an the AIN-76A diet containing 7.9 mmol/kg choline chloride and water sweetened with 50 mM saccharine, resulting in an average daily choline intake of 1.3 mmol/(kg·day). The supplemented group received AIN-76A diet containing 7.9 mmol/kg choline chloride and water containing 25 mM choline chloride sweetened with 50 mM saccharine, resulting in an average daily choline intake of 4.6 mmol/(kg·day). No significant differences in the amounts of diet or water consumed by the different groups were observed (data not shown). After the treatment period, all dams consumed the control AIN-76A diet (containing 7.9 mmol/kg choline chloride) and saccharine-free water ad libitum. Pups remained with their mother after birth. On postnatal day (P) 22, two rats of the same sex were weaned per cage and given free access to water and the control AIN-76A diet. On P1, P3, P7, P17, P27, P35, and P90, rats were randomly selected and the striatum, hippocampus, and cortex were dissected and stored at -80°C for use in AChE and ChAT assays. On P17 and P27, fresh hippocampi were dissected on ice and sliced (0.4 mm thick) with a McIlwain tissue chopper for immediate use in HACU and potassium-evoked ACh release experiments. Both male and female rats were used for the determinations of AChE, ChAT, and HACU. No significant differences between the sexes were observed in these assays, and combined data from both sexes are reported. ACh release experiments were performed on males only.

AChE and ChAT assays
AChE and ChAT activities were determined in rat brain homogenates by a modification of the method of Fonnum (19). The tissue was sonicated on ice in a homogenizing buffer [0.25 M sucrose, 3% Triton X-100, 10 mM 4-(2-hydroyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM ethylenediamine-tetraacetic acid (EDTA), pH 7.2]. The ChAT assay was performed using 100 µg (P1, P3, P7) or 50 µg (P17, P27, P35, P90) of the homogenate protein, which was incubated for 30 min at 37°C in 200 µl of a buffer [50 mM Na-phosphate, 300 mM NaCl, 10 mM EDTA, 0.025 mM acetyl-CoA, 5 mM choline, 0.1 mM eserine, and [3H-acetyl]CoA (5 µCi), pH 7.4]. The reaction was terminated with ice-cold 1.5% tetraphenylboron in a 3-heptanone solution. The tubes were vortex mixed, centrifuged, and the radioactivity of an aliqouot of the organic phase (containing [3H]ACh) was determined by liquid scintillation spectrophotometry. The results are expressed as nmol ACh formed per mg protein per 30 min based on the specific radioactivity of [3H-acetyl]CoA.

AChE activity was measured using 2 µg of the homogenate protein, which was incubated for 30 min at 37°C in a reaction mixture (50 mM Na-phosphate, 300 mM NaCl, 10 mM EDTA, pH 7.4) containing 0.1 µCi [3H-acetyl]choline per tube (final concentration of 426 µM ACh). The reaction was terminated with ice-cold 1.5% tetraphenylboron in a 3-heptanone solution. The tubes were vortex mixed, centrifuged, and the radioactivity in an aliquot of the aqueous phase (containing [3H]acetate) was determined by liquid scintillation spectrophotometry. The results are expressed as nmoles ACh hydrolyzed per milligram protein/30 min based on the specific radioactivity of [3H-acetyl]choline.

Synthesis of ACh from choline transported by high-affinity choline uptake
Hippocampal slices obtained on P17 and P27 were equilibrated in an ice-cold HEPES-buffered saline solution (HBS), in mM: 145 NaCl, 5 KCl, 1 MgSO4, 1 CaCl2, 10 glucose, 10 HEPES, 0.1 neostigmine, pH 7.4] for 1 h. During this equilibration period, HBS was replaced every 15 min. Slices were then transferred into cell strainers placed in a 12-well cell culture cluster (six slices per well) and incubated in fresh HBS for 10 min at 37°C. The slices were then incubated for 10 min at 37°C in HBS containing 1 µM [14C]choline with or without 10 µM hemicholinium-3, a specific inhibitor of HACU. The slices were washed with ice-cold HBS; ACh and choline were extracted by adding methanol:1 N formic acid:chloroform: water (1: 0.1: 2: 1 by volume). The samples were vortex mixed and centrifuged for 10 min at 2000 rpm. The aqueous phase was collected, dried under a vacuum, and reconstituted in 200 µl of water. ACh and choline were purified by high-performance liquid chromatrography (HPLC) using a column purchased from BAS (West Lafayette, Ind.) [1.5 ml flow rate, mobile phase (22 mM phosphate buffer, pH 8.5, containing 0.005% Kathon Reagent)] and their mass was determined by a kit purchased from BAS containing AChE and choline oxidase based on the method of Potter et al. (20). After separation by HPLC, samples were collected in vials in 0.38 min intervals, using a fraction collector, and the radioactivity of [14C]choline and [14C]ACh was determined by liquid scintillation spectrophotometry. To obtain a measure of [14C]choline and [14C]ACh derived from HACU specifically, the amount of radioactivity associated with these compounds after incubation in hemicholinium-3-containing medium was subtracted from that incorporated in the control medium. The values of the mass of choline and ACh and of the radioactivity of [14C]choline and [14C]ACh were used to calculate the specific radioactivities of these compounds.

Potassium-evoked acetylcholine release
Hippocampal slices were transferred into cell strainers (10 slices per well) and incubated at 37°C for 1 h in a physiological salt solution (PSS) (in mM: 135 NaCl, 5 KCl, 1 CaCl2, 0.75 MgCl2, 10 glucose, 10 HEPES, pH 7.4). The cell strainers containing slices were transferred into fresh PSS solution every 15 min. The slices were then incubated at 37°C for 5-min periods sequentially in a normal (designated NaPSS) and depolarizing (designated KPSS) solution as follows: NaPSS/KPSS/NaPSS/KPSS (NaPSS), in mM: 135 NaCl, 5 KCl, 1 CaCl2, 0.75 MgCl2, 10 glucose, 10 HEPES, 0.02 neostigmine bromide, pH 7.4; KPSS (in mM): 100 NaCl, 40 KCl, 1 CaCl2, 0.75 MgCl2, 10 glucose, 10 HEPES, 0.02 neostigmine bromide, pH 7.4. The media were collected, centrifuged to remove debris, and ACh and choline were determined by HPLC as above. The slices were extracted and ACh content was determined by HPLC as above.

Protein assay
Protein was assayed by the method of Smith et al. (21).

Statistical analysis
Data were analyzed by analysis of variance (ANOVA). If significant effects were found, data were further analyzed by Tukey's multiple comparison test or Fisher's Least significant difference test. Only statistically significant differences (P<0.05) are described in the Results section. Analyses were performed with the statistical program Systat (SPSS Inc., Chicago, Ill.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AChE
AChE activity in the rat hippocampus was dependent on age and the availability of prenatal choline ( Fig. 1). AChE activity increased gradually with age until about P35 (22). Hippocampal AChE activity was similar among the groups on P1. AChE activity was increased in homogenates from prenatally choline-deficient rats on P17, P27, and P35 relative to the choline-supplemented group (Fisher's test). The prenatally choline-supplemented animals had on average the lowest AChE activity throughout development. The largest difference in AChE activity between treatment groups occurred on P27 when AChE activity was 35% higher in prenatally choline-deficient and 21% lower in the prenatally choline-supplemented groups compared to controls, resulting in a 70% higher AChE activity in the prenatally choline-deficient relative to the prenatally choline-supplemented rats. By P90, the effect of prenatal choline availability on hippocampal AChE diminished. A similar developmental pattern of AChE activity was observed in the striatum and cortex; however, prenatal choline availability had no effect on AChE activity in these brain regions (data not shown).



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  		Figure 1. Acetylcholinesterase activity in the hippocampus. AChE was measured in hippocampal homogenates as described in Methods. The data are reported as means ± SEM. A two-way ANOVA for Age (F6,164=29.86) and Diet (F2,164=9.35) revealed significant main effects of Age (P<0.001), Diet (P<0.001), and an Age x Diet interaction (F12,164=2.71; P<0.01). Post hoc analysis of the overall Diet effect by Tukey test revealed that prenatal choline supplementation reduced AChE activity compared to control and choline-deficient groups (P<0.01, P<0.001, respectively). Post hoc analysis by the Tukey test for all possible comparisons revealed that on P27 AChE activity was highest in prenatally choline-deficient group vs. control and supplemented groups ({dagger}P<0.05; P<0.001, respectively).

ChAT
ChAT activity in rat hippocampal homogenates was dependent on age and the availability of prenatal choline. Consistent with previous studies (21–25), ChAT activity increased with age, with a large increase (approximately fivefold) in activity occurring from P7 to P17 (data not shown). On P17 and P27, ChAT activity was highest in the prenatally choline-deficient and lowest in the prenatally choline-supplemented hippocampus. These differences were small but statistically significant ( Fig. 2). Changes in ChAT activity were transient and did not occur at other ages (P1, P3, P7, P35, P90).



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  		Figure 2. Choline acetyltransferase activity in the hippocampus. ChAT was measured in hippocampal homogenates as described in Methods. The data are reported as means ± SEM. A two-way ANOVA for Age (P17 and P27) and Diet revealed significant effects of Age (F1,51=18.82; P<0.001) and Diet (F2,51=3.32; P<0.05). Post hoc analysis of the overall Diet effect by Tukey test revealed that ChAT activity was higher in the prenatally choline-deficient group than in the prenatally choline-supplemented group (P<0.05).

Synthesis of ACh from choline transported by HACU
The amount of ACh synthesized from choline transported by HACU was determined on P17 and P27, the two ages at which ChAT and AChE activities were affected by prenatal choline availability. At these ages, cholinergic nerve terminals in the hippocampus of prenatally choline-deficient group incorporated the most [14C]choline radioactivity into [14C]ACh, whereas the prenatally choline-supplemented group incorporated the least ( Fig. 3A). The effect of prenatal choline availability on the formation of [14C]ACh in hippocampal cholinergic neurons was largest on P27, when the prenatally choline-supplemented group formed about 25% and 50% the amount of [14C]ACh compared to the prenatally choline-deficient and control groups, respectively.



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  		Figure 3. Acetylcholine content and synthesis from choline transported by HACU in hippocampal slices. A) Slices from P17 and P27 hippocampi were incubated for 10 min at 37°C with 1 µM [14C]choline in the presence or absence of 10 µM hemicholinium-3, a specific inhibitor of HACU. ACh was extracted, purified by HPLC, and its radioactivity was determined as described in Methods. The data are reported as dpm of [14C]ACh (mean±SEM) accumulated in the absence of hemicholinium-3 minus that accumulated in its presence, as an index of ACh synthesis from choline taken up by HACU. A two-way ANOVA for Age and Diet on the formation of [14C]ACh revealed significant effects of Diet (F2,44=15.06), Age (F1,44=49.47), and an Age x Diet interaction (F2,44=10.98) (P<0.001 for all comparisons). Post hoc analysis of the overall Diet effect by Tukey test revealed that the amount of [14C]choline incorporated into [14C]ACh was increased in the prenatally choline-deficient groups compared to prenatally choline-supplemented and control groups (*P<0.001). Post hoc analysis of all possible comparisons by the Tukey test revealed that the amount of [14C]choline incorporated into [14C]ACh was increased in the prenatally choline-deficient groups compared to prenatally choline-supplemented and control groups on P27 ({dagger}P<0.001). B) ACh content in slices incubated as in panel A in the presence of hemicholinium-3 was detemined by HPLC, as described in Methods, and the data are presented as mean ± SEM. A two-way ANOVA for Age (F1,45=169.76) and Diet (F2,45=7.64) on ACh content revealed significant effects of Age (P<0.001), Diet (P<0.01), and an Age x Diet interaction (F2,45=4.61; P<0.05). Post hoc analysis of the overall Diet effect by the Tukey test revealed that the amount of ACh in hippocampal slices was decreased in the prenatally choline-deficient groups compared to the prenatally choline-supplemented and control groups (*P<0.01). Post hoc Tukey test analysis of all possible comparisons revealed that the amount of ACh in hippocampal slices on P27 was decreased in the prenatally choline-deficient group compared to the prenatally choline-supplemented and control groups ({dagger}P<0.01). C) Specific radioactivity of [14C]ACh was obtained by dividing the values in panel A by those in panel B. The data are reported as mean ± SEM. A two-way ANOVA for Age (F1,42=6.44) and Diet (F2,42=11.96) on the specific radioactivity of [14C]ACh revealed significant effects of Age (P<0.05) and Diet (P<0.001) only. Post hoc analysis of the overall Diet effect by Tukey test revealed that the specific radioactivity of [14C]ACh was increased in the prenatally choline-deficient groups compared to the prenatally choline-supplemented and control groups (*P<0.001).

In the same hippocampal slices, ACh content increased from P17 to P27. Hippocampal ACh content on P17 and P27 was similar in prenatally choline-supplemented and control groups ( Fig. 3B). However, the hippocampal ACh content was reduced in the prenatally choline-deficient group on both P17 and P27 compared with the other two groups. The reduction of ACh content after prenatal choline deficiency was more robust on P27 than on P17.

To obtain an index of ACh turnover, we determined the specific radioactivity of [14C]ACh in the slices. The specific radioactivity of [14C]ACh was lower on P27 compared to P17 ( Fig. 3C). Further, the specific radioactivity of [14C]ACh was increased (two- to fivefold) at both ages in the hippocampal slices of prenatally choline-deficient rats compared to the control and prenatally choline-supplemented groups.

Potassium-evoked ACh release
Hippocampal slices released ACh at rest, and this release was more than doubled during the first period of incubation in a depolarizing medium ( Fig. 4). Only slices obtained from P27 animals were capable of sustaining this high rate of ACh release during the second period of depolarization, whereas slices of the P17 animals released approximately 30% more ACh during the second depolarization period than when at rest. The spontaneous release of ACh was similar among treatment groups at both P17 and P27. Prenatal choline supplementation enhanced the first potassium-evoked ACh releases in P17 and P27 hippocampal slices ( Fig. 4). Prenatal choline supplementation also enhanced the second potassium-evoked ACh release on P27, but not at age P17. ACh release was similar for control and prenatally choline-deficient groups.



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  		Figure 4. Depolarization-evoked acetylcholine release in hippocampal slices. Spontaneous and depolarization-evoked ACh release was measured, as described in Methods, using slices prepared from P17 and P27 hippocampi. Data are reported as means; error bars are omitted for clarity; coefficients of variation in the data were 12–40%. Individual two-way ANOVAs were performed for the two ages. A two-way ANOVA for Depolarization and Diet on ACh release on P17 revealed a significant effect of Depolarization (F2,39=48.12; P<0.001) and a Diet by Depolarization interaction (F4,39=3.51; P<0.05). ACh release in all three conditions (spontaneous, first depolarization, second depolarization) was different when compared by Tukey test (P<0.05 for all comparisons). P17 hippocampal slices from prenatally choline-supplemented groups had an increased first depolarization-evoked ACh release compared to control and prenatally choline-deficient groups (*P<0.05 for both comparisons by Tukey test). A two-way ANOVA for Depolarization and Diet on ACh relase in P27 hippocampal slices revealed significant effects of Diet (F2,45=14.03; P<0.001), Depolarization (F2,45=67.38; P<0.001), and a Diet by Depolarization interaction (F4,45=6.36; P<0.001). Spontaneous ACh release was lower compared to the amount of ACh released during the first and second depolarizations (P<0.001 for both comparisons by Tukey test). P27 hippocampal slices from the prenatally choline-supplemented group had an increased first depolarization-evoked ACh release compared to the prenatally choline-deficient and control groups (*P< 0.01 by Fisher's test for both comparisons). P27 hippocampal slices from the prenatally choline-supplemented rats showed an increased second depolarization-evoked ACh release compared to both the control and prenatally choline-deficient groups (*P<0.05 and {dagger}P<0.001, respectively by Tukey test).

The intracellular ACh content after the second ACh release in the slices from prenatally choline-deficient animals was lower on both P17 and P27 by about 25% compared to control and prenatally choline-supplemented groups ( Fig. 5).



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  		Figure 5. Acetylcholine content of hippocampal slices. ACh was measured in hippocampal slices by HPLC (as described in Methods) after the second depolarization period described in Fig. 4. Data are presented as means ± SEM. A two-way ANOVA for Age and Diet revealed a significant effect of Age (F1,28=6.47; P<0.05) and Diet (F2,28=3.79; P<0.05) only. Post hoc analysis of the overall Diet effect by Fisher's test revealed that the prenatally choline-deficient group had reduced ACh content compared to the control and choline-supplemented groups (*P<0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The availability of choline during the second half of fetal development alters the developmental pattern of the hippocampal cholinergic system. The changes observed appear well after the termination of treatment, and are particularly apparent during the third and fourth postnatal weeks. The current study is different in this respect from previous work performed in the adult on the effects of dietary choline availability on the brain cholinergic system immediately after treatment (26–30). In general, prenatally choline-deficient animals are characterized by elevations in AChE and ChAT activities, and increased synthesis of ACh from choline transported by HACU, concomitant with reductions in hippocampal ACh content and a relative inability to sustain depolarization-evoked ACh release. Together, the results indicate that the hippocampus of prenatally choline-deficient animals is characterized by accelerated ACh turnover (i.e., accelerated rate of synthesis, degradation, and choline reutilization by HACU, as indicated by the high specific radioactivity of newly synthesized ACh). The latter observation is consistent with the data showing that hippocampal HACU is activated by depolarization (31). This pattern of changes suggests that the hippocampal cholinergic system of these animals is organized so that it is capable of fast ACh recycling and high efficiency of choline reutilization for ACh synthesis, possibly in order to maintain normal cholinergic neurotransmission. This may indicate an adaptive response to the reduced availability of choline in utero. In contrast, prenatally choline-supplemented animals show less pronounced changes in their hippocampal cholinergic system; however, the direction of those changes is consistent with the above model. AChE and ChAT activities, and ACh synthesized from choline transported by HACU, were lowest in prenatally choline supplemented rats. However, depolarization-evoked ACh release was highest in these animals. The latter result, together with the reduced AChE activity, suggests that intrasynaptic ACh concentrations and dwell times are increased, resulting in enhanced cholinergic neurotransmission. The observations that ACh turnover in prenatally choline-supplemented animals is relatively slow (as indicated by low specific radioactivity of ACh newly synthesized from exogenous choline), but that cholinergic neurotransmission is well maintained (as evidenced by robust ACh release), suggest that the pool of choline used for the synthesis of ACh in these animals may include that stored in membrane phosphatidylcholine (29, 32) and may be generated by the hydrolysis of phosphatidylcholine catalyzed by PLD (33, 34). Consistent with the latter possibility, hippocampal PLD activity was twofold higher in prenatally choline-supplemented rats relative to control animals (18).

The mechanism of choline-induced changes in the hippocampus remains unknown. The period of E11–E17 in the rat correlates with the peak of neurogenesis (see ref 35 for a review), including that of the basal forebrain in general (36) and cholinergic neurons of this brain region in particular (37, 38); exogenous choline is necessary for this process. Conversely, choline deficiency causes apoptosis of cultured cells (39, 40), and alterations in hippocampal apoptotic patterns have been reported in animals subjected to treatments identical to those used in the current study (40). Thus, it is likely that the organization of intrinsic hippocampal neurons as well as cholinergic septohippocampal neurons is influenced by choline availability during the E11–E17 treatment. These organizational changes influence the way that the cholinergic septohippocampal pathway is established during the postnatal period. Data presented here show multiple neurochemical changes in the cholinergic system. Some of those changes may result from alterations in the numbers of cholinergic nerve terminals in the hippocampus or from changes in the activities of enzymes and macromolecules within those terminals, or both. The second possibility is more consistent with the results. For example, ChAT activity is lowest while the amount of ACh release is highest in the prenatally choline-supplemented rats, a result that would be difficult to explain by postulating changes in the numbers of cholinergic nerve terminals. Thus, choline availability in utero appears to result in multiple adaptive metabolic responses in the developing hippocampus.

In prenatally choline-deficient animals, the adaptation results in efficient recycling of choline, whereas in prenatally choline-supplemented rats, choline recycling is lower and there seems to be higher reliance on membrane-stored choline for ACh synthesis. These adaptations seem appropriate for the periods when choline availability is altered (i.e., prenatally), but they are long-lasting,—observed for as long as 1 month (P27) after termination of the treatment (E17). Later in life the differences between groups diminish ( Fig. 1), indicating that brain organization may readjust as all animals consume the control diet. The molecular mechanisms governing these adaptations are not known. How does the developing brain sense changes in choline availability? In a previous study, the choline supplementation paradigm described here caused 40% elevations in the content of fetal brain phosphocholine (41), a metabolite that could be considered a storage pool of choline and postulated to be necessary for cell division initiated by certain growth factors (42, 43). In addition to serving as a precursor of the neurotransmitter ACh (which may act as a growth factor during development) (44), the choline moiety is also present in two other signaling molecules: platelet-activating factor (PAF) and sphingosylphosphocholine (45). The functions of the latter compound in the brain remain to be established; however, the importance of PAF for brain development recently became apparent when it was shown that a mutation in a subunit of PAF acetylhydrolase is responsible for the human genetic disease Miller-Dieker lissencephaly (46), a disorder characterized by smooth brain hemispheres and abnormal neuronal migration. It will be interesting to determine whether the levels of those compounds and/or the signaling pathways initiated by those compounds are altered by choline availability in the rat model described here.

Prenatal choline supplementation facilitates spatial (11–13) and temporal (14, 15) memory functions across the life span, whereas prenatal choline deficiency has been shown to impair attention performance and accelerate age-related declines in temporal processing (15). Given the key role of the cholinergic system in spatial memory and attention (47–52), our results suggest that changes in the hippocampal cholinergic system may constitute part of the mechanism responsible for the long-lasting cognitive changes evoked by the availability of choline in utero.

Previous studies have demonstrated considerable plasticity of the septohippocampal cholinergic system in response to injury, growth factor availability, and training (53–64). The complex adaptive response of this neurochemical system to prenatal choline availability constitutes a novel example of developmental plasticity in the nervous system governed by the supply of a single nutrient.


   ACKNOWLEDGMENTS
 
These studies were supported by grant AG09525 from the National Institute on Aging.


   FOOTNOTES
 
1 Correspondence: Boston University School of Medicine, 85 East Newton St., Rm. M1009, Boston, MA 02118, USA. E-mail: jbluszta{at}bu.edu

2 Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; ANOVA, analysis of variance; ChAT, choline acetyltransferase; E, embryonic day; EDTA, ethylene-diamine-tetraacetic acid; HACU, high-affinity choline uptake; HBS, HEPES-buffered saline solution; HEPES, 4-(2-hydroyethyl)-1-piperazineethanesulfonic acid; P, postnatal day; PAF, platelet-activating factor; PLD, phospholipase D; PSS, physiological salt solution; HPLC, high-performance liquid chromatography.

Received for publication November 10, 1997. Accepted for publication November 21, 1997.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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