Developmental Periods of Choline Sensitivity Provide an Ontogenetic Mechanism for Regulating Memory Capacity and Age-Related Dementia

In order to determine brain and behavioral sensitivity of nutrients that may serve as inductive signals during early development, we altered choline availability to rats during 7 time frames spanning embryonic day (ED) 6 through postnatal day (PD) 75 and examined spatial memory ability in the perinatally-treated adults. Two sensitive periods were identified, ED 12–17 and PD 16–30, during which choline supplementation facilitated spatial memory and produced increases in dendritic spine density in CA1 and dentate gyrus (DG) regions of the hippocampus while also changing the dendritic fields of DG granule cells. Moreover, choline supplementation during ED 12–17 only, prevented the memory decline normally observed in aged rats. These behavioral changes were strongly correlated with the acetylcholine (ACh) content of hippocampal slices following stimulated release. Our data demonstrate that the availability of choline during critical periods of brain development influences cognitive performance in adulthood and old age, and emphasize the importance of perinatal nutrition for successful cognitive aging.


INTRODUCTION
Maternal nutrition during pregnancy and lactation is important for the health of the fetus and the neonate, and although the contribution of nongenetic maternal factors to the cognitive development of the offspring is highly signifi cant, the requirements of specifi c nutrients for brain development are poorly understood (Devlin et al., 1997;Feil, 2006;Lucas et al., 1990Lucas et al., , 1992. A recent report of the Food and Nutrition Board: Institute of Medicine of the National Academy of Sciences (1998) has, for the fi rst time, issued specifi c guidelines for choline intake by pregnant and lactating women (see Zeisel, 2000aZeisel, ,b, 2005Zeisel and Niculescu, 2006). Here we show that when choline availability is augmented during early development two critical periods can be identifi ed in rats: ED 12-17 and PD 16-30. During these periods, choline supplementation produced facilitation of spatial memory that persisted throughout the entire lifespan and prevented the memory decline normally observed in aged rats (McCann et al., 2006;Meck and Williams, 2003).
One of the most consistent fi ndings in the gerontological literature on cognition is an age-related decline in spatial learning and memory abilities (Bruce and Herman, 1986;de Toledo-Morrell et al., 1984;Evans et al., 1984;Tanila et al., 1997). In the rat, the brain areas necessary for the expression of spatial memory are well characterized, and age-related changes in a number of anatomical, electrophysiological, and neurochemical measures in the hippocampus and frontal cortex have been correlated with performance (Barnes, 1979;Gallagher and Rapp, 1997;Lanahan et al., 1997;Meck et al., 1989). Because choline serves as a precursor of phospholipid components of membranes, including those of neurons and glia, an adequate supply of this essential nutrient may be crucial in early development, as brain cells divide and grow, myelin is laid down, and synapses form (Albright et al., 1999a(Albright et al., ,b, 2003Zeisel and Blusztajn, 1994). Choline is also a selective agonist of α7 nicotinic acetylcholine receptors (Alkondon et al., 1997(Alkondon et al., , 1999Cheng et al., 2006) and provides a precursor for ACh, the neurotransmitter of cholinergic neurons that enervate the hippocampus and cortex and plays an important role in cognitive and sensorimotor function (e.g., Berger-Sweeney, 2003;Blusztajn and Wurtman, 1983;Meck, 2002;Meck and Williams, 2003;Nag and Berger-Sweeney, 2007;Teather and Wurtman, 2005;Zeisel, 2000a).
Our study was designed to determine the developmental window(s) for sensitivity to choline-induced facilitation of spatial memory in adult rats and to examine whether memory facilitation by prenatal choline supplementation lasts into old age using behavioral tasks specifi cally developed for this purpose (Meck et al., 1988(Meck et al., , 1989. The experimental design included an untreated control group and seven treatment groups that were exposed to choline supplementation at various time frames during pre-or postnatal development. The results indicate that there are 2 critical periods, ED 12-17 and PD 16-30 for choline-induced facilitation of spatial memory. Additional results reported here using both longitudinal and cross-sectional methods demonstrate that signifi cant age-related impairments in spatial memory performance are observed for the control rats (see Beatty et al., 1995;Bierley et al., 1986 for a discussion of the advantages of employing both cross-sectional and longitudinal designs in Golgi morphometric analysis of spine density Single-section Golgi impregnation was used to examine the morphologic characteristics of pyramidal cells from the CA1 region of the hippocampus and the lateral-dorsal thalamus, as well as granule cells from the dentate gyrus. Twenty behaviorally naïve rats (approximately 7 months of age) were randomly selected from the CONTROL, ED 12-17, PD 1-15, and PD 16-30 treatment groups for morphometric analysis. Rats (n = 5/group) were deeply anesthetized with an overdose of sodium pentobarbital and transcardially perfused with 120 ml of 4.0% paraformaldehyde in 0.1 M phosphate buffer and 1.5% picric acid (v/v). Brains were postfi xed and stored overnight in the same solution. After postfi xation, a modifi ed version of the single-section Golgi impregnation procedure was used to process brains (Gabbott and Somogyi, 1984;Woolley and Gould, 1994). Serial coronal sections (150 µ thick) were cut on an oscillating tissue slicer in a bath of 3.0% potassium dichromate in distilled water. The sections were incubated overnight at room temperature in individual wells containing 3.0% potassium dichromate. The following day, the sections were rinsed and mounted onto ungelatinized slides, a coverslip was glued over the sections at the four corners, and the slide assembly was placed in a Coplin jar containing 1.5% silver nitrate in distilled water. After 48 h, the slide assemblies were dismantled and the sections removed from the slides. The sections were rinsed in distilled water, dehydrated in ethanol, cleared in xylenes, and mounted onto ungelatinized glass slides. Slides were coverslipped with Histomount and allowed to dry before quantitative analysis.
Quantifi cation techniques developed to study dendritic branching and spine density as a function of sex, age, and environmental enrichment were employed (e.g., Juraska et al., 1985Juraska et al., , 1989Kolb et al., 2003). Spine density analysis was conducted blind to experimental condition. For CA1 pyramidal neurons, spine density was measured on apical dendrites of stratum radiatum and basal dendrites of stratum oriens. Quantitative analysis was conducted on tissue stained dark with Golgi impregnation that was uniform throughout the section. Six Golgi-impregnated pyramidal neurons discernible from nearby impregnated cells were selected from each rat. These neurons were located within the CA1 region of the dorsal hippocampal formation and were required to have no breaks in staining along its dendrites. Measurement occurred at least 50 µm away from the soma for apical dendrites and 30 µm for basal dendrites on secondary and tertiary branches. Five segments between 10 and 20 µm in length and in the same plane of focus were chosen. In some cases, the segments were from the same branch. Granule cells were drawn from the upper and lower limbs of the fascia dentata of the hippocampus with approximately equal selection from each of these areas. Six neurons for each rat were chosen evenly from each of the sampling regions of both hemispheres within the limitations of the sections obtained. Criteria for selection of neurons were based on completeness of staining and traceability of dendrites. Counting required focusing in and out with the fi ne adjustment of the microscope. Only spines that were distinct from the dendritic branch were counted. Spine density was calculated by dividing the number of spines on a segment by the length of the segment and was expressed as the number of spines per 10 µm of dendrite. Densities of spines on fi ve segments of a cell were averaged for a cell mean, and the six cells from each animal were averaged for an animal mean. Spine density values using this method are underestimates, because spines protruding either above or beneath the dendritic shaft are not accounted for (Woolley and Gould, 1994). In addition, although the quantifi cation of spine densities was designed to be consistent with earlier studies of environmental enrichment, we assume that the results would be substantially the same using unbiased stereology (e.g., Rusakov and Stewart, 1995).

Golgi morphometric analysis of dendritic fi elds
In addition to spine counts, granule cells from the dentate gyrus were traced in three dimensions at a magnifi cation of 40×, with the aid of a 3-D Eutectic Neuron Tracing System (Eutectic Electronics, Inc., Raleigh, NC) using unbiased sampling methods (Capowski, 1989). As described above, granule cells were selected from the upper and lower limbs of the fascia dentata of the hippocampus with approximately equal selection from each of these areas. Six neurons for each rat were randomly chosen from each of the sampling regions of both hemispheres within the limitations of the sections obtained. From the time of killing of the rats, all histological, drawing, and scoring procedures were carried out on rats coded so as not to reveal treatment condition in order to preclude experimenter bias. In order to determine specifi c changes in the dimensions of the dendritic fi elds of neurons modifi ed by perinatal choline supplementation measurements of the vertical width and transverse spread of the molecular layer for granule cells, which is delineated by the hippocampal fi ssure, were obtained in a manner similar to Rihn and Claiborne (1990).

Behavioral assessment of spatial memory
The acquisition of rats' spatial memory performance as indexed by the number of choices made while fulfi lling the requirement of visiting each of the baited radial-maze arms (choices to criterion) is plotted as a function of blocks of 3 daily sessions in Figure 1. The general pattern observed in the number of errors was an improvement over blocks of sessions, at approximately equal rates for all groups of rats. Remarkably, two groups of rats showed fewer errors relative to the untreated control group. These rats were supplemented with choline either during the periods of ED 12-17 or PD 16-30. Moreover, supplementation with choline during the ED 12-17 period caused a greater improvement of performance than did the supplementation during PD 16-30. At no other time frame studied did choline supplementation alter performance. These data show that adult cognitive performance is sensitive to choline availability during two critical periods of early brain development, ED 12-17 and PD 16-30.
For additional analysis, the overall choice performance of rats in the ED 12-17, PD 16-30, and control groups was broken down into episodic and reference memory components (Eichenbaum, 2002). An episodic memory error (also referred to in the animal literature a working memory error -see Olton and Papas, 1979) was defi ned as a re-entry into an arm already visited during that trial. A reference memory error was defi ned as an entry into arms that are consistently unbaited. Reliable facilitation of both types of spatial memory was shown by rats from the ED 12-17 and PD 16-30 treatment groups. Additional analyses indicated that rats in these two treatment groups made signifi cantly more correct choices before committing their fi rst working memory error than rats in any of the remaining groups (P-values < 0.05, data not shown), suggesting that they were able to hold more items in memory accurately. The latency to complete the fi rst 8 choices (minimum number of choices required to fi nd all baited arms) serves as a measure of the rats' motivation independent of memory processes (e.g., Meck et al., 1988Meck et al., , 1989. During the 12 sessions of training, the overall mean latency taken to complete the fi rst 8 choices was 50.7 ± 1.4 s and did not differ reliably as a function of treatment group, P > 0.05. Because there is evidence that high levels of cognitive performance in adulthood may indicate the presence of a "cognitive reserve", a term used to describe the capacity to delay age-related cognitive decline  (Control,, which did not reliably differ from each other. The ED 12-17 group required signifi cantly fewer choices to complete the maze than the PD 16-30 group, P-values < 0.05. A signifi cant effect of choline treatment was also observed for both episodic and reference memory errors in the ED 12-17 and the PD 16-30 treatment groups relative to controls; F(7,67) = 12.6, P < 0.01 and F(7,67) = 17.3, P < 0.01, respectively.
Results from both the longitudinal and the cross-sectional studies revealed signifi cant age-related impairments in spatial memory for the untreated control rats, while rats given prenatal choline supplementation showed enhanced memory when young and little or no decline in spatial memory as a function of age. The main effects of prenatal choline supplementation on the acquisition/reacquisition of spatial memory performance in the 12-arm radial maze during the longitudinal and cross-sectional studies are presented in Figures 2A,B, respectively. For comparison purposes, steady-state performance averaged over the fi nal 5 blocks of training is illustrated for male and female rats in Figures 2C,D for the longitudinal and cross-sectional studies, respectively.  (Alexander et al., 1997;Butler et al., 1996), we next determined the longevity of this choline-induced memory enhancement. In order to assess the long-term infl uence of prenatal choline availability, rats were exposed to choline supplementation during ED 12-17 or were untreated, and then were trained for 30 consecutive days on a radial-arm maze task at 2 months of age, 14 months of age, and again at 26 months of age in a longitudinal design. In order to control for the repeated experience effects of longitudinal studies, a cross-sectional design was also used in which radial-arm maze performance was assessed in separate groups of young (2-3 months) and aged (27-28 months) rats that had been given prenatal choline supplementation during ED 12-17 or given no added choline.

Morphometric analyses of neurons: alterations in spine density
Dendritic spines are the primary source of synaptic contact in the mammalian brain. A number of studies have reported increases in synaptogenesis after behavioral training (e.g., Van Reempts et al., 1992;Wenzel et al., 1980) and similarly, use-dependent measures of synaptic plasticity such as long-term potentiation (LTP) are associated with changes in hippocampal synapse number and/or structure (Desmond and Levy, 1986;Geinisman, 2000;Yuste and Bonhoeffer, 2001). More recently, it was reported that trace conditioning did not alter the number of axospinous synapses in the hippocampus, but did increase the number of multiple synapse boutons, a condition under which one presynaptic bouton synapses with two or more dendritic spines (Geinisman et al., 2001). This effect was evident on apical dendrites, but basal dendrites were not examined.
There is general agreement that spatial memory is dependent on the integrity of the hippocampus (Broadbent et al., 2004). It has also been shown that reversible inactivation of the lateral dorsal thalamus disrupts hippocampal place representation and impairs spatial learning (e.g., Mizumori et al., 1994). In the present study we were interested in morphometric changes in the hippocampus and lateral dorsal thalamus associated with perinatal choline supplementation in behaviorally naïve animals. Light microscopic examination of Golgi-impregnated tissue from control and experimentally treated brains revealed reliable and consistent staining of pyramidal and granule cells. In all cases, hippocampal and thalamic neurons showed a large number of spines covering their dendrites. These spines were typically short and in many instances showed a terminal swelling or enlargement. Spine density measures as a function of treatment condition (Control, ED 12-17, PD 0-15, and PD 16-30 periods of choline supplementation) and brain region (apical and basal dendrites of CA1 pyramidal cells and DG granule cells) are illustrated in Figures 3A-C. Spine densities as a function of treatment condition for pyramidal neurons in the lateral-dorsal nucleus of the thalamus are shown in Figure 3D. The major observation was that spine densities were signifi cantly increased in each hippocampal region as a function of the ED 12-17 and PD 16-30 periods

Morphometric analyses of dentate granule cells: alterations in dendritic fi elds
Representative neural tracings of dentate granule cells for rats in the ED 12-17 and CON treatment groups are illustrated in Figures 4A,B, respectively. The mean (±SEM) transverse spread and vertical width of the molecular layer of dentate granule cells is plotted as a function of treatment condition in Figure 4C. ANOVAs conducted on the transverse spread measure revealed a signifi cant effect of treatment condition, F(3,19) = 52.55, P < 0.0001. Fisher PLSD post-hoc contrasts revealed the CON vs. ED 12-17, CON vs. PD 16-30, ED 12-17 vs. PD 1-15, ED 12-17 vs. PD 16-30, and the PD 1-15 vs. PD 16-30 comparisons to be signifi cant at the P < 0.05 level. These results indicate a larger transverse spread of the molecular layer of dentate granule cells for rats exposed to choline supplementation during the ED 12-17 and PD 16-30 time frames compared to untreated control rats and rats given choline supplementation during the PD 1-15 time frame. In contrast, ANOVAs conducted on the vertical width of the molecular layer of granule cells revealed a signifi cant, but opposite effect of treatment condition, F(3,19) = 26.61, P < 0.0001. Fisher PLSD post-hoc contrasts revealed the CON vs. ED 12-17, CON vs. PD 16-30, ED 12-17 vs. PD 1-15, and the PD 1-15 vs. PD 16-30 comparisons to be signifi cant at the P < 0.05 level. These results indicate that in contrast to the transverse spread measure, the vertical width of the molecular layer of dentate granule cells for rats exposed to choline supplementation during the ED 12-17 and PD 16-30 time frames was smaller compared to untreated control rats and rats given choline supplementation during the PD 1-15 time frame.

Neurochemical correlates of memory enhancement
Hippocampal cholinergic neurotransmission has been implicated in the mechanisms of learning and memory (Fibiger, 1991;Gais and Born, 2004;Hasselmo and McGaughy, 2004;Hasselmo and Schnell, 1994;Meck et al., 1987;Rogers and Kesner, 2004;Power, 2004;Sarter and Parikh, 2005). Previous studies have also shown that the availability of choline during the ED 12-17 period causes multiple changes of the hippocampus in young adult rats including protection from the neuropathological response to status epilepticus (Holmes et al., 2002;Wong-Goodrich et al., 2008;Yang et al., 2000), increased neurogenesis (Glenn et al., 2007), and heightened responsiveness to cholinergic stimulation (e.g., Cermak et al., 1998;Jones et al., 1999;Mellott et al., 2004;Montoya et al., 2000). Consequently, we measured ACh release and content using hippocampal slices obtained from the 27 months old rats at the completion of behavioral training. The amount of ACh release at rest and under depolarizing conditions was not affected by the prenatal choline treatment (data not shown). However, ACh content was signifi cantly higher in hippocampal slices obtained from choline-supplemented rats as illustrated in Figure 5A, suggesting that this prenatal nutritional treatment modifi ed the hippocampal cholinergic system for the entire lifespan. Moreover, there was a signifi cant negative correlation between hippocampal ACh content and the number of errors made by the male and female rats during the fi nal sessions in the radialarm maze task as shown in Figures 5B,C, respectively. These fi ndings are consistent with those reported by Blusztajn et al. (1998) for young adult rats indicating that the higher ACh content observed in prenatal choline supplemented rats allows them to maintain an undiminished level of ACh release for a longer period of time following repeated depolarizations.

Critical periods for metabolic imprinting by the availability of choline
The major fi nding is that the brain appears to be particularly vulnerable to alterations in the availability of choline during two periods: ED 12-17 and PD 15-30. The most probable explanation for this pattern of sensitivity to choline is that prior to ED 12 maternal diet and body stores are suffi cient to provide adequate amounts of choline for the growing embryos; however, after ED 12, supplementation of the diet is necessary for optimal brain development. During this period the brain grows rapidly due to massive division of the neuronal and glial progenitor cells. Choline is necessary for cell division and it has been demonstrated that supplementation with choline during the ED 12-17 period increases the number of mitoses in brain (Albright et al., 1999a,b). Following birth, choline is supplied to pups via milk. Rat milk, which contains approximately 6 mmol/l of free choline and choline esters (Holmes-McNary et al., 1996;Garner et al., 1995) is a rich source of this nutrient. We estimate that the average daily choline consumption of a suckling pup is 3 mmol/kg. This is more than two-fold of the amount of choline consumed by dams on our control diet (1.3 mmol/kg). Thus, it is likely that the amount of choline pups obtain from their diet of milk during PD 1-15 is suffi cient for brain development and further supplementation would be expected to have no effect. Later on, during the period of PD 16-30 when pups consume both milk and solid diet, choline supplementation becomes benefi cial again. This suggests that the choline supply via milk and/or diet is inadequate for the developing brain. It is important to note that maternal choline pools become markedly diminished by pregnancy and lactation, and dietary supplementation with choline restores them . Thus, choline supplementation during pregnancy and lactation does not constitute a pharmacological manipulation, but rather can be viewed as rehabilitation of a naturally occurring defi ciency state that develops due to the extra demands for choline presented by the fetus and the suckling neonate. The PD 1-30 time frame is characterized by the establishment of neuronal projections, synaptogenesis, and myelination. Choline, as a component of structural phospholipids of membranes, is necessary for all of these processes. It appears that the long-term benefi cial effects of choline supplementation on memory ability are confi ned to the pre-PD 30 period, suggesting that sensitivity to the supply of choline terminates approximately at the time when brain development is mostly complete. The long-term behavioral and neurochemical actions of perinatal choline supplementation reported here are supported by other fi ndings revealing that perinatal choline supplementation produces a complex pattern of anatomical, behavioral, chemical, and electrophysiological changes in the brains of young adult rats (e.g., Brandner, 2002;Guo-Ross et al., 2003;Holler et al., 1996;Jones et al., 1999;Li et al., 2004;Meck andWilliams, 1997a, b, c, 1999;Mohler et al., 2001;Pyapali et al., 1998;Ricceri and Berger-Sweeney, 1998;Tees, 1999;Williams et al., 1998;Yang et al., 2000). Further work is necessary to fully understand how the effects of a single nutrient are translated into improved memory function that lasts a lifetime. At present, it appears that the imprinting of hippocampal metabolism of choline by its availability during gestation is a major factor in the enhancement of spatial memory and its protection from agerelated memory dysfunction (Blusztajn, 1998;Blusztajn et al., 1998).

Functional implications of spine density
Synaptic plasticity provides the basis for most models of learning, memory, and development in neural circuits. The neural mechanisms by which memory capacity is determined and how this substrate is modifi ed as memories are acquired and stored in the mammalian brain are assumed to involve modifi cations in synaptic scaling, spike-timing, and synaptic redistribution (Abbott and Nelson, 2000). The most extensively examined region in which plastic events are thought to occur is the hippocam-pal formation, a brain region involved in the acquisition of spatial and temporal relations (e.g., Fortin et al., 2002;Meck et al., 1984;Shapiro and Eichenbaum, 1999). Although there are many instances of changes in hippocampal synaptic neurotransmission in response to learning (e.g., McNaughton and Morris, 1987;Power et al., 1997), there are few examples of changes in structural plasticity that involve either the production of new synapses or a reorganization of existing synapses that can be directly related to learning and memory capacity in the adult (Bailey and Kandel, 1993;Moser, 1999).
Dendritic spines, small protrusions on the shaft of dendrites in the mammalian brain, represent a means whereby new contacts between cells can be established and existing contacts strengthened. As such, it has long been suggested that dendritic spines are involved in the formation of new memories (Kasai et al., 2003). Because most spines are associated with excitatory synapses, an increase in their number could translate into a signifi cant increase in excitatory neurotransmission in the hippocampus (Andersen et al., 1966;Harris and Kater, 1994), which is often considered an integral step in memory formation. It has also been proposed that existing spines may relocate from non-activated boutons and synapse with those activated by training, at least on apical dendrites (Geinisman et al., 2001). Although there are reports that environmental experience can affect dendritic spines (Anderson et al., 1996;Fiala et al., 1978;Kleim et al., 1996Kleim et al., , 2002Knafo et al., 2001;Leuner et al., 2003), convincing evidence distinguishing effects based on specifi c developmental, environmental, or learning-induced changes is still lacking.
Training on a hippocampal-dependent task of spatial maze learning has been associated with a transient increase in dendritic spine density (O'Malley et al., 2000), although others did not observe a change (Rusakov et al., 1997). There is also indirect evidence associating spines with learning; exposure to a complex spatial environment enhanced spines and, in a separate group of animals, enhanced performance in the water maze (Moser et al., 1994(Moser et al., , 1997. Importantly, the baseline levels of spine density and increases due to prenatal choline supplementation reported here are comparable to those reported for similarly aged male rats exposed to isolated vs. complex environments (Kolb et al., 2003). Training on another hippocampal-dependent task (Beylin et al., 2001), trace eyeblink conditioning, was associated with changes in synaptic structure, but spine number was not assessed (Geinisman et al., , 2001. Consequently, it remains to be determined to what extent changes in spine density produced by environmental enrichment and/or perinatal choline supplementation (see Tees, 1999) might serve as a format for the reorganization of synaptic connections. The current fi ndings indicate an increase in both apical and basal dendritic spines in the CA1 region of the adult hippocampus as well as an increase in the spine density of the DG as a function of perinatal choline supplementation in the absence of explicit behavioral training. These increases in spine density do not refl ect the pattern of selective increases in CA1 basal spine density and reorganization observed following both hippocampal-dependent and hippocampal-independent eyeblink conditioning preparations (Leuner et al., 2003) and suggest a more general role in memory capacity.
The current data indicate that a relatively permanent increase in spine density accompanies perinatal choline supplementation when administered during specifi c time periods. This result does not indicate that an increase in spine density in the hippocampus is necessary for hippocampal-dependent learning to occur, especially because spine density has previously been observed to be enhanced following a variety of training conditions that do not depend upon this brain structure (Beylin et al., 2001;Leuner et al., 2003). In these earlier experiments, the increase in spine density did not appear to be a result of enhanced arousal or the stress of training, although some studies have reported that exposure to an acute stressful event also increased spine density in area CA1 of the hippocampus (e.g., Shors et al., 1992Shors et al., , 2001. Adult spatial memory performance as well as hippocampal spine density exhibited an "On-Off-On" pattern of sensitivity as a function of the time frame of perinatal choline supplementation. In particular, spine densities were observed to increase signifi cantly above control levels in the CA1 and DG regions of the hippocampus during the ED 12-17 and the PD 16-30 treatment conditions. This result suggests greater numbers of synaptic connections as a function of the time frames for choline supplementation and is supportive of an increase in the capacity of the neural networks and related mechanisms of LTP thought to subserve learning and memory (e.g., Malenka and Nicoll, 1999). In this regard, the present data are supportive of the observations that the threshold for induction and the magnitude of LTP following theta-burst stimulation trains in area CA1 were signifi cantly enhanced in slices from choline supplemented rats compared to controls. In contrast, the slices from choline defi cient rats showed signifi cantly lower levels of LTP than controls and were less stable in baseline electrophysiological indices (Jones et al., 1999;Pyapali et al., 1998). Other studies have reported greater excitatory responsiveness, reduced slow after-hyperpolarizations, and enhanced afterdepolarizing potentials in stimulated adult hippocampal CA1 pyramidal cells following prenatal choline supplementation (Li et al., 2004). Taken together, these electrophysiological data support the proposal that the availability of choline to the fetus during specifi c periods of neurogenesis and synaptogenesis in the hippocampus is critical to the development of neural mechanisms responsible for memory capacity and the expression of LTP in the adult.

Implications of changes in dendritic fi elds
Broader transverse spreads as well as narrower vertical widths of the molecular layer of dentate granule cells were associated with choline supplementation during the ED 12-17 and PD 16-30 time frames. Interestingly, it has been previously shown by Rihn and Claiborne (1990) that even though dendritic tree shapes for granule cells are similar in young (PD 14-19) and older (PD 50-60) rats there is a strong developmental trend for the spread of the dendritic tree in the transverse plane of the hippocampus to decrease with age. In addition, while dendritic segment numbers decreased and total dendritic length remained constant, the average vertical width of the molecular layer was observed to increase by approximately 50% from PD 14 to PD 60. A direct comparison of our dendritic measures and neuronal drawings of granule cells with those presented by Rihn and Claiborne (1990) indicate that the morphometric data from rats in the ED 12-17 and the PD 16-30 treatment groups strongly parallel the morphometric data reported earlier for Sprague-Dawley rats at PD 14-19. Similarly, the morphometric data from our rats in the CON and the PD 1-15 treatment groups strongly parallel the morphometric data reported earlier for PD 50-60 rats in terms of the transverse spread and vertical width measures of the molecular layer of dentate granule cells. Taken together, these data suggest that perinatal choline supplementation during the ED 12-17 and PD 16-30 time frames may block some aspects of the age-related changes normally observed in dendritic growth and regression in rat dentate granule cells during late postnatal development. These effects may be related to the observation that choline supplementation in utero from ED 12-17 (300 mg/kg/day choline chloride, p.o. to the dam) results in elevated levels of hippocampal NGF, an increase in the size of diagonal band neurons immunoreactive for the low affi nity neurotrophin receptor (p75), and in p75 mRNA (McKeon-O'Malley et al., 2003;Sandstrom et al., 2002).
Even though the dentate gyrus in the rat begins to form prenatally, the majority of its development occurs during the fi rst three postnatal weeks (Stanfi eld and Cowan, 1988). Dendritic segment number of granule neurons in the suprapyramidal blade of the hippocampus may increase up to PD 14, and steadily decrease after that time. Beginning at about PD 14, a number of signifi cant changes take place in the rat dentate gyrus (Cowan et al., 1980). The major afferents to the molecular layer reach their adult patterns of lamination at about this time (Fricke and Cowan, 1977), and most inhibitory interneurons in the granule cell layer and hilar region mature by PD 18. The granule cell axons (the mossy fi bers) attain their adult size and shape between PD 15 and 21, and at PD 15 it is fi rst possible to elicit reliable physiological responses in CA3 pyramidal neurons from granule cell stimulation.

Neurochemical correlates of spatial memory performance
When the dynamics of the hippocampal cholinergic synapse were examined in prenatal choline supplemented, defi cient, and control weaningage rats, it was discovered that prenatal choline availability altered choline and ACh turnover in addition to ACh tissue content Meck and Williams, 2003). The synapse of the defi cient rat was characterized by elevated acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities, and increased synthesis of ACh from choline transported by sodium-dependent high-affi nity choline uptake (SDHACU). ACh content was reduced and hippocampal slices were unable to sustain depolarization-evoked ACh release in adult rats that experienced a brief period of prenatal choline defi ciency. Taken together, these fi ndings indicate that in the hippocampus of prenatal choline-defi cient rats ACh turnover is accelerated (i.e., there is more rapid synthesis, degradation, and choline reutilization by SDHACU). In contrast, prenatally cholinesupplemented rats showed elevated ACh tissue content and changes in metabolic pathways that were opposite to the effects observed in the defi cient rat. That is, choline-supplemented rats have cholinergic synapses that have adapted to maintain function by developing reduced turnover and recycling due to the higher availability of ACh, whereas the prenatal choline defi cient rat has adapted by increasing ACh turnover and recycling to compensate for reduced tissue levels. Our behavioral data suggest that this metabolic adaptation is an effective compensation for the choline defi cient rat when task demands are low, but if cognitive load is increased (e.g., by massing trials), the system fails and memory deficits are seen. In contrast, the choline supplemented rat is able to maintain levels of stimulated ACh release in conjunction with increased memory performance under massed-trial conditions due to a release from proactive interference (Meck andWilliams, 1999, 2003). The conclusion is that these behavioral and neurochemical data demonstrate that alterations in choline availability during the prenatal period can "imprint" cholinergic synapses such that they operate quite differently in the adult as a function of prenatal choline availability. These fi ndings suggest that similar long-term metabolic changes at the synaptic level could result from genetic alteration in choline or methyl metabolism during the prenatal period that correlate with adult spatial memory performance (e.g., Glenn et al., 2007;Meck et al., 1989;Nafee et al., 2008;Niculescu et al., 2006;Waterland and Michels, 2007;Wong-Goodrich et al., 2008).

Inoculation against age-related decline in cognitive function
Age-related learning and memory defi cits have been well documented in humans and other animals. One proposal is that an age-related changes in neuronal responsiveness to cholinergic and glutaminergic stimulation is responsible for impaired memory ability in aged animals (Akaike and Rhee, 1997;Cheng et al., 2006;Engstrom et al., 1993;Williams et al., 2007). To date, no effective treatment exists to prevent this type of cognitive deterioration in old age. Our data show that supplementation with choline during several critical periods of early develop constitutes such a treatment. We hypothesize that choline supplementation during these vulnerable periods may alter brain organization in such a way that it has more "cognitive reserve" that can be used as the brain ages and loses connectivity. In addition, the time frames for spatial memory/hippocampal sensitivity to perinatal choline availability may correspond to prenatal periods of neurogenesis and postnatal periods of dendritic remodeling of cholinergic neurons in the basal forebrain (Meck and Williams, 2003;Williams et al., 1998). Choline supplementation given during these periods appears to enhance spatial memory of adult rats and to inoculate them against age-related decline in memory seen in untreated controls. It will be important to determine if our observations obtained with a rat model are applicable to humans. Human babies are exposed to diets with varying levels of free choline and other choline compounds. Postnatally, human infants may drink breast milk or a milk substitute and infant formulas vary widely in their choline content (Holmes-McNary et al., 1996;Zeisel and Blusztajn, 1994). The current studies indicate that alterations in the dietary availability of a single nutrient, choline, can alter brain development and have benefi cial effects on cognitive function throughout the entire lifespan in both males and females (Cheng et al., 2008).