Prenatal choline availability alters the context sensitivity of Pavlovian conditioning in adult rats

The effects of prenatal choline availability on Pavlovian conditioning were assessed in adult male rats (3–4 mo). Neither supplementation nor deprivation of prenatal choline affected the acquisition and extinction of simple Pavlovian conditioned excitation, or the acquisition and retardation of conditioned inhibition. However, prenatal choline availability significantly altered the contextual control of these learned behaviors. Both control and choline-deprived rats exhibited context specificity of conditioned excitation as exhibited by a loss in responding when tested in an alternate context after conditioning; in contrast, choline-supplemented rats showed no such effect. When switched to a different context following extinction, however, both choline-supplemented and control rats showed substantial contextual control of responding, whereas choline-deficient rats did not. These data support the view that configural associations that rely on hippocampal function are selectively sensitive to prenatal manipulations of dietary choline during prenatal development.There is increasing evidence that variations in maternal dietary choline intake during the second half of pregnancy cause structural, biochemical, and physiological changes in basal forebrain neurons and their projections to the hippocampal complex as well as long-term cognitive changes in the offspring (e.g., Meck and Williams 2003; McCann et al. 2006; Meck et al. 2008). We know, for instance, that the adult offspring of pregnant rats supplemented with 4.5 times the amount of choline in the standard laboratory diet display improved memory capacity and precision on the radial-arm maze (e.g., Meck et al. 1988, 1989; Meck and Williams 1997b, 1999; Tees 1999a), Morris water maze (e.g., Tees 1999b; Tees and Mohammadi 1999; Yang et al. 2000; Brandner 2002), as well as facilitation of sustained attention and interval timing (e.g., Meck and Williams 1997a, c; Mohler et al. 2001; Cheng et al. 2006, 2008a, b; Cheng and Meck 2007) compared with offspring of dams fed a standard diet. Choline deficiency during the same developmental time frame, embryonic days (ED) 12–17, results in impaired performance on some, but not all, of these behavioral measures (e.g., Meck and Williams 1999, 2003). Furthermore, perinatal choline supplementation can alter behavior following a variety of developmental disorders, including the alleviation of abnormalities associated with fetal alcohol syndrome in rats (Thomas et al. 2000, 2004, 2007; Wagner and Hunt 2006), attenuation of some of the motor deficits observed in a Mecp21^lox mouse model of Rett syndrome (Nag and Berger-Sweeney 2007), and the improvement of sensory gating in a DBA/2 mouse model of schizophrenia that exhibits reduced numbers of hippocampal a7 nicotinic receptors (Stevens et al. 2008).These choline-induced alterations in cognitive function are accompanied by changes in the size and shape of basal forebrain cholinergic neurons (e.g., Williams et al. 1998; McKeon-O’Malley et al. 2003); modifications in acetylcholine turnover and choline transporter expression in the septum and hippocampus (Cermak et al. 1999; Mellott et al. 2007b); modulation of hippocampal neurogenesis, gene expression, phospholipase D activity, NGF levels, and MAPK and CREB activation (e.g., Holler et al. 1996; Sandstrom et al. 2002; Mellott et al. 2004, 2007a; Glenn et al. 2007); changes in dendritic fields and spine density in CA1 and dentate gyrus (DG) regions of the hippocampus (Meck et al. 2008); as well as modification of the neuropathological response to status epilepticus (e.g., Holmes et al. 2002; Wong-Goodrich et al. 2008a) and thresholds for eliciting long-term potentiation (LTP) in the hippocampus (Pyapali et al. 1998; Jones III et al. 1999). Together, these findings suggest that alterations in choline availability during early development may have specific impact on the ontogeny and later functioning of basal forebrain cholinergic neurons as well as efferent neurons involved in hippocampal LTP (Montoya et al. 2000). These findings also predict that behaviors that rely on the hippocampus are likely to be most affected by this dietary manipulation.Although choline is well known as the precursor for the neurotransmitter acetylcholine, it may be especially crucial to young or developing mammals for a number of other reasons (see Blusztajn and Wurtman 1983; Blusztajn 1998; Zeisel 2000, 2004, 2005). It is the precursor of certain phospholipids (e.g., phosphatidylcholine, sphingomyelin, and plasmenylcholine), which constitute the bulk of phospholipids in all biological membranes. Thus, there may be a particularly high demand for choline during prenatal and neonatal periods associated with rapid neurogenesis and synaptogenesis. Choline can also be enzymatically oxidized to betaine (mostly in peripheral tissues) and the methyl groups of betaine can then be used to resynthesize methionine from homocysteine. Changes in methionine availability alter the methylation of regulatory sequences of genes and of histones, leading to alterations in the patterns of gene expression (e.g., Waterland and Michels 2007; Nafee et al. 2008). Choline is also the precursor of two signaling molecules, platelet-activating factor, and sphingosylphosphorylcholine. Changes in choline availability may also alter membrane synthesis, methylation, and signaling broadly throughout the brain and periphery as well as more restricted effects on cholinergic neuronal pathways (e.g., Zeisel and Blusztajn 1994; Meck and Williams 2003).One common distinction in the Pavlovian-conditioning literature is between tasks that are sensitive to manipulation of the hippocampal formation from those that are not (e.g., Ross et al. 1984; Meck 1988; Schmajuk and Buhusi 1997; Holland et al. 1999). For example, simple excitatory Pavlovian conditioning is typically found to be unaffected by lesions of the hippocampus, while conditional discriminations in which animals must rely on combinations of predictive cues to respond correctly are disrupted by hippocampal damage (e.g., Jarrard and Davidson 1990, 1991). If prenatal choline availability is altering the development of cholinergic neurons in the basal forebrain that project to the hippocampus (see Meck and Williams 2003), our dietary manipulation might only be expected to affect conditioning tasks that require hippocampal involvement, not relatively simple tasks such as excitatory conditioning which do not rely on the hippocampus (e.g., Green and Woodruff-Pak 2000).In the current series of experiments, we examined the effects of prenatal choline supplementation and deficiency using a series of appetitive Pavlovian-conditioning tasks, all of which require associative learning. Our rationale was to determine whether variations in choline availability during prenatal development altered the learning of a simple association between the conditioned (CS) and unconditioned (US) stimuli (e.g., noise → food sequence), or if the dietary manipulation primarily affected conditioning tasks that require more complex relational processing and intact septal-hippocampal function (e.g., context A = tone → food; context B = noise → no food).In order to assess the importance of prenatal choline availability on associative learning, we investigated basic aspects of appetitive Pavlovian conditioning, i.e., conditioned excitation and extinction (e.g., Pavlov 1927) in experiment 1. In this paradigm, rats first receive repeated trials in which the CS occurs just before the presentation of the US, i.e., in a noise → food sequence. During this initial phase of training, the rat develops an increasing tendency to perform the conditioned response (CR) in the presence of the CS indicating that it expects the occurrence of the US. Typically, the probability of the CR increases in a negatively accelerating fashion until it reaches an asymptotic level. If the CS is then repeatedly presented in the absence of the reinforcing US (i.e., noise → no food), then the CR gradually declines; this is referred to as extinction of the CR (Gallistel and Gibbon 2000).One behavioral phenomenon that has been shown to be sensitive to hippocampal manipulation is the discriminative use of contextual cues to control conditioned responding (e.g., Holland and Bouton 1999). Typically, when CS-US pairings occur in one training environment or context, there is a small loss of responding to the CS if it is subsequently presented to the animal in the presence of a different set of contextual cues (e.g., Lovibond et al. 1984; Hall and Honey 1990; Honey et al. 1990; Kaye and Mackintosh 1990). However, this typical decrement in responding with a context switch is not observed in rats with electrolytic or aspiration lesions of the hippocampus (e.g., Good et al. 1997).In order to assess the effects of prenatal choline availability on contextual control of conditioned responding, we employed a renewal design (e.g., Bouton and Bolles 1979) in experiment 2. In this design, rats receive conditioning in one physical context (context A) prior to extinction in either the same context or a context different from that in which they received the initial CS-US pairings (context B). Finally, all of the rats are retested in the original conditioning context (i.e., context A). Bouton and colleagues (e.g., Bouton and Bolles 1979; Frohardt et al. 2000) have found that when rats that are conditioned in context A followed by extinction training in context B are later returned to the original training context for the final testing phase, they show a substantial recovery of the initial CR. Presumably, stimuli contained within the original training context act as reminder cues in this ABA condition, retrieving the memory for the initial acquisition phase (A) of the experiment during the final test phase as opposed to the more recent extinction phase (B). Rats in the AAA condition have no effective cues to discriminate the different phases of the experiment and as a consequence cannot selectively retrieve a specific memory from the sequence. In contrast, test session responding for the ABA condition should be more similar to the low levels observed at the end of the initial extinction phase due to the availability of differential contextual cues. This renewal design is particularly useful in that it provides for the potential to observe treatment effects in both the extinction and the renewal test phases of the experiment. Specifically, either the loss of responding with a context switch during extinction or the response recovery in the renewal test (or both) may be affected by prenatal choline availability. More importantly, those two effects may be due to either the same mechanism (e.g., processing of contextual stimuli) or two different mechanisms (e.g., context conditioning and memory retrieval)—potentially resulting in nonlinear effects of prenatal choline availability across the two experimental phases.A second basic type of associative learning, conditioned inhibition, in which the animal learns to predict the absence of an important event, was described by Pavlov (1927). A typical conditioned-inhibition task consists of training with two types of intermixed trials: On reinforced trials, one CS is followed by reinforcement (e.g., noise → food). On other trials, the same CS is paired with a second stimulus in the absence of the reinforcement (i.e., light/noise → no food). It is presumed that under these training conditions animals learn that the noise predicts the occurrence of the food, while light, the “conditioned inhibitor,” comes to predict the absence of food. That is, light “inhibits” the learned response to noise alone.A relatively small number of studies have examined the neural substrates of inhibitory learning. Aspiration lesions of the hippocampus, for example, impaired a relatively complex phenomenon called “blocking” of excitatory conditioning, but not the learning of conditioned inhibition (e.g., Solomon 1977; Chan et al. 2001). These data suggest that the hippocampal complex is not required for learning conditioned inhibition. Thus, in order to further assess whether prenatal choline availability affects basic associative learning, experiment 3 was designed to evaluate conditioned inhibition in supplemented (SUP), deficient (DEF), and control (CON) rats. In this experiment, rats were given randomly mixed presentations of reinforced and nonreinforced trial types. As training proceeds, the rats should learn to respond more on reinforced trials than on nonreinforced trials. After acquisition of the discrimination, the rats were presented with a retardation test phase in which the inhibitory light CS was paired with food. Rescorla (1969) described this retardation test as one of the critical measures of conditioned inhibition. Presumably, if the CS is a true inhibitor and predicts the absence of reinforcement at the outset of the retardation test, then acquisition of conditioned responding to the cue should be relatively slow during this phase of testing. Consequently, tests of conditioned inhibition should distinguish among prenatal choline treatment groups if inhibitory mechanisms are strengthened or weakened by prenatal choline availability.