Acquisition of response thresholds for timed performance is regulated by a calcium-responsive transcription factor, CaRF.

Interval timing within the seconds-to-minutes range involves the interaction of the prefrontal cortex and basal ganglia via dopaminergic-glutamatergic pathways. Because the secreted protein brain-derived neurotrophic factor (BDNF) is able to modulate dopamine release as well as glutamatergic activity, we hypothesized that BDNF may be important for these timing mechanisms. Recently, the calcium-responsive transcription factor (CaRF) was identified as an important modulator of BDNF expression in the cerebral cortex. In this study, a strain of Carf knockout mice was evaluated for their ability to acquire the 'Start' and 'Stop' response thresholds under sequential and simultaneous training conditions, using multiple (15-second and 45-second) or single (30-second) target durations in the peak-interval procedure. Both Carf(+/-) and Carf(-/-) mice were impaired in their ability to acquire timed response thresholds relative to Carf(+/+) mice. Additionally, control mice given microinjections of BDNF antisense oligodeoxynucleotide to inhibit protein expression in the prefrontal cortex showed timing impairments during acquisition similar to Carf mice. Together, these results suggest that the inhibitory processes required to update response thresholds and exert temporal control of behavior during acquisition may be dependent on CaRF regulation of genes including Bdnf in cortico-striatal circuits.

Quinpirole-induced sensitization to noisy/sparse periodic input: temporal synchronization as a component of obsessive-compulsive disorder.

Quinpirole-sensitized rats were tested on a discrete-trials 40-s peak-interval procedure using lever pressing as the instrumental response. Although there was no evidence of rhythmical activity in lever pressing, periodic output was observed in a secondary response (food-cup entries) during the inter-trial interval following the delivery of reinforcement on fixed-interval trials, but not during unreinforced probe trials. This repetitive pattern of behavior with a 40-s period points to the primacy of reinforcement as a time marker and an increased tendency to synchronize to noisy and sparse periodic input as a result of reduced inhibitory control in cortico-striatal circuits following chronic quinpirole administration. Parallels between quinpirole-induced rhythmical behavior and the repetitive motor habits frequently observed in obsessive-compulsive disorder are discussed.

Paying attention to time as one gets older.

Age-related changes in attention and interval timing as a function of time of day were examined using a temporal bisection task with single and compound auditory and visual stimuli. Half of the participants in each age group were tested in the morning, and half were tested in the afternoon. Duration judgments were found to be shorter for visual signals than for auditory signals. This discrepancy was greater in the morning than in the afternoon and larger for the older than for the younger adults. Young adults showed equal sensitivity to signal duration for single and compound trials and higher sensitivity in the afternoon than in the morning for both signal modalities. In contrast, older adults showed impaired sensitivity on compound trials and the greatest sensitivity overall to single visual trials in the morning. These results suggest that age-related reductions in attentional resources may cause older adults to focus on signals that require controlled attention during specific phases of the circadian cycle.

Differential effects of auditory and visual signals on clock speed and temporal memory.

The effects of signal modality on duration classification in college students were studied with the duration bisection task. When auditory and visual signals were presented in the same test session and shared common anchor durations, visual signals were classified as shorter than equivalent duration auditory signals. This occurred when auditory and visual signals were presented sequentially in the same test session and when presented simultaneously but asynchronously. Presentation of a single modality signal within a test session, or both modalities but with different anchor durations did not result in classification differences. The authors posit a model in which auditory and visual signals drive an internal clock at different rates. The clock rate difference is due to an attentional effect on the mode switch and is revealed only when the memories for the short and long anchor durations consist of a mix of contributions from accumulations generated by both the fast auditory and slower visual clock rates. When this occurs auditory signals seem longer than visual signals relative to the composite memory representation.

Prenatal choline exposure alters hippocampal responsiveness to cholinergic stimulation in adulthood.

Manipulation of dietary choline levels during gestation results in enduring neurobehavioral changes in offspring that last into adulthood. Alterations of hippocampal function and memory are among the most striking changes. Depending upon the measures assessed, prenatal choline supplementation tends to promote excitatory synaptic efficacy in hippocampal circuits while prenatal choline deficiency diminishes it. However, the mechanisms underlying these changes remain unclear. Transverse hippocampal slices were prepared from adult offspring of dams fed choline supplemented, choline deficient, or control diets. We assessed paired-pulse inhibition, and excitatory synaptic responsiveness before and after activation of cholinergic receptors with Carbachol. Prenatally choline deficient animals yielded significantly fewer electrophysiological viable hippocampal slices than did animals from either of the other two treatment groups. Among the slices tested, there were no differences in paired pulse inhibition between the treatment groups. However, transient cholinergic activation resulted in a prolonged enhancement of the amplitude of the population EPSP (pEPSP) response in slices from prenatally choline supplemented animals. These results suggest that GABA receptor-mediated inhibition remains intact after prenatal choline manipulations, and that enhancement of the excitatory responsiveness of hippocampal circuits in slices from prenatally choline supplemented rats may be related in part to an increase in cholinergic tone within the CA1 circuit.

Timing for the absence of a stimulus: the gap paradigm reversed.

Contrary to data showing sensitivity to nontemporal properties of timed signals, current theories of interval timing assume that animals can use the presence or absence of a signal as equally valid cues as long as duration is the most predictive feature. Consequently, the authors examined rats' behavior when timing the absence of a visual or auditory stimulus in trace conditioning and in a "reversed" gap procedure. Memory for timing was tested by presenting the stimulus as a reversed gap into its timed absence. Results suggest that in trace conditioning (Experiment 1), rats time for the absence of a stimulus by using its offset as a time marker. As in the standard gap procedure, the insertion of a reversed gap was expected to "stop" rats' internal clock. In contrast, a reversed gap of 1-, 5-, or 15-s duration "reset" the timing process in both trace conditioning (Experiment 2) and the reversed gap procedure (Experiment 3). A direct comparison of the standard and reversed gap procedures (Experiment 4) supported these findings. Results suggest that attentional mechanisms involving the salience or content of the gap might contribute to the response rule adopted in a gap procedure.

Neuropsychological mechanisms of interval timing behavior.

Interval timing in the seconds-to-minutes range is believed to underlie a variety of complex behaviors in humans and other animals. One of the more interesting problems in interval timing is trying to understand how the brain times events lasting for minutes with millisecond-based neural processes. Timing models proposing the use of coincidence-detection mechanisms (e.g., the detection of simultaneous activity across multiple neural inputs) appear to be the most compatible with known neural mechanisms. From an evolutionary perspective, coincidence detection of neuronal activity may be a fundamental mechanism of timing that is expressed across a wide variety of species. BioEssays 22:94-103, 2000.

Choline supplementation during prenatal development reduces proactive interference in spatial memory.

Previous research has demonstrated that increasing dietary choline during early development can have long-lasting effects on cholinergic (Ch) function that are correlated with improvement of spatial memory ability in rats. The present study is designed to further our understanding of these organizational changes in brain and behavior by examining the effects of spaced vs. massed trials. A third of the rats (n=10) were supplemented with choline chloride prenatally by adding it to the drinking water of their dams. Another third were made deficient of choline during early development by removing choline from the dams diet. The remaining rats served as untreated controls. Postnatally, the offspring were maintained on a choline-sufficient diet and at 120 days of age they began 12-arm radial maze training. The maze data revealed two major effects of early choline availability: (1) Both choline-supplemented and choline-deficient rats performed more accurately than control littermates when trials were spaced. These differences in spatial ability did not appear to be a function of differential response or cue-use strategies. (2) Choline-supplemented rats showed little proactive interference when trials were massed; whereas control rats demonstrated moderate levels and choline-deficient rats exhibited high levels of proactive interference as a function of massed trials. These data suggest that the behavioral consequences of early dietary availability of choline may involve the modification of the discriminative abilities used to attend to stimuli that demarcate the end of one trial and the start of another as well as the capacity for remembering the locations that have been visited during a trial.

Choline availability to the developing rat fetus alters adult hippocampal long-term potentiation.

Supplementation with choline during pregnancy in rats causes a long-lasting improvement of visuospatial memory of the offspring. To determine if the behavioral effects of choline are related to physiological changes in hippocampus, the effect of perinatal choline supplementation or deficiency on long-term potentiation (LTP) was examined in hippocampal slices of 6-8 and 12-14 month old rats born to dams consuming a control, choline-supplemented, or a choline-free diet during pregnancy. Stimulating and recording electrodes were placed in stratum radiatum of area CA1 to record extracellular population excitatory postsynaptic potentials (pEPSPs). To induce LTP, a theta-like stimulus train was generated. The amplitude of the stimulus pulses was set at either 10% or 50% of the stimulus intensity which had induced the maximal pEPSP slope on the input/output curve. We found that at both ages, a significantly smaller percentage of slices from perinatally choline-deficient rats displayed LTP after 10% stimulus intensity (compared with control and choline-supplemented rats), and a significantly larger percentage of slices from choline-supplemented rats displayed LTP at 50% stimulus intensity (compared with control and choline-deficient rats). Results reveal that alterations in the availability of dietary choline during discrete periods of development lead to changes in hippocampal electrophysiology that last well into adulthood. These changes in LTP threshold may underlie the observed enhancement of visuospatial memory seen after prenatal choline supplementation and point to the importance of choline intake during pregnancy for development of brain and memory function.

The use of timing behaviors in animals and humans to detect drug and/or toxicant effects.

Behavioral paradigms applicable for use in both human and nonhuman subjects for investigating aspects of timing behavior are presented with a view towards exploring their strengths, weaknesses, and utility in a variety of experimental situations. Tri-peak, peak interval, differential reinforcement of low rate responding, and temporal response differentiation procedures are highlighted. In addition, the application of timing tasks in preclinical and clinical settings is discussed: pharmacological manipulations are providing information on the neurotransmitters involved and species differences; normative data for children are being developed; and noninvasive imaging procedures are being employed in adult human subjects to explore the involvement of specific brain areas.

Prenatal availability of choline alters the development of acetylcholinesterase in the rat hippocampus.

Choline (Ch) supplementation during embryonic days (ED) 12-17 enhances spatial and temporal memory in adult and aged rats, whereas prenatal Ch deficiency impairs attention performance and accelerates age-related declines in temporal processing. To characterize the neurochemical and neuroanatomical mechanisms that may mediate these behavioral effects in rats, we studied the development [postnatal days (PD) 1, 3, 7, 17, 27, 35, 90, and 26 months postnatally] of acetylcholinesterase (AChE) activity in hippocampus, neocortex and striatum as a function of prenatal Ch availability. We further measured the density of AChE-positive laminae (PD27 and PD90) and interneurons (PD20) in the hippocampus as a function of prenatal Ch availability. During ED11-ED17 pregnant Sprague-Dawley rats received a Ch-deficient, control or Ch-supplemented diet (average Ch intake 0, 1.3 and 4.6 mmol/kg/day, respectively). Prenatal Ch deficiency increased hippocampal AChE activity as compared to control animals in both males and females from the 2nd to 5th week postnatally. Moreover, prenatal Ch supplementation reduced hippocampal AChE activity as compared to control animals over the same developmental period. There was no effect of prenatal Ch status on either cortical or striatal AChE activity at any age measured, and by PD90 the effect of Ch on hippocampal AChE was no longer observed. In order to localize the early changes in hippocampal AChE activity anatomically, frozen coronal brain sections (PD20, PD27, PD90) were stained histochemically for AChE. Consistent with biochemical results, the AChE staining intensity was reduced in PD27 hippocampal laminae in the Ch-supplemented group and increased in the Ch-deficient group compared to control animals. There was no effect of the diet on hippocampal AChE staining intensity on PD90. In addition, the prenatal Ch availability was found to alter the size and density of AChE-positive PD20 interneurons. These results show that prenatal Ch availability has long-term consequences on the development of the hippocampal cholinergic system.

Reinforcement-induced within-trial resetting of an internal clock.

Rats were trained on a modified peak-interval timing procedure in which three response levers were individually associated with different criterion durations (10, 30 and 90 s) following the onset of a tone stimulus. Delivery of response-dependent reinforcement for each duration was independent of both the responding and the delivery of reinforcement for the other durations, such that the tone stimulus stayed on during food delivery, and no change in the primed reinforcement times occurred. We report here that the delivery of reinforcement at an earlier criterion duration produces a rightward shift in the temporal response functions for later durations by the amount of time that has already elapsed in the trial. This reset in temporal accumulation produces large discrepancies between the programmed and expected (peak) times of reinforcement and suggests that rats are unable to make conditional reinforcement-based discriminations of interval duration.

Transdermal nicotine effects on attention.

Nicotine has been shown to improve attentiveness in smokers and attenuate attentional deficits in Alzheimer's disease patients, schizophrenics and adults with attention-deficit/hyperactivity disorder (ADHD). The current study was conducted to determine whether nicotine administered via transdermal patches would improve attentiveness in non-smoking adults without attentional deficits. The subjects underwent the nicotine and placebo exposure in a counterbalanced double-blind manner. Measures of treatment effect included the Profile of Mood States (POMS), Conners' computerized Continuous Performance Test (CPT) of attentiveness and a computerized interval-timing task. The subjects were administered a 7 mg/day nicotine transdermal patch for 4.5 h during a morning session. Nicotine significantly increased self-perceived vigor as measured by the POMS test. On the CPT, nicotine significantly decreased the number of errors of omission without causing increases in either errors of commission or correct hit reaction time. Nicotine also significantly decreased the variance of hit reaction time and the composite measure of attentiveness. This study shows that, in addition to reducing attentional impairment, nicotine administered via transdermal patches can improve attentiveness in normal adult non-smokers.

Coupled temporal memories in Parkinson's disease: a dopamine-related dysfunction.

Dysfunction of the basal ganglia and the brain nuclei interconnected with them leads to disturbances of movement and cognition, including disordered timing of movement and perceptual timing deficits. Patients with Parkinson's disease (PD) were studied in temporal reproduction tasks. We examined PD patients when brain dopamine (DA) transmission was impaired (OFF state) and when DA transmission was reestablished, at the time of maximal clinical benefit following administration of levodopa + apomorphine (ON state). Patients reproduced target times of 8 and 21 sec trained in blocked trials with the peak interval procedure, which were veridical in the ON state, comparable to normative performance by healthy young and aged controls (Experiment 1). In the OFF state, temporal reproduction was impaired in both accuracy and precision (variance). The 8-sec signal was reproduced as longer and the 21-sec signal was reproduced as shorter than they actually were (Experiment 1). This "migration" effect was dependent upon training of two different durations. When PD patients were trained on 21 sec only (Experiment 2), they showed a reproduction error in the long direction, opposite to the error produced under the dual training condition of Experiment 1. The results are discussed as a mutual attraction between temporal processing systems, in memory and clock stages, when dopaminergic regulation in the striatum is dysfunctional.

Hypertrophy of basal forebrain neurons and enhanced visuospatial memory in perinatally choline-supplemented rats.

The effects of choline supplementation during two time-frames of early development on radial-arm maze performance and the morphology of basal forebrain neurons immunoreactive for the P75 neurotrophin receptor (NTR) in male and female Sprague-Dawley rats were examined. In the first experiment, rats were supplemented with choline chloride from conception until weaning. At 80 days of age, subjects were trained once a day on a 12-arm radial maze for 30 days. Compared to control littermates, supplemented rats made fewer working and reference memory errors; however, the memory enhancing effects of choline supplementation were greater in males than females. A morphometric analysis of NTR-immunoreactive cell bodies at three levels through the medial septum/diagonal band (MS/DBv) of these rats revealed that perinatal choline supplementation caused the somata of cells in the MS/DBv to be larger by 8-15%. In a second experiment, choline supplementation was restricted to embryonic days 12-17. A developmental profile of NTR immunoreactive cell bodies in the MS/DBv of 0-, 8-, 16-, 30- and 90-day old male and female rats again revealed that cell bodies were larger in choline-supplemented rats than controls. As in the behavioral studies, the effect of choline supplementation was greater in male than female rats. These data are consistent with the hypothesis that supplementation with choline chloride during early development leads to an increase in the size of cell bodies of NTR-immunoreactive cells in the basal forebrain and that this change may contribute to long-term improvement in spatial memory.

Prenatal dietary choline supplementation decreases the threshold for induction of long-term potentiation in young adult rats.

Choline supplementation during gestation in rats leads to augmentation of spatial memory in adulthood. We hypothesized that prenatal (E12-E17) choline supplementation in the rat would lead to an enhancement of hippocampal synaptic plasticity as assessed by long-term potentiation (LTP) at 3-4 mo of age. LTP was assessed blindly in area CA1 of hippocampal slices with first suprathreshold (above threshold for LTP generation in control slices) theta-burst stimulus trains. The magnitude of potentiation after these stimuli was not different between slices from control and prenatally choline supplemented animals. Next, threshold (reliably leading to LTP generation in control slices) or subthreshold theta-burst stimulus trains were applied to slices from control, prenatally choline-supplemented, and prenatally choline-deprived rats. Threshold level stimulus trains induced LTP in slices from both the control and choline-supplemented rats but not in those from the choline-deficient rats. Subthreshold stimulus trains led to LTP induction in slices from prenatally choline-supplemented rats only. These observations indicate that prenatal dietary manipulation of the amino acid, choline, leads to subsequent significant alterations of LTP induction threshold in adult animals.

Scalar expectancy theory and peak-interval timing in humans.

The properties of the internal clock, temporal memory, and decision processes used to time short durations were investigated. The peak-interval procedure was used to evaluate the timing of 8-, 12-, and 21-s intervals, and analyses were conducted on the mean response functions and on individual trials. A distractor task prevented counting, and visual feedback on accuracy and precision was provided after each trial. Mean response distributions were (a) centered at the appropriate real-time criteria, (b) highly symmetrical, and (c) scalar in their variability. Analysis of individual trials indicated more memory variability relative to response threshold variability. Taken together, these results demonstrate that humans show the same qualitative timing properties that other animals do, but with some quantitative differences.

Characterization of the facilitative effects of perinatal choline supplementation on timing and temporal memory.

Perinatal choline supplementation can improve performance on a variety of spatial memory tasks in adulthood. In order to extend these studies, we have investigated the effects of perinatal choline supplementation on the performance of a peak-interval timing task in which a 20 s temporal criterion was trained for a visual signal in adult (3-6 months old) rats. Following 5 weeks of baseline training, rats received systemic injections of nicotine (0.1, 0.2, or 0.4 mg/kg, s.c.) or saline prior to testing on the peak-interval timing task. The results indicated that perinatal choline supplementation increased rats' sensitivity to the 20 s temporal criterion during baseline training and facilitated the clock speed enhancing effects of nicotine during drug testing. The present study extends the types of long-term cognitive enhancement produced by perinatal choline supplementation to include the temporal processing domain and relates these effects to modifications in cholinergic function.

Simultaneous temporal processing is sensitive to prenatal choline availability in mature and aged rats.

Rats were trained at 2-4 months and at 24-26 months of age on a peak-interval timing procedure in which auditory and visual stimuli signaled two different fixed-interval schedules of reinforcement (15 and 30 s) that were presented simultaneously in a hierarchical fashion. Compared with control rats, increases in the probability of attention to the 15 s signal were observed for both the choline-supplemented and the choline-deficient rats. In contrast, an increase in attention to the 30 s signal was only observed for the choline-supplemented rats, whereas choline-deficient rats exhibited a decrease in attention that increased with age. Proportional rightward shifts in the remembered times of reinforcement emerged for the 24-26-month-old rats in the choline-deficient and control groups, but not in the choline-supplemented group. These results indicate that prenatal choline supplementation facilitates cognitive function across the lifespan, whereas prenatal choline deficiency impairs divided attention and accelerates age-related declines in temporal processing.

Perinatal choline supplementation increases the threshold for chunking in spatial memory.

Chunking and perinatal choline supplementation each provide rats with alternative memory processing advantages. Evidence from radial-arm maze performance of adult (2- to 5-month-old) rats indicates that chunking of multiple food types (sunflower seeds, Noyes pellets and rice puffs) emerges for stable, differentiable baiting patterns as a function of the memory load (6, 12, 18 or 24 maze arms). The number of maze arms appeared to determine both the level of task difficulty at which rats began to implement a chunking strategy as well as when they were unable to successfully implement such a strategy due to the excess memorial demands of the task. In comparison to control rats, rats treated perinatally with choline supplementation displayed a horizontal rightward shift of the response function that related level of clustering of like-food types to the number of maze arms. These results indicate a higher threshold for implementing a chunking strategy in rats treated perinatally with choline supplementation, possibly due to a choline-induced increase in memory capacity.