reversible inactivations of rat medial prefrontal ... - Semantic Scholar

Neuroscience 139 (2006) 865– 876

REVERSIBLE INACTIVATIONS OF RAT MEDIAL PREFRONTAL CORTEX IMPAIR THE ABILITY TO WAIT FOR A STIMULUS N. S. NARAYANAN,a,b N. K. HORSTa,b AND MARK LAUBACHa,c*

delay-dependent speeding is thought to be due to a process of temporal anticipation based on the increasing probability that the stimulus will occur during the foreperiod (Näätänen, 1970, 1971, 1972). Learning to perform a simple RT task with variable foreperiods would therefore involve learning the timing of trigger stimuli. Excitotoxic lesions of prefrontal regions in rats increase premature responding in simple RT tasks (Broersen and Uylings, 1999; Risterucci et al., 2003). For example, Risterucci et al. (2003) found a profound increase in premature responses following lesions of the prefrontal cortex. The effects of the lesions were temporary; that is, after about six behavioral sessions following surgery, the animals’ behavior returned to normal. Moreover, changing the timing of the trigger stimuli reinstated increased premature responding. In the present study, we attempt to replicate and extend the results of Risterucci et al. (2003) by using muscimol (Lomber 1999; Martin and Ghez, 1999) to reversibly inactivate the rat prefrontal cortex during simple RT tasks. Reversible inactivations allowed us to use a within-subjects design instead of the between-subjects design that is required in lesion studies. In addition, we used a novel compound for reversible inactivation, fluorescent muscimol, which enabled us to visualize the anatomical extent of the inactivations. This project focused on dorsomedial prefrontal cortex (dmPFC), which includes dorsal prelimbic (area 32) and anterior cingulate areas (Cg1 or area 24a; Swanson, 1999), and is distinct from ventral areas of medial prefrontal cortex (including ventral prelimbic and infralimbic areas; Sesack et al., 1989; Heidbreder and Groenewegen, 2003; Vertes, 2004; Milad et al., 2004; Gabbott et al., 2005). The dmPFC is involved in functions such as working memory, attention, response initiation, and management of autonomic control and emotion (Groenewegen and Uylings, 2000; Uylings et al., 2003). These functions are also subserved by primate prefrontal cortex (Fuster, 2000; Miller and Cohen, 2001). Additionally, primate prefrontal regions have been implicated in RT performance (Niki and Watanabe, 1979; Sawaguchi, 1987; Naito et al., 2000; Stuss et al., 2005). Risterucci et al. (2003) interpreted the effects of lesions of rodent prefrontal cortex in simple RT tasks as a deficit in response preparation, which might be due to impairments in attentional processing, time estimation, or inhibitory control of response execution. However, in the task used by these authors, these processes are not separable. To address these issues, we reversibly inactivated dmPFC during performance of RT tasks with variable or fixed

a The John B. Pierce Laboratory, 290 Congress Avenue, New Haven, CT 06519, USA b Interdepartmental Neuroscience Program, Yale School of Medicine, New Haven, CT 06520, USA c

Department of Neurobiology, Yale School of Medicine, New Haven, CT 06510, USA

Abstract—In simple reaction time tasks, lesions of rat dorsomedial prefrontal cortex impair the ability to wait for trigger stimuli and result in increased premature responding. This effect could be due to impairments in attending to trigger stimuli, estimating the timing of trigger stimuli, or inhibitory control of the motor response. Here, we examined these issues by reversibly inactivating dorsomedial prefrontal cortex during simple reaction time tasks with variable or fixed foreperiods. There were three consistent effects of dorsomedial prefrontal cortex inactivation: 1) increased premature responding, 2) increased variability in the timing of premature responses, and 3) speeded response latencies, especially on trials with short foreperiods in tasks with variable foreperiods. We observed these effects independent of differences in foreperiod duration, foreperiod variability, and stimulus probabilities. Therefore, dorsomedial prefrontal cortex appears not to be involved in attending to the trigger stimulus or in time estimation. Instead, we suggest that dorsomedial prefrontal cortex is critical for inhibiting responses before the maximum foreperiod duration, i.e. the “deadline” [Ollman RT, Billington MJ (1972) The deadline model for simple reaction times. Cognit Psychol 3:311–336], after which the rat should respond even if the trigger stimulus has not occurred. © 2006 Published by Elsevier Ltd on behalf of IBRO. Key words: reaction time, foreperiod, attention, preparation, timing, waiting.

In a typical simple reaction time (RT) task, subjects must withhold responses while waiting for a trigger stimulus over a variable delay period, or foreperiod, after which they must respond as fast as possible (Luce, 1986). As rats learn such a task, they struggle to withhold responses during the foreperiod prior to the stimulus (Laubach et al., 2000). Eventually, rats learn to wait for the stimulus. They also come to demonstrate knowledge about the timing of stimuli in the task by responding the fastest on trials with long foreperiods (e.g. Brown and Robbins, 1991). Such *Correspondence to: M. Laubach, The John B. Pierce Laboratory, 290 Congress Avenue, New Haven, CT 06519, USA. Tel: ⫹1-203-4016202; fax: ⫹1-203-624-4950. E-mail address: [email protected] (M. Laubach). Abbreviations: dmPFC, dorsomedial prefrontal cortex; RT, reaction time. 0306-4522/06$30.00⫹0.00 © 2006 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.11.072

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foreperiods. A fixed foreperiod RT task should require reduced attentional processing relative to a RT task with variable foreperiods, as there is no uncertainty about the occurrence of the trigger stimuli. Therefore, if dmPFC mediates attentional processing, we would expect to find that dmPFC inactivation produces more premature responses in the variable foreperiod RT task relative to the fixed foreperiod RT task. By contrast, if dmPFC is not involved in attending to the impending trigger stimulus, we would expect to find that bilateral dmPFC inactivation results in equivalent premature responding in both tasks.

EXPERIMENTAL PROCEDURES Experiment 1: effects of dmPFC inactivation on simple RT performance with variable foreperiods Subjects. Eleven rats (aged 5–10 months; Brown-Norway⫽6; Long-Evans⫽5) were trained to perform simple RT tasks with variable foreperiods. Rats were motivated by water restriction, while food was available ad libitum. Rats received 10 –15 ml of water during each behavioral session. Additional water (5–10 ml) was provided 1–3 h after each behavioral session in the home cage. Rats were maintained at ⬃90% of their free-access body weights during the course of these experiments, and received one day of free access to water per week. All procedures were approved by the Animal Care and Use Committee at the John B. Pierce Laboratory and conformed to the standards of the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were taken to minimize the number of animal subjects in this study and reduce subject pain and suffering. Brown-Norway rats are a common model system for studies of aging (Sprott and Austad, 1995; Grill and Riddle, 2002). The animals used here were from a study comparing age-related issues in learning RT tasks (M. Laubach and C. Akirav, unpublished observations). The use of two strains did not impact the results we obtained in the present study. No differences were observed between the Long-Evans and Brown-Norway rats, and data summaries were collapsed across strains (see Results). Behavioral apparatus. Operant chambers (MedAssociates, St. Albans, VT, USA) were equipped with a lever, a drinking tube, and a speaker driven to produce an 8 kHz tone at 70 dBA, using audio equipment either from Tucker-Davis Technologies (Alachua, FL, USA) or manufactured in the Instruments Shop at the Pierce Laboratory. The audio equipment was controlled by custom-written scripts for the Data Acquisition Toolbox for Matlab (Mathworks, Natick, MA, USA). Each chamber contained a house light, which was turned on at the beginning of each session, and a fan, which produced broadband noise at approximately 60 dBA. Behavioral arenas were housed in sound-attenuating chambers (MedAssociates). Water was delivered via a pump (MedAssociates) connected to a standard metal drinking tube (Ancare, Bellmore, NY, USA) via Tygon tubing. Each conditioned response in the task activated the pump at 0.03 ml/s for 1–2 s, depending on the stage of training. Behavior was monitored during all sessions via a closed circuit video camera (Allied Electronics, Fort Worth, TX, USA), mounted in each behavioral arena, and was recorded to video tape in some sessions. Finally, in some sessions, a strain gauge (Omega Engineering, Stamford, CT, USA) was used to measure lever force during a behavioral session. Behavioral training. Rats had restricted access to water in the home cage for three days, during which they were provided with limited access (20 ml of water) at the time of day when behavioral training was subsequently carried out. A conditioning

session was then run in which an audible click stimulus (generated via a mechanical relay, Potter & Brumfield, Winston-Salem, NC, USA) was paired with activation of the water pump, which was active for 3 s every 5–20 s over 50 min. Next, animals were shaped by the method of successive approximations to depress the response lever (⬃0.15 N), which was located immediately below the drinking tube in the first training session. Each press of the lever activated the pump for 2.5 s (Volume per trial: 0.075 ml), during which time additional lever presses were not rewarded. In subsequent sessions, the lever was moved gradually to the right of the drinking tube to encourage the rats to press the lever with the right paw and the pump was activated by the release of the lever, instead of the press. Finally, the animals were trained to perform a “temporally conditioned” lever-press task, in which they were required to press on the lever over increasing periods of time (i.e. from 100 ms to 800 ms in steps of 100 ms after every three consecutive conditioned responses) and to release the lever within 2 s in order to collect a liquid reward. Response durations less than the target interval or longer than the target plus 2 s were followed by a timeout period (4 – 8 s). Rats learned to make sustained lever presses on at least 60% of trials in 5–15 sessions. Over the course of training, pump time was decreased to 1.0 s (volume per trial: 0.03 ml). Simple RT task with variable foreperiods. Each trial in the simple RT task was initiated by the rat depressing the lever and ended when the lever was released. The period between trial initiation (lever press) and when reward became available, indicated by a trigger stimulus, was called the foreperiod. Temporally conditioned responses occurred when the rats successfully maintained a lever press for the full foreperiod duration and released the lever with short response latencies (i.e. within a response window of 1000 ms after the trigger stimulus). These responses were followed by liquid rewards. Premature responses occurred when rats did not maintain a lever press for the full foreperiod. Late responses occurred when the lever was released after the response window. These latter two types of trials were followed by a timeout period, in which all experimental stimuli were extinguished for 4 – 8 s. Lever pressing during the timeout period counted as a Timeout Lever Press, which reset the timer controlling the duration of the timeout period. After initial training, six rats (Brown-Norway) were trained to perform the simple RT task with variable foreperiods (Fig. 2A) between 500 ms and 1000 ms in four steps (500 ms, 666 ms, 833 ms, and 1000 ms, 0 –25 ms jitter; foreperiod mean⫾STD: 750⫾215 ms; the foreperiod duration was chosen pseudo-randomly on each trial). At the end of the foreperiod, the trigger stimulus (8000 Hz tone, 70 dBA) was presented for 100 ms and rats had to release the lever within 1000 ms of stimulus onset to obtain a reward. Five rats (Long-Evans) were initially trained to perform a simple RT task with longer foreperiods that ranged between 900 and 1400 ms in four steps (900 ms, 1066 ms, 1233 ms, and 1400 ms, 0 –25 ms jitter; foreperiod mean⫾STD: 1150⫾215 ms; the foreperiod duration was chosen pseudo-randomly on each trial) and tested with dmPFC inactivation in this task. They were then retrained over 10 sessions in the simple RT task with foreperiods between 500 and 1000 ms before being tested again with dmPFC inactivation. Therefore, these five rats had four total infusions per site (two saline, two muscimol, 10 days between muscimol infusions), while other rats in this study had only two infusions per site (saline and muscimol). Surgery. Anesthesia was initiated with ⬃4% halothane and maintained with intraperitoneal injections of either pentobarbital (50 mg/kg) or intramuscular injections of ketamine (100 mg/kg) and diazepam (10 mg/kg). A surgical level of anesthesia was maintained over the course of surgery with supplements (30 mg/ kg) of ketamine every 45 min–1 h. Under aseptic conditions, the


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Fig. 1. Reversible inactivation of bilateral dmPFC. (A) Location of cannulae placed bilaterally in the dmPFC of all nineteen animals. Three animals had cannulae targeting rostral anterior cingulate cortex (white circles), while 16 others had cannulae targeting dorsal prelimbic cortex (black circles). Dorsal prelimbic cortex coordinates were used for animals in all other experiments; injectors protruded 0.2 mm beyond cannulae tips. Template adapted from Swanson (1999); top section ⫹3.2 anteroposterior from bregma, bottom section ⫹2.8 anteroposterior from bregma. (B) Infusion of fluorescent muscimol (bright region) via cannulae into the dmPFC of one animal performing in experiment 2. See Fig. 5B.

scalp was retracted, and the skull was leveled between bregma and lambda. Craniotomies were made bilaterally above the target cannulae sites. Twenty-six-gauge cannulae (Plastics One, Roanoke, VA, USA) with stylettes cut flush to the cannulae tips were lowered slowly into bilateral dmPFC (targeting dorsal prelimbic cortex; coordinates from bregma: anteroposterior ⫹3.2, mediolateral ⫾1.4, dorsoventral ⫺3.7 at 12° in the frontal plane as measured stereotaxically; Fig. 1A). After guide cannulae were placed, craniotomies were sealed with cyanoacrylate (SloZap, Pacer Technologies, Rancho Cucamonga, CA, USA) accelerated by ZipKicker (Pacer Technologies), and methyl methacrylate (i.e. dental cement; AM Systems, Port Angeles, WA, USA). Rats were given

Drug infusion. After at least one week of recovery from surgery, rats were water restricted and behavioral sessions were resumed. After demonstrating stable post-operative RT performance (3 days’ behavior at ⬎60% conditioned responses), rats were acclimated to the infusion procedure by performing behavioral tasks 45 min after being lightly anesthetized for ⬍10 min with 4% halothane. Although there was some damage associated with placement of guide cannulae in dmPFC (Fig. 1B), there was no effect on subsequent behavior in the RT tasks. In order to reversibly inactivate brain regions, we used muscimol, a GABA-A agonist (Lomber, 1999; Martin and Ghez, 1999). Testing with muscimol was done as follows: on day 1 rats were tested with halothane and saline infusion into the cannulae (control sessions), on day 2 rats were tested with halothane and muscimol (or fluorescent muscimol) infusion into the cannulae (dmPFC inactivation sessions), and on day 3 the rats were tested without any manipulation (recovery sessions). We observed no deleterious effects in the recovery sessions (see Results). Infusions of muscimol were made with a 33-gauge injector (Plastics One) that protruded 0.2 mm from the tip of the guide cannula. Prior to injections, rats were lightly anesthetized using halothane (as above), injectors were inserted into the guide cannula, and 0.5 ␮l of infusion fluid was delivered per site at a rate of 15 ␮l/h (0.25 ␮l/min; Martin and Ghez, 1999) via a syringe infusion pump (KDS Scientific, Holliston, MA, USA). The infusion fluid was sterile saline (0.9%; Phoenix Scientific, St. Joseph, MO, USA) or muscimol at 1 ␮g/␮l (Sigma-Aldrich, St. Louis, MO, USA). Fluid was infused via 0.38 mm diameter polyethylene tubing (Intramedic, New York, NY, USA) joined to the injector on one end and to a 10 ␮l Hamilton syringe (Hamilton, Reno, NV, USA) on the other end. Injections were confirmed by monitoring movement of fluid in the tubing via a small bubble. After injection was complete, the injector was left in place for 2 min to allow diffusion of fluid. Rats were tested in the RT task 45 min after infusion. This amount of time was sufficient to allow for full recovery from the halothane anesthesia, for muscimol to reach its maximal effectiveness in inhibiting cortical activity, and to allow for stationary behavioral effects. Previous physiological studies have suggested that the effects of muscimol begin almost immediately and are stable for many hours (Hikosaka and Wurtz, 1985; Krupa et al., 1999). Rats were infused first in either right or left dmPFC (chosen randomly), and then infused in the contralateral location; consequently, the contralateral infusion was completed 4 min (2 min for infusion, and then 2 min to allow diffusion) after the initial infusion was complete. Preliminary experiments with infusion of Evans-Blue dye or with infusion of fluorescent muscimol suggested that at 45 min and at 1 h and 15 min post-infusion (for fluorescent muscimol experiments; Fig. 1B), infused volume was equivalent bilaterally in dmPFC; that is, it did not vary with whether right or left dmPFC was initially infused. Therefore, based on our visualization of fluorescent muscimol spread at 1 h and 15 min post-infusion, we believe that we have inactivated comparable regions of cortex bilaterally in dmPFC at the time of behavioral testing. Histology. Once experiments were complete, rats were anesthetized with 100 mg/kg sodium pentobarbital and then transcardially perfused with either 10% formalin or 4% paraformaldehyde. Brains were sectioned on a freezing microtome, mounted on subbed slides, and stained for Nissl with Thionin or with fluorescent Nissl (Molecular Probes, Eugene, OR, USA). Data analysis and statistics. Distributions of response latencies were heavily skewed. Therefore, we used the geometric mean (or “geomean,” i.e. the square root of the mean of squared response latencies) and the mean absolute deviation from the

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geometric mean (or “geomad,” i.e. the mean difference between each response latency and the geomean) to estimate the central tendencies of response latencies. In order to plot distributions of response times, kernel density estimation was used (ksdensity.m in the Matlab Statistics Toolbox, The Mathworks; see Silverman (1986) for a review of kernel density methods and Van Zandt (2000) for how these methods are commonly applied to RT data). The spread of these estimates was generated via bootstrapping (1000 iterations). Differences in distributions of response latencies were assessed using repeated-measures ANOVA. Significance values for the ANOVAs were carried out using a “permutation test” approach (Good, 2000). We pseudo-randomly permuted the independent variable of interest (e.g. DRUG) 1000 times and computed F values for each permutation. An empirical probability of the results being obtained due to chance was computed as the ratio of the number of F values larger than that obtained in the actual data to the number of randomization data sets.

Experiment 2: effects of dmPFC inactivation on fixed foreperiod RT performance with trigger stimuli on 50% of trials Subjects. Eight rats (aged 5–10 months; Brown-Norway⫽5; Long-Evans⫽3) were trained to perform fixed foreperiod RT tasks. Rats were housed, handled, and motivated as described for experiment 1. Behavioral apparatus and training. The behavioral apparatus, instruments, and initial training procedures were identical to those described for experiment 1. After initial training, rats were trained to sustain lever presses in the absence of trigger stimuli for 800 ms, and then subsequently trained in the fixed foreperiod RT task with trigger stimuli on 50% of trials. Fixed foreperiod RT task with trigger stimuli on 50% of trials. Each trial in the fixed foreperiod RT task was initiated by the rat depressing the lever and ended when the lever was released. The foreperiod used in this task was 1200 ms. On 50% of trials, a trigger stimulus was presented at the end of the foreperiod (TRIG trials); on remaining trials, the end of the foreperiod was unsignaled (TIME trials). As in experiment 1, temporally conditioned responses occurred when the rats successfully maintained a lever press for the full foreperiod duration and released the lever with short response latencies (within 600 ms after the end of the foreperiod), premature responses occurred when rats did not maintain a lever press for the full foreperiod, and late responses occurred when the lever was released after the response window. These latter two types of trials were followed by a timeout period, in which all experimental stimuli were extinguished for 4 – 8 s. Each session began with a foreperiod of 100 ms and the foreperiod increased by 100 ms with every conditioned response until the rats reached a foreperiod of 1100 ms, after which foreperiods were fixed at 1200 ms. Surgery. Anesthesia and surgeries were performed as described in experiment 1. In five animals (Brown-Norway), guide cannulae were placed bilaterally in dmPFC using the coordinates in experiment 1. As our fluorescent muscimol studies revealed slight spread of infused fluid dorsally to cannula tip (Fig. 1B), we controlled for this spread of infused fluid in three animals (LongEvans) that had guide cannulae placed bilaterally in rostral anterior cingulate cortex (Fig. 1A; coordinates from bregma: anteroposterior ⫹3.2, mediolateral ⫾1.4, dorsoventral ⫺1.7 at 12° in the frontal plane). These coordinates allowed for inactivations of both the prelimbic cortex and the more dorsal regions of dmPFC by varying the length of the injector cannula (see next section). Drug infusion. Drug infusions were performed as described in experiment 1. For animals with cannulae in rostral anterior cingulate cortex, infusions of muscimol were made into anterior

cingulate cortex with an injector cannula that protruded 0.2 mm from the tip of the guide cannula. In these animals, subsequent infusions of muscimol were made into dorsal prelimbic cortex with an injector cannula that protruded 2.0 mm from the tip of the guide cannula one week after initial infusions. At the completion of experiments fluorescent muscimol (0.5 ␮l at a concentration 1 ␮g/ ␮l; BODIPY TMR-X Muscimol, Molecular Probes) was infused into bilateral dmPFC of three animals (Brown-Norway) (Fig. 1B) 45 min prior to testing in the fixed foreperiod RT task. Animals performed the RT task for 30 min and were then deeply anesthetized and transcardially perfused as in experiment 1. These three rats had three total infusions per site (saline, muscimol and fluorescent muscimol, with infusions made every 10 days), the three animals with cannulae in bilateral rostral anterior cingulate had four total infusions per site (saline and muscimol into dmPFC; 10 days later, saline and muscimol into rostral anterior cingulate), while the remaining two rats in this experiment had two total infusions per site (saline, muscimol). Histology. Animals were perfused and brains were processed as in experiment 1. Localization of cortical inactivation using fluorescent muscimol. Fluorescent material was rehydrated after fixation in PBS, mounted on slides with 90% glycerol, and visualized using a Zeiss research microscope equipped FITC filters and a CCD camera (Carl Zeiss, Thornwood, NY, USA). Exposure times were adjusted to maximize image quality between 2 and 8 s, and did not affect visualized area of fluorescence. Images were reassembled (by direct insertion without manipulation into red and green color channels) using Adobe Photoshop (Adobe Systems, San Jose, CA, USA). Measurements of drug spread were made from images of equally spaced 50 ␮m sections.

RESULTS Cannulae and drug localization Approximate locations of the tips of injection cannulae from the eight rats in experiment 2 are shown in Fig. 1A. Rats in experiments 1 and five rats in experiment 2 were implanted in dmPFC. Three rats in experiment 2 had cannulae implanted in rostral anterior cingulate cortex, and drug was infused into dmPFC via an injector projecting 2 mm from the tip of the injection cannula. To quantify the region of cortex that, when inactivated, resulted in altered behavioral performance, fluorescent muscimol was infused bilaterally into dmPFC of three rats 45 min before testing in a fixed foreperiod RT task (see experiment 2 below), and animals were killed immediately after 30 min of behavior (Fig. 1B). Performance following infusion of fluorescent muscimol was comparable to that following infusion of standard muscimol (subject 1, whose sections are shown in Fig. 1B: 25% conditioned responses in muscimol sessions, 22% conditioned responses in fluorescent muscimol session; subject 2: 22.9% conditioned responses in muscimol sessions, 38% conditioned responses in fluorescent muscimol sessions; subject 3: 32% conditioned responses in muscimol sessions, 34% conditioned responses in fluorescent muscimol sessions). Fluorescent muscimol was primarily observed around the cannulae tip and tract and observed to spread over a volume of cortex involving the dorsomedial regions of PFC (dorsal prelimbic and rostral anterior cingulate cortex).


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Experiment 1: effects of dmPFC inactivation on simple RT performance with variable foreperiods

Fig. 2. Experiment 1: effects of dmPFC inactivation on simple RT performance with variable foreperiods. (A) Sequence of events in simple RT task. Rats initiated trials by pressing a lever until the end of the foreperiod that was signaled by a trigger stimulus. Rats had to release the lever within a designated response window to collect a liquid reward (Conditioned responses). If the lever was released before the trigger stimulus (Premature responses) or after the response window (Late responses), rats experienced a timeout period. (B) Premature responses (mean⫾S.E.M.) for 11 rats trained to perform the simple RT tasks with variable foreperiods. (C) Distributions of response durations for premature responses in saline (gray line, light

Previous studies have demonstrated that lesions of dmPFC result in increased premature responding during simple RT performance (Risterucci et al., 2003). The goal of the present study was to replicate and extend these results with reversible inactivation of dmPFC. Eleven rats with cannulae implanted bilaterally in dmPFC were tested with muscimol in a simple RT task with variable foreperiods (Fig. 2A). In preoperative sessions, 26⫾2% of rats’ responses were premature (i.e. before the trigger stimuli). In postoperative control sessions (saline infusions), 32⫾3% of responses were premature, unchanged from preoperative sessions (t(1,14)⫽1.4, P⬍0.18; preoperative sessions for five rats are at longer foreperiods and excluded from preoperative comparisons; no differences in premature responding were seen between these rats after retraining and the other six rats: paired t(1,10)⫽0.97, P⬍0.35; see Experimental Procedures). With dmPFC inactivated, premature responses increased to 47⫾5%, significantly more than in control sessions (paired t(1,10)⫽2.9, P⬍0.02). In recovery sessions, 28⫾3% of responses were premature, similar to control (paired t(1,10)⫽1.8, P⬍0.11) and preoperative sessions (paired t(1,14)⫽0.32, P⬍0.75; see Fig. 2B). These data replicate the increased premature responding reported by Risterucci et al. (2003) for neurochemical lesions of dmPFC. In addition to increased premature responding, dmPFC inactivation altered the distributions of premature response latencies relative to control sessions (Control: 480⫾50 ms; Muscimol: 310⫾40 ms; rmANOVA: F(1,1553)⫽35.60, P⬍ 0.001). In control sessions, premature responses either occurred early (⬍100 ms), or occurred around the time of the mean foreperiod (750 ms). However, in sessions with dmPFC inactivated, premature responses occurred with no temporal pattern throughout the foreperiod (Fig. 2C). Conditioned response latencies were also affected with dmPFC inactivated. In control sessions, conditioned response latencies were speeded on trials with the longest foreperiods compared with trials with the shortest foreperiods (500 ms foreperiods: 400⫾48 ms; 1000 ms foreperiods: 290⫾70 ms; rmANOVA: F(1,554)⫽36.171, P⬍0.001). This phenomenon of speeding of response latencies with longer foreperiods (delay-dependent speeding) has been observed in previous studies in rats (e.g. Brown and Robbins, 1991; Risterucci et al., 2003) and human subjects (e.g. Näätänen, 1970). With dmPFC inactivated, response latencies were speeded on trials with short foreperiods (380⫾30 ms; rmANOVA: F(1,282)⫽11.302, P⬍0.001; Fig. 3A) and unchanged on trials with long foreperiods (270⫾50 ms;

gray band) and muscimol sessions (black line, dark gray band). Distributions were estimated using kernel density analysis and the variance was estimated using a bootstrap method. Inactivation of dmPFC altered the timing of premature responding; instead of responding at the time associated with the mean foreperiod (as they did in control sessions), rats with dmPFC inactivated made premature responses throughout the foreperiod. Asterisk indicates significant differences between groups (paired t-test, P⬍0.05).

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Fig. 3. Conditioned response latencies in experiment 1. Response latencies for (A) short (500 ms) and (B) long (1000 ms) foreperiods for saline and muscimol sessions. Colors and distributions are as in Fig. 3C. (C) Response latency as a function of foreperiod in the simple RT task. Geometric mean and geometric absolute deviation (vertical lines) are plotted for each level of the foreperiod (500, 670, 830, and 1000 ms) for saline (gray) and muscimol (black) sessions. (D) Response latency as a function of foreperiod in the simple RT task with longer foreperiods (900 –1400 ms). Asterisk indicates a significant interaction between foreperiod and drug in an rmANOVA (P⬍0.05). Data in A–C are from six animals trained explicitly for the simple RT task with foreperiods between 500 –1000 ms, and data in D are from five animals trained to perform a simple RT task with variable foreperiods between 900 –1400 ms.

rmANOVA: F(1,271)⫽0.116, P⬍0.75; Fig. 3B). As a result, inactivation of dmPFC eliminated the slowing of response latencies that occurred on trials with short foreperiods, thereby decreasing delay-dependent speeding. This effect resulted in a significant interaction between length of the foreperiod and cortical inactivation (rmANOVA: F(1,554)⫽ 5.700, P⬍0.03; Fig. 3C). Note that these data are for the six rats exclusively trained in the simple RT task with foreperiods of 500 –1000 ms. Five rats trained with longer foreperiods (900 –1400 ms; see Experimental Procedures) also had a significant interaction between the length of the foreperiod and cortical inactivation both for 900 –1400 ms foreperiods (rmANOVA: F(1,653)⫽21.75, P⬍0.001) and 500 –1000 ms foreperiods (rmANOVA: F(1,648)⫽10.75, P⬍0.001) due to altered delay-dependent speeding (Fig. 3D). These five rats were initially tested with dmPFC inactivation in a simple RT task with 900 –1400 ms foreperiods to investigate the role of dmPFC in time estimation during RT performance. If dmPFC is involved in time estimation, inactivation should produce more premature responses at 900 –1400 ms foreperiods relative to 500 –1000 ms fore-

periods because rats must estimate a longer mean foreperiod (1150 ms vs 750 ms). As in the simple RT task with 500 –1000 ms foreperiods, rats performed more premature responses in 900 –1400 ms foreperiod sessions with dmPFC inactivated compared with control sessions (39⫾ 5% in control sessions compared with 56⫾6% in sessions with dmPFC inactivated; paired t(1,4)⫽6.68, P⬍0.003). However, these rats made similar numbers of premature responses in sessions with dmPFC inactivated at 500 – 1000 ms and 900 –1400 ms foreperiods (paired t(1,4)⫽ 0.48, P⬍0.66). Such equivalent performances across different foreperiod durations indicate that the dmPFC is not involved explicitly in time estimation. Experiment 2: effects of dmPFC inactivation on fixed foreperiod RT performance with trigger stimuli on 50% of trials Experiment 1 found that bilateral inactivation of dmPFC produced increased premature responding during simple RT performance. In order to test if this effect was due to impairments in attending to the stimulus, time estimation,


Fig. 4. Experiment 2: effects of dmPFC inactivation on fixed foreperiod RT performance with trigger stimuli on 50% of trials. (A) Rats had to maintain a lever-press over a fixed foreperiod (1200 ms) and release the lever within 600 ms to receive a reward. On 50% of trials, a trigger stimulus was presented at the end of the foreperiod (TRIG); on the remaining trials, the end of the foreperiod was not signaled (TIME). (B) Premature responses (mean⫾S.E.M.) for eight rats trained to perform the fixed foreperiod RT task. (C) Distribution of premature

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or inhibitory control, we trained eight rats to perform a fixed foreperiod RT task in which trigger stimuli were presented on only 50% of trials (Kornblum, 1973; Fig. 4A). As in experiment 1, dmPFC inactivation in the fixed foreperiod RT task produced increased premature responding. In preoperative sessions, 32⫾3% of rats’ responses were premature. In control (saline) sessions, 30⫾ 2% of responses were premature, unchanged from preoperative levels (paired t(1,7)⫽0.76, P⬍0.47). With dmPFC inactivated, premature responses increased to 57⫾6%, significantly increased from control sessions (paired t(1,7)⫽ 4.73, P⬍0.002). In recovery sessions, 35⫾3% of responses were premature, similar to control (paired t(1,7)⫽1.18, P⬍0.25) and preoperative sessions (paired t(1,7)⫽0.68, P⬍0.52; Fig. 4B). In order to control for dorsal spread of infused fluid, three rats had cannulae implanted in rostral anterior cingulate cortex targeting the dorsal regions of dmPFC. By varying injector lengths, different regions of dmPFC could be targeted (see Experimental Procedures). In these animals, inactivating dmPFC by infusing muscimol into rostral anterior cingulate cortex produced increased premature responding (25⫹2% of responses were premature in control sessions compared with 71⫾5% in sessions with rostral anterior cingulate cortex inactivated, increased in three of three rats). The same effect was observed when inactivating dmPFC by infusing muscimol into dorsal prelimbic cortex (26⫹6% of responses were premature in control sessions compared with 48⫾11% in sessions with dorsal prelimbic cortex inactivated, increased in three of three rats; note that this site is in dorsal prelimbic cortex ventral to the rostral anterior cingulate site; see Fig. 1A). Finally, in each of these three rats, rostral anterior cingulate cortex inactivation produced more premature responding than dorsal prelimbic cortex inactivation, though because of the small sample, this effect was not significant (paired t(1,2)⫽2.4, P⬍0.13). If dmPFC is involved in attending to the stimulus, more premature responses would be expected in a task with variable foreperiods (experiment 1) than a task with a fixed foreperiod (experiment 2) because with a fixed foreperiod, there is less uncertainty about when the trigger stimulus will occur. However, in experiment 2, rats made similar amounts of premature responses compared with experiment 1 (t(1,17)⫽1.2, P⬍0.24), suggesting that dmPFC is not involved directly in attending to the stimulus. The distributions of premature response latencies in the fixed foreperiod task were altered by dmPFC inactivation (Control: 690⫾60 ms; Muscimol: 350⫾50 ms; rmANOVA: F(1,972)⫽166.70, P⬍0.001; Fig. 4C). In control sessions, the premature responses increased nearing the end of the foreperiod. With dmPFC inactivated, the rats responded in an apparently random manner throughout the normal

response latencies in saline and muscimol sessions. Colors and distributions are as in Fig. 3C. As in Fig. 3C, rats with dmPFC inactivated made many premature responses around the midpoint of the set of foreperiods (four of eight animals) and also responded early in the foreperiod (⬍0.2 s; four of eight animals).

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range of the foreperiod (e.g. the coefficient of variation for premature response latencies was ⬃50% larger in muscimol versus saline sessions), as in experiment 1. Following inactivation of dmPFC, response latencies of conditioned responses were slightly shorter than response latencies in control conditions (Fig. 5B and C; Control: 1460⫾10 ms; Muscimol: 1350⫾20 ms; rmANOVA: F(1,1171)⫽5.41, P⬍0.02). These results suggest that rats are able to respond faster with dmPFC inactivated. This task enables comparison between trials guided by external (sensory) and internal (timing) factors in RT performance on TRIG vs TIME trials respectively (Kornblum, 1973). If dmPFC were involved primarily in attending to the stimulus, inactivation of dmPFC would produce a decrease in conditioned responses on trials with stimuli and not on trials without stimuli. Conversely, if dmPFC were involved primarily in estimating the passage of time, inactivation of dmPFC would produce a decrease in conditioned responses on trials without stimuli and not on trials with stimuli. Remarkably, there were no significant differences in the percentage of conditioned responses with and without trigger stimuli in control and muscimol sessions (57⫾7% TRIG trials vs. 54⫾5% on TIME trials in control sessions; paired t(1,7)⫽1.66, P⬍0.13; 10⫾14% TRIG trials vs. 14⫾12% on TIME trials in muscimol sessions, paired t(1,7)⫽0.97, P⬍0.36; decreases in conditioned responses are driven by increased premature responding as detailed above; Fig. 5A). Moreover, there was no difference in distributions of conditioned response latencies on TRIG and TIME trials (rmANOVA: F(1,1260)⫽0.033, P⬍0.85) and no interaction of these distributions with cortical inactivation (rmANOVA: F(1,1260)⫽0.035, P⬍0.85; Fig. 5B and C). Taken together, these results suggest that dmPFC is involved in inhibiting responses during the foreperiod before either the stimulus has occurred or the foreperiod has been estimated. Nonspecific effects of dmPFC inactivation With dmPFC inactivated in both experiments, rats pressed the lever more often during the timeout period (or intertrial interval) with dmPFC inactivated than in control sessions (0.9⫾0.1 timeout lever presses relative to timeout periods in control sessions; this ratio was 2.0⫾0.2 with dmPFC inactivated, paired t(1,18)⫽4.00, P⬍0.0002). Also in both experiments, rats completed less trials with dmPFC inactivated than in control sessions (275⫾28 trials in control sessions and 135⫾25 trials with dmPFC inactivated; paired t(1,18)⫽4.29, P⬍0.001). Finally, there was no difference in the percentage of premature responses between strains (i.e. Long-Evans vs. Brown-Norway) in control sessions (t(1,17)⫽1.03, P⬍0.31) or with dmPFC inactivated (t(1,17)⫽0.68, P⬍0.50), though Long-Evans rats tended to perform more trials in control sessions only (371⫾35 trials vs 205⫾27 trials for Brown-

Fig. 5. Performance on TRIG vs TIME trials for the fixed foreperiod task. (A) Percentage of conditioned responses on TRIG vs. TIME trials in saline and muscimol sessions. No significant differences were found

between trial types. Distribution of response latencies for conditioned responses on (B) TRIG and (C) TIME trials in saline and muscimol sessions. Colors and distributions as in Fig. 3C.


Norway rats; t(1,17)⫽3.87, P⬍0.001; in dmPFC inactivation sessions Long-Evans rats performed 145⫾49 trials vs. 128⫾22 trials for Brown-Norway rats; t(1,17)⫽1.10, P⬍ 0.28).

DISCUSSION In the present study, we reversibly inactivated the dmPFC of rats performing simple RT tasks with variable foreperiods and a fixed foreperiod RT task with trigger stimuli on 50% of trials. In both experiments, despite differences in foreperiod duration, foreperiod variability, and stimulus probabilities, there were three consistent effects of dmPFC inactivation: 1) increased premature responding, 2) increased variability in the timing of premature responses, and 3) speeded response latencies, especially on trials with short foreperiods in tasks with variable foreperiods. While other studies have reported increased premature responding following lesions of dmPFC (Broersen and Uylings, 1999; Risterucci et al., 2003), the latter two effects above are, to our knowledge, novel results in the animal literature on RT performance. Inactivation of dmPFC results in increased premature responding The increased premature responding found in this study could be due to disruptions of preparatory processing (Risterucci et al., 2003). Preparatory set has traditionally been defined as “a state of readiness to receive a stimulus that has not yet arrived or a state of readiness to make a movement” (Evarts et al., 1984). One role of dmPFC in preparatory processing thus might be related to selective attention to the forthcoming trigger stimulus. If dmPFC was indeed involved in attending to the trigger stimulus, we would have expected to find an increasing effect of inactivation in tasks with fixed and variable foreperiods respectively. Greater attentional effort would be required when the timing of the trigger stimulus was variable as opposed to being fixed. However, we found that the effects of inactivation of dmPFC were equivalent in the fixed and variable foreperiod tasks. As such, it is unlikely that dmPFC functions in a process of selective attention, at least not in the task designs used in our experiments. Alternatively, dmPFC might be involved in estimating the timing of the trigger stimulus (Matell and Meck, 2000; MacDonald and Meck, 2004). Recent evidence has suggested that rats with lesions of cingulate cortex were unable to use temporal information to plan their responses in a five-choice serial RT task (Passetti et al., 2002). However, in experiment 1 we found no difference in the effects of inactivations of dmPFC in sessions with 500 –1000 ms variable foreperiods and relatively longer (900 –1400 ms) variable foreperiods. Such sessions should require different levels of time estimation because rats must estimate different lengths of the foreperiod. For example, deficits in time estimation in human subjects become apparent using time intervals of 1000 ms and longer (Näätänen et al., 1974). Furthermore, in experiment 2, we found no specific deficit on trials without stimuli relative to trials with stimuli.

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Given these results, dmPFC appears to be involved beyond pure time estimation. Finally, dmPFC might be involved in the motor aspects of preparing the response (Evarts et al., 1984). Data shown in Fig. 3, in which delay-dependent speeding was eliminated by decreasing response latencies on trials with short foreperiods and increasing latencies on trials with long foreperiods, indicate that if dmPFC is involved in preparation for movement, its role is more likely to be inhibitory than excitatory. Indeed, the increased production of short premature responses (⬍250 ms; Figs. 2C, 4C, early peaks in distribution of premature responses) following inactivation of dmPFC may be “premature motor reactions” (Näätänen, 1971) that are distinct from “premature expectant responses” associated with anticipatory behavior. An increase in such short premature motor reactions with dmPFC inactivated suggests that dmPFC was involved in motor preparation. However, based on speeded response latencies in experiment 1 and 2, we conclude that rather than enhancing motor excitability, dmPFC seems to be involved in reducing the drive to respond (Näätänen, 1971, pp. 324 –326). dmPFC and the response “deadline” Optimal RT performance is achieved if the subject knows when the trigger stimulus is likely to occur. That is, the subject must estimate the momentary likelihood of the trigger stimulus during the foreperiod. This information can be computed by knowing how much time has elapsed during the foreperiod and the longest possible waiting time (i.e. the upper limit of the foreperiod). One classic model for simple RT performance, called the “deadline” model (Ollman and Billington, 1972), suggests that subjects make anticipation responses at the upper limit of the foreperiod if the trigger stimulus has not occurred at an earlier moment in time. A similar rule for estimating the upper limit on stimulus times is inherent in many models of RT performance (Raab, 1962; Ollman and Billington, 1972; Kornblum, 1973; Ratcliff et al., 1999; Los et al., 2001; Miller and Ulrich, 2003; Hackley and Valle-Inclán, 2003). Given the results described here, rat dmPFC might be involved in this kind of computation. If the ability to inhibit responses prior to the response deadline was eliminated by inactivation of dmPFC, one would expect the rat to respond at random times during the foreperiod. A prediction of this model is that when trigger stimuli occurred prior to the response deadline, rats should respond to this stimulus more rapidly than under normal conditions due to a lack of temporally conditioned response inhibition. In both experiment 1 and experiment 2, we observe speeding of response times, particularly at short foreperiods (500 ms) that occurred prior to the response deadline. dmPFC and motivation In the present study, we observe a marked increase in premature responding and an altered distribution of premature responses. However, on 33⫾4% of trials, animals were still able to successfully wait during the foreperiod

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Fig. 6. Temporal inhibitory control of RT performance. (A) Kornblum’s (1973) model of simple RT performance, in which temporal factors (such as time estimation) and sensory factors (such as the conditioned stimulus) compete over a response generator (such as motor cortex). Our data suggest that dmPFC is not involved specifically with these processes; rather, it is involved in inhibiting premature responses during the foreperiod. (B) An inhibitory process mediated by dmPFC would oppose the excitatory drive to respond, which might be mediated by brain areas involved in time estimation, response preparation, and/or the incentive value of the trigger stimulus. Early in the foreperiod, inhibitory processes would be dominant. As a result, response latencies would be longer or more variable on trials with short foreperiods. Later in the foreperiod, as the deadline approaches, excitatory factors would be dominant and result in apparent “delay-dependent speeding” of the response latencies.

and perform a conditioned response. This suggests that our inactivation of dmPFC was incomplete (Fig. 1B), or that other areas are involved in suppressing premature responses (Baunez and Robbins, 1997, 1999). An alternative explanation, suggested by the decrease in completed trials, is that dmPFC inactivations altered the motivational state of the animal. Such an effect would impair the ability to sustain a lever press during the foreperiod. It is possible that increased timeout responding with dmPFC inactivated contributed to the decrease in the number of completed trials. In our task, timeout lever presses reset the timeout period (see Experimental Procedures), thereby reducing the time available for trial initiation. Moreover, a decrease in motivation would be expected to slow response latencies; however, our results indicate that response latency is faster with mPFC inactivated. Our findings suggest that the effect of mPFC inactivation on rats’ ability to perform temporally sustained lever presses may be independent of the decreased number of completed trials performed per session. That is, the effects we observed cannot be explained solely by changes in the animals’ motivation to perform the RT task. A model for dmPFC in RT performance Our results suggest that rat dmPFC is not directly involved in time estimation, attending to the trigger stimulus, or in motor aspects of response preparation. Instead, dmPFC appears to encode how long the rat must maintain these processes before executing an anticipatory response. Furthermore, as inactivations of dmPFC speeded response latencies and increased premature responding, the role of dmPFC appears to be inhibitory. Premature responses made with dmPFC inactivated might be viewed as “impulsive,” and could be due to a general increase in behavioral activity. However, we found no evidence for increased

behavioral activity in our experiments. Instead, the effects of dmPFC inactivation were specific to the relationship between the foreperiod and the animals’ response time, i.e. the ability of animals to wait for trigger stimuli. Based on these results, we propose a model in which dmPFC exerts inhibitory control over responding during RT performance (Fig. 6A). In our model, internal factors (such as time estimation) and external factors (such as the conditioned stimulus) compete over a response generator (such as motor cortex). Our data suggest that dmPFC is not involved specifically with these processes; rather, it is involved in inhibiting premature responses during the foreperiod. We propose that during a trial in the simple RT task (Fig. 6B) an inhibitory process mediated by dmPFC would oppose the excitatory drive to respond. The excitatory drive might be mediated by brain areas involved in time estimation (of short temporal epochs) (e.g. cerebellum; Ivry and Spencer, 2004; Mauk and Buonomano, 2004) and/or the incentive value of the trigger stimulus (orbitofrontal cortex and basolateral amygdala: Schoenbaum and Roesch, 2005; Joel et al., 2005; perirhinal/posterior-insular cortex: Kyuhou et al., 2003). Early in the foreperiod, inhibitory processes mediated by dmPFC would be dominant. As a result, response latencies would be longer or more variable on trials with short foreperiods. Later in the foreperiod, as the deadline approaches, excitatory factors would be dominant and result in apparent “delay-dependent speeding” of the response latencies. A more explicit test of this model of prefrontal control in simple RT tasks will require new experiments in which the temporal limits of waiting behavior are established for laboratory rats, as proposed by Ollman and Billington (1972), e.g. a long foreperiod in which rats make anticipation responses on 50% of trials, a short foreperiod in which rats make anticipation responses on a small percentage of


trials (⬍10%), and a time-production trial in which rats are rewarded for responding at the deadline. Such tasks, combined with neurophysiological stimulation and recordings, are needed to more fully characterize the role of dmPFC in simple RT tasks and the interactions of dmPFC with components of the motor system, such as motor cortex and basal ganglia. Acknowledgments—This work was supported by funds from the Defense Advanced Research Projects Agency, the American Federation for Aging Research, the Tourette Syndrome Association, and the John B. Pierce Laboratory for M.L., support from an NIH training grant to the Yale Medical Scientist Training Program for N.S.N., and support from an NIH training grant to the Yale Interdepartmental Neuroscience Program for N.K.H. We thank Eyal Kimchi and Dr. Lawrence Marks for helpful comments on this manuscript. We thank Carol Akirav for assistance in training animals and the Instruments Shop at the John B. Pierce Laboratory (John Buckley, Tom D’Alessandro, Michael Fritz, Ronald Goodman, and Angelo DiRubba) for outstanding technical support. We also thank Dr. David Krupa (CD Neural Engineering, Ithaca, NY, USA) for advice on using muscimol to carry out reversible inactivation studies. Finally, we thank Dr. Vincent Pieribone, Dr. Steven Segal, and Mary Whitman for assistance with fluorescence microscopy.

REFERENCES Baunez C, Robbins TW (1997) Bilateral lesions of the subthalamic nucleus induce multiple deficits in an attentional task in rats. Eur J Neurosci 9:2086 –2099. Baunez C, Robbins TW (1999) Effects of transient inactivation of the subthalamic nucleus by local muscimol and APV infusions on performance on the five-choice serial reaction time task in rats. Psychopharmacology (Berl) 141:57– 65. Broersen LM, Uylings HB (1999) Visual attention task performance in Wistar and Lister hooded rats: response inhibition deficits after medial prefrontal cortex lesions. Neuroscience 94:47–57. Brown VJ, Robbins TW (1991) Simple and choice reaction time performance following unilateral striatal dopamine depletion in the rat. Impaired motor readiness but preserved response preparation. Brain 114(Pt 1B):513–525. Evarts E, Shinoda Y, Wise SP (1984) Neurophysiological approaches to higher brain functions. New York: Wiley. Fuster JM (2000) Prefrontal neurons in networks of executive memory. Brain Res Bull 52:331–336. Gabbott PL, Warner TA, Jays PR, Salway P, Busby SJ (2005) Prefrontal cortex in the rat: Projections to subcortical autonomic, motor, and limbic centers. J Comp Neurol 492:145–177. Good PI (2000) Permutation tests: A practical guide to resampling methods for testing hypotheses. New York: Springer. Grill JD, Riddle DR (2002) Age-related and laminar-specific dendritic changes in the medial frontal cortex of the rat. Brain Res 937:8 –21. Groenewegen HJ, Uylings HB (2000) The prefrontal cortex and the integration of sensory, limbic and autonomic information. Prog Brain Res 126:3–28. Hackley SA, Valle-Inclán F (2003) Which stages of processing are speeded by a warning signal? Biol Psychol 64:27– 45. Heidbreder CA, Groenewegen HJ (2003) The medial prefrontal cortex in the rat: evidence for a dorso-ventral distinction based upon functional and anatomical characteristics. Neurosci Biobehav Rev 27:555–579. Hikosaka O, Wurtz RH (1985) Modification of saccadic eye movements by GABA-related substances. I. Effect of muscimol and

875

bicuculline in monkey superior colliculus. J Neurophysiol 53:266 – 291. Ivry RB, Spencer RM (2004) The neural representation of time. Curr Opin Neurobiol 2:225–232. Joel D, Doljansky J, Schiller D (2005) ‘Compulsive’ lever pressing in rats is enhanced following lesions to the orbital cortex, but not to the basolateral nucleus of the amygdala or to the dorsal medial prefrontal cortex. Eur J Neurosci 21:2252–2262. Kornblum S (1973) Simple reaction time as a race between signal detection and time estimation: A paradigm and a model. Percept Psychophysics 13:108 –112. Krupa DJ, Ghazanfar AA, Nicolelis MA (1999) Immediate thalamic sensory plasticity depends on corticothalamic feedback. Proc Natl Acad Sci U S A 96:8200 – 8205. Kyuhou S, Matsuzaki R, Gemba H (2003) Perirhinal cortex relays auditory information to the frontal motor cortices in the rat. Neurosci Lett 353:181–184. Laubach M, Wessberg J, Nicolelis MA (2000) Cortical ensemble activity increasingly predicts behaviour outcomes during learning of a motor task. Nature 405:567–571. Lomber SG (1999) The advantages and limitations of permanent or reversible deactivation techniques in the assessment of neural function. J Neurosci Methods 86:109 –117. Los SA, Van Den Heuvel CE (2001) Intentional and unintentional contributions to nonspecific preparation during reaction time foreperiods. J Exp Psychol Hum Percept Perform 27:370 –386. Luce D (1986) Response times: their role in inferring elementary mental organization. Oxford, UK: Oxford University Press. MacDonald CJ, Meck WH (2004) Systems-level integration of interval timing and reaction time. Neurosci Biobehav Rev 28:747–769. Martin JH, Ghez C (1999) Pharmacological inactivation in the analysis of the central control of movement. J Neurosci Methods 86:145– 159. Matell MS, Meck WH (2000) Neuropsychological mechanisms of interval timing behavior. Bioessays 22:94 –103. Mauk MD, Buonomano DV (2004) The neural basis of temporal processing. Annu Rev Neurosci 27:307–340. Milad MR, Vidal-Gonzalez I, Quirk GJ (2004) Electrical stimulation of medial prefrontal cortex reduces conditioned fear in a temporally specific manner. Behav Neurosci 118:389 –394. Miller EK, Cohen JD (2001) An integrative theory of prefrontal cortex function. Annu Rev Neurosci 24:167–202. Miller J, Ulrich R (2003) Simple reaction time and statistical facilitation: a parallel grains model. Cognit Psychol 46:101–151. Näätänen R (1970) The diminishing time-uncertainty with the lapse of time after the warning signal in reaction-time experiments with varying foreperiods. Acta Psychol 34:399 – 419. Näätänen R (1971) Non-aging fore-periods and simple reaction time. Acta Psychol 35:316 –327. Näätänen R (1972) Time uncertainty and occurrence uncertainty of the stimulus in a simple reaction time task. Acta Psychol 36: 492–503. Näätänen R, Muranen V, Merisalo A (1974) Timing of expectance peak in simple reaction time situation. Acta Psychol (Amst) 38: 461– 470. Naito E, Kinomura S, Geyer S, Kawashima R, Roland PE, Zilles K (2000) Fast reaction to different sensory modalities activates common fields in the motor areas, but the anterior cingulate cortex is involved in the speed of reaction. J Neurophysiol 83:1701–1709. Niki H, Watanabe M (1979) Prefrontal and cingulate unit activity during timing behavior in the monkey. Brain Res 171:213–224. Ollman RT, Billington MJ (1972) The deadline model for simple reaction times. Cognit Psychol 3:311–336. Passetti F, Chudasama Y, Robbins TW (2002) The frontal cortex of the rat and visual attentional performance: dissociable functions of distinct medial prefrontal subregions. Cereb Cortex 12:1254 – 1268.

876


Raab DH (1962) Statistical facilitation of simple reaction times. Trans N Y Acad Sci 24:574 –590. Ratcliff R, Van Zandt T, McKoon G (1999) Connectionist and diffusion models of reaction time. Psychol Rev 106:261–300. Risterucci C, Terramorsi D, Nieoullon A, Amalric M (2003) Excitotoxic lesions of the prelimbic-infralimbic areas of the rodent prefrontal cortex disrupt motor preparatory processes. Eur J Neurosci 17: 1498 –1508. Sawaguchi T (1987) Properties of neuronal activity related to a visual reaction time task in the monkey prefrontal cortex. J Neurophysiol 58:1080 –1099. Schoenbaum G, Roesch M (2005) Orbitofrontal cortex, associative learning, and expectancies. Neuron 47:633– 636. Sesack SR, Deutch AY, Roth RH, Bunney BS (1989) Topographical organization of the efferent projections of the medial prefrontal cortex in the rat: an anterograde tract-tracing study with Phaseolus vulgaris leucoagglutinin. J Comp Neurol 290:213–242.

Silverman BW (1986) Density estimation for statistics and data analysis. London, UK: Chapman and Hall. Sprott RL, Austad SN (1995) Animal models for aging research. In: Handbook of the biology of aging (Schneider EL, Rowe JW, eds), pp 3–24. San Diego, CA: Academic Press. Stuss DT, Alexander MP, Shallice T, Picton TW, Binns MA, Macdonald R, Borowiec A, Katz DI (2005) Multiple frontal systems controlling response speed. Neuropsychologia 43:396 – 417. Swanson LW (1999) Brain maps: structure of the rat brain: a laboratory guide with printed and electronic templates for data, models, and schematics. New York: Elsevier. Uylings HB, Groenewegen HJ, Kolb B (2003) Do rats have a prefrontal cortex? Behav Brain Res 146:3–17. Van Zandt T (2000) How to fit a response time distribution. Psychon Bull Rev 7:424 – 465. Vertes RP (2004) Differential projections of the infralimbic and prelimbic cortex in the rat. Synapse 51:32–58.

(Accepted 30 November 2005) (Available online 24 February 2006)