Cholinergic interneurons in the dorsal striatum play an important role in the acquisition of duration memory.

The present study aimed to examine the effect of cholinergic interneuron lesions in the dorsal striatum on duration-memory formation. Cholinergic interneurons in the dorsal striatum may be involved in the formation of duration memory since they are among the main inputs to the dorsal striatal muscarinic acetylcholine-1 receptors, which play a role in the consolidation of duration memory. Rats were sufficiently trained using a peak-interval 20 s procedure and then infused with anti-choline acetyltransferase-saporin into the dorsal striatum to cause selective ablation of cholinergic interneurons. To make the rats acquire new duration-memories, we trained them with a peak interval 40 s after lesion. Before lesion, the peak times (an index of duration memory) for sham-lesioned and lesioned groups were similar at approximately 20 s. In the peak interval 40 s session, the peak times for the sham-lesioned and lesioned groups were approximately 30 and 20 s, respectively. After additional peak interval 40 s sessions, the peak times of both groups were shifted to approximately 40 s. Those results suggest that the cholinergic interneuron lesion delayed new duration-memory acquisition. Subsequent experiments showed that cholinergic interneuron lesions did not retard the shift of peak time to the original target time (20 s). Following experiment without changing the target time after lesion showed that cholinergic interneuron lesions did not change their peak times. Our findings suggest that cholinergic interneurons in the dorsal striatum are involved in new duration-memory acquisition but not in the utilization of already acquired duration memory and interval timing.

Intra-dorsal striatal acetylcholine M1 but not dopaminergic D1 or glutamatergic NMDA receptor antagonists inhibit consolidation of duration memory in interval timing.

The striatal beat frequency model assumes that striatal medium spiny neurons encode duration via synaptic plasticity. Muscarinic 1 (M1) cholinergic receptors as well as dopamine and glutamate receptors are important for neural plasticity in the dorsal striatum. Therefore, we investigated the effect of inhibiting these receptors on the formation of duration memory. After sufficient training in a peak interval (PI)-20-s procedure, rats were administered a single or mixed infusion of a selective antagonist for the dopamine D1 receptor (SCH23390, 0.5 µg per side), N-methyl-D-aspartic acid (NMDA)-type glutamate receptor (D-AP5, 3 µg), or M1 receptor (pirenzepine, 10 µg) bilaterally in the dorsal striatum, immediately before initiating a PI-40 s session (shift session). The next day, the rats were tested for new duration memory (40 s) in a session in which no lever presses were reinforced (test session). In the shift session, the performance was comparable irrespective of the drug injected. However, in the test session, the mean peak time (an index of duration memory) of the M1 + NMDA co-blockade group, but not of the D1 + NMDA co-blockade group, was lower than that of the control group (Experiments 1 and 2). In Experiment 3, the effect of the co-blockade of M1 and NMDA receptors was replicated. Moreover, sole blockade of M1 receptors induced the same effect as M1 and NMDA blockade. These results suggest that in the dorsal striatum, the M1 receptor, but not the D1 or NMDA receptors, is involved in the consolidation of duration memory.

The dorsal hippocampus is required for the formation of long-term duration memories in rats.

Interval timing-the perception of durations mainly in seconds or minutes-is a ubiquitous behavior in organisms. Animal studies have suggested that the hippocampus plays an essential role in duration memory; however, the memory processes involved are unclear. To clarify the role of the dorsal hippocampus in the acquisition of long-term duration memories, we adapted the "time-shift paradigm" to a peak-interval procedure. After a sufficient number of training with an initial target duration (20 s), the rats underwent "shift sessions" with a new target duration (40 s) under a muscimol (0.5 µg per side) infusion into the bilateral dorsal hippocampus. The memory of the new target duration was then tested in drug-free "probe sessions," including trials in which no lever presses were reinforced. In the probe sessions, the mean response rate distribution of the muscimol group was located leftward to the control group, but these two response rate distributions were superimposed on the standardized time axis, suggesting a scalar property. In the session-by-session analysis, the mean peak time (an index of timing accuracy) of the muscimol group was lower than that of the control group in the probe sessions, but not in the shift sessions. These findings suggest that the dorsal hippocampus is required for the formation of long-term duration memories within the range of interval timing.

Striatal dopamine D1 receptors control motivation to respond, but not interval timing, during the timing task.

Dopamine plays a critical role in behavioral tasks requiring interval timing (time perception in a seconds-to-minutes range). Although some studies demonstrate the role of dopamine receptors as a controller of the speed of the internal clock, other studies demonstrate their role as a controller of motivation. Both D1 dopamine receptors (D1DRs) and D2 dopamine receptors (D2DRs) within the dorsal striatum may play a role in interval timing because the dorsal striatum contains rich D1DRs and D2DRs. However, relative to D2DRs, the precise role of D1DRs within the dorsal striatum in interval timing is unclear. To address this issue, rats were trained on the peak-interval 20-sec procedure, and D1DR antagonist SCH23390 was infused into the bilateral dorsocentral striatum before behavioral sessions. Our results showed that the D1DR blockade drastically reduced the maximum response rate and increased the time to start responses with no effects on the time to terminate responses. These findings suggest that the D1DRs within the dorsal striatum are required for motivation to respond, but not for modulation of the internal clock speed.

A Task for Assessing the Impact of a Partner on the Speed and Accuracy of Motor Performance in Rats.

To our knowledge, no study has examined the effect of mere presence on accuracy of performance in animals. Therefore, we developed an experimental task to measure rats' motor performance (speed and accuracy) in a social condition. Rats were trained to run on a runway and pull down a lever at the end of the runway. In testing, rats performed the task solitarily (single) or in the presence of a confederate rat beyond the lever (pair or a social condition). As indices of the performance speed, we measured the time needed to start running, run through the runway, and pull down the lever. As the index of performance accuracy, we counted the number of trials in which rats could pull down the lever during their first attempt. One-way and two-way repeated-measure analyses of variance were used to analyze the data. This run-and-pull task enabled us to examine the effect of the presence of another conspecific on both speed and accuracy of motor performance in one experiment. The results showed that rats performed the task faster but less accurately in pair sessions than in single sessions. This protocol would be a valid animal model to examine the effect of mere presence on speed and accuracy of motor performance in rats.

Basolateral amygdala inactivation eliminates fear-induced underestimation of time in a temporal bisection task.

We examined interval timing - time perception in the seconds-to-minutes range - of the fear-inducing stimulus and the role of the amygdala in this phenomenon. Rats were initially trained to perform a temporal bisection task, in which their responses to levers A and B were reinforced following 2-s and 8-s tones, respectively. After acquisition, the rats were also presented with tones of intermediate durations and pressed one of the two levers to indicate whether the tone duration was closer to 2 or 8 s. Subsequently, the rats underwent differential fear conditioning, in which one frequency tone (conditioned stimulus; CS+) was paired with an electric foot shock, whereas another frequency tone (CS-) was presented alone. The rats were then infused with artificial cerebrospinal fluid (aCSF) or the GABAA agonist muscimol into the bilateral basolateral amygdala (BLA) before performing the bisection task with CS+ and CS-. In rats infused with aCSF, the psychophysical function shifted rightward in CS+ relative to that in CS-. Moreover, the point of subjective equality of the CS+ was higher than that of CS-, suggesting that the duration of the fear -CS was perceived as shorter than that of the neutral CS. However, muscimol infusion into the BLA abolished this difference, suggesting that BLA inactivation suppresses the effect of the fear -CS. Our results demonstrate that normal BLA activity is essential for fear-induced underestimation of time.

Effects of the mere presence of conspecifics on the motor performance of rats: Higher speed and lower accuracy.

Many studies on humans and animals have shown that the mere presence of another individual or individuals accelerates the motor performance speed of the subject individual. However, it has not been well investigated whether the mere presence of another individual affects the accuracy of motor performance in animals. In this study, we developed a novel task (run-and-pull task) to simultaneously investigate both the speed and accuracy of motor performance in rats and examined the effect of the mere presence of another rat on the task performance of the subject rat. Rats were first trained in isolation to run a runway and then pull a lever on the terminal end of the runway. After training, the subject rats were required to perform the task in isolation (Single) or in front of a non-competitive confederate rat without direct interaction (Pair). The results showed that the latency to start running and to pull the lever were shorter and the accuracy of the lever-pull movement was lower in the Pair condition than in the Single condition. These findings suggest that the mere presence of another individual increased the speed and decreased the accuracy of the motor performance of rats.

Insular cortex inactivation generalizes fear-induced underestimation of interval timing in a temporal bisection task.

In this study, we investigated: (1) the effect of fear on interval timing-time perception in the seconds-to-minutes range-and (2) the role of the insular cortex in the modulation of this effect. Rats were first trained on a temporal bisection task in which their response to a lever A was reinforced following a 2.00-s tone, whereas their response to a lever B was reinforced following an 8.00-s tone. After acquisition, the rats were also presented with intermediate-duration tones and pressed one of two levers to indicate whether tone duration was closer to 2.00 or 8.00s. Subsequently, the rats underwent differential fear conditioning in which one pitch tone (conditioned stimulus; CS+) was paired with an electric foot shock, while the other pitch tone (CS-) was presented alone. Either artificial cerebrospinal fluid (aCSF) or the GABAA agonist muscimol was then infused into the rats' bilateral insular cortex before the animals were tested on the bisection task using the CS+and CS- tones. We found that in the rats infused with aCSF, the point of subjective equality (PSE) of the CS+ was higher than that for CS-, suggesting that the duration for CS+ was perceived to be shorter than that of CS-. However, muscimol eliminated the difference in PSE between CS+ and CS- by generalizing of the effect from CS+to the CS-. Taken together, our results show that normal activity in the insular cortex is involved in fear-induced modulation of interval timing.

Post-acquisition hippocampal blockade of the NMDA receptor subunit GluN2A but not GluN2B sustains spatial reference memory retention.

While it has been shown that the blockade of N-methyl-d-aspartate type glutamate receptors (NMDARs) impairs memory acquisition, recent studies have reported that the post-acquisition administration of NMDAR antagonists suppresses spatial memory decay. These findings suggest that NMDARs are important not only for the acquisition of new memories but also for the decay of previously acquired memories. The present study investigated the contributions of specific NMDAR subunits to spatial memory decay using NVP-AAM077 (NVP), an NMDAR antagonist that preferentially binds to GluN2A subunits, and the selective GluN2B blocker Ro 25-6981 (Ro). Following Morris water maze training (four trials/day for four days), NVP and/or Ro were subchronically infused into the rat hippocampus for five days. Seven days after training, NVP-treated rats and NVP/Ro-treated rats explored the target area significantly more than the control and Ro-treated rats. These results demonstrate that post-acquisition treatment with NVP, but not Ro, suppresses the forgetting of previously acquired spatial memories. The NVP-treated rats more persistently explored the target area in the second test, which was conducted one day after the first, while the NVP/Ro-treated rats did not, which suggest that Ro treatment downregulates memory retention. In conclusion, the present results indicate that the NMDAR GluN2A and GluN2B subunits contribute to spatial memory deterioration and maintenance, respectively.

[Post-training N-methyl-D-aspartate receptor blockade facilitates retention of acquired spatial memory in rats].

We investigated the effect of a post-training chronic infusion of N-methyl-D-aspartate (NMDA) receptor blocker on retention of spatial reference memory in rats. In Experiment 1, we trained 4 groups of rats for 4 days (4 trials/ day) in the Morris water maze task. In a single probe trial after retention intervals of 1, 7, 14, and 28 days, the 1-day group showed more goal crossings than shown by the other 3 groups. In Experiment 2, a chronic infusion of the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (D-AP5) or a control vehicle into the lateral ventricle was initiated 1 day after the training session, and continued for 6 days. In the subsequent probe trial (7 days after training), the rats that had received the D-AP5 infusion showed significantly more goal crossings than the controls. These findings suggest that an NMDA receptor blockade following acquisition facilitates retention of spatial reference memory.

Post-acquisition hippocampal NMDA receptor blockade sustains retention of spatial reference memory in Morris water maze.

Several studies have demonstrated that the hippocampal N-methyl-D-aspartate type glutamate receptors (NMDARs) are necessary for the acquisition but not the retention of spatial reference memory. In contrast, a few studies have shown that post-acquisition repetitive intraperitoneal injections of an NMDAR antagonist facilitate the retention of spatial reference memory in a radial maze task. In the present study, we investigated the role of hippocampal NMDARs in the retention of spatial reference memories in Morris water maze. In Experiment 1, 24 h after training (4 trials/day for 4 days), D-AP5 was chronically infused into the hippocampus of rats for 5 days. In the subsequent probe test (seven days after training), we found that rats infused with D-AP5 spent a significantly longer time in the target quadrant compared to chance level, whereas rats in the control group did not. In Experiment 2, D-AP5 was infused into the hippocampus 1 (immediate) or 7 (delayed) days after the training session. In the probe test, following the retention interval of 13 days, immediate infusion facilitated the performance in a manner similar to Experiment 1, whereas the delayed infusion did not. These findings suggest that hippocampal NMDARs play an important role in the deterioration of spatial reference memory.

Intra-ventral tegmental area or intracerebroventricular orexin-A increases the intra-cranial self-stimulation threshold via activation of the corticotropin-releasing factor system in rats.

Although orexin-A peptide was recently found to inhibit the brain reward system, the exact neural substrates for this phenomenon remain unclear. The aim of the present study was to investigate the role of orexin neurons in intra-cranial self-stimulation behavior and to clarify the pathways through which orexin-A inhibits the brain reward system. Immunohistochemical examination using Fos, a neuronal activation marker, revealed that the percentage of activated orexin cells was very low in the lateral hypothalamus even in the hemisphere ipsilateral to self-stimulation, suggesting that orexin neurons play only a small part, if any, in performing intra-cranial self-stimulation behavior. Intra-ventral tegmental area administration of orexin-A (1.0 nmol) significantly increased the intra-cranial self-stimulation threshold. Furthermore, the threshold-increasing effects of intra-ventral tegmental area or intracerebroventricular orexin-A were inhibited by administration of the nonspecific corticotropin-releasing factor receptor antagonist, d-Phe-CRF(12-41) (20 µg). Following intra-ventral tegmental area infusion of orexin-A, the percentage of cells double-labeled with corticotropin-releasing factor and Fos antibodies increased in the central nucleus of the amygdala but not in the bed nucleus of the stria terminalis, and brain microdialysis analyses indicated that dopamine efflux in both the central nucleus of the amygdala and bed nucleus of the stria terminalis were enhanced. Taken together, the present findings suggest that intra-ventral tegmental area or intracerebroventricular administration of orexin-A exerts its threshold-increasing effect via subsequent activation of the corticotropin-releasing factor system.

Hippocampal acetylcholine efflux increases during negative patterning and elemental discrimination in rats.

The purpose of this paper is to examine whether hippocampal acetylcholine (ACh) efflux increases during negative patterning (NP) discrimination tasks. For these tasks, a rat's response was rewarded when either a single stimulus A (tone) or stimulus B (light) was presented, but was not rewarded when the compound stimulus AB (tone+light) was presented to the NP group of rats. An elemental discrimination (E) task was given to another group (E group). In the E group, the rat's response was rewarded when one of two stimuli (e.g., tone) was presented, but not rewarded when the other stimulus (e.g., light) was presented. After reaching a learning criterion, a guide cannula was implanted into dorsal hippocampus under anesthesia. In test sessions, rats were given the same task as before the guide cannula implantation, and ACh efflux was measured. Hippocampal ACh efflux increased during both NP and E tasks. In addition, the magnitude of increase was higher in the NP group than in the E group. Thus, over all our results demonstrate that task difficulty is a critical factor that relates to the difference in ACh efflux in the hippocampus.

Medial prefrontal cortex and precision of temporal discrimination: a lesion, microinjection, and microdialysis study.

In this paper, we examined the role of the medial prefrontal cortex in temporal discrimination in three experiments using rats. Experiment 1 attempted to dissociate the roles of the medial precentral (PrCm) area from the prelimbic and infralimbic (PL-IL) area in temporal discrimination using fixed-interval (FI) schedule. The gradient of response rate distribution became more moderate by a lesion of the PrCm, but not by a lesion of the PL-IL. In experiment 2, the efflux of acetylcholine (ACh) in the PrCm area during temporal discrimination tasks was compared to that during non-temporal discrimination tasks. ACh efflux was not different between these two tasks. In experiment 3, microinjection of the anticholinergic drug scopolamine (10 microg) into the PrCm area made the gradient of response rate distribution moderate. These findings suggest that reduced activity of the ACh system within the PrCm area impairs the precision of temporal discrimination, even though enhancement of this system is not indispensable for performing temporal discrimination.