Exploratory analyses of postanesthetic effects of desflurane using behavioral test battery of mice.

Halogenated ethers, such as desflurane, sevoflurane, and isoflurane, are known to exert an array of effects besides sedation. However, the postanesthetic effects of desflurane remain undiscovered as no study has explored these effects systematically. Phenotypic screening using behavioral test batteries is a powerful method to identify such effects. In the present study, we behaviorally phenotyped desflurane-treated mice to investigate postanesthetic effects. We applied comprehensive behavioral test batteries measuring sensorimotor functions, anxiety, depression, sociability, attention, and learning abilities, starting 7 days after anesthesia performed with 8.0% desflurane for 6 h. Although our previous study revealed postanesthetic effects of isoflurane in adult mice, in the current study, desflurane-treated mice exhibited no such effects in any behavioral test. To further examine whether desflurane affect behavior in more early time point, we built up a new additional test battery, which carried out 1 day or 3 days after exposure to desflurane. Mice treated with desflurane 1 day before testing showed more slips than other two groups in the first trial, suggesting mild acute side effects of desflurane on motor coordination. These results suggest the safety of desflurane in clinical settings and imply that postanesthetic effects are unique to each halogenated ether.

Neonatal isolation augments social dominance by altering actin dynamics in the medial prefrontal cortex.

Social separation early in life can lead to the development of impaired interpersonal relationships and profound social disorders. However, the underlying cellular and molecular mechanisms involved are largely unknown. Here, we found that isolation of neonatal rats induced glucocorticoid-dependent social dominance over nonisolated control rats in juveniles from the same litter. Furthermore, neonatal isolation inactivated the actin-depolymerizing factor (ADF)/cofilin in the juvenile medial prefrontal cortex (mPFC). Isolation-induced inactivation of ADF/cofilin increased stable actin fractions at dendritic spines in the juvenile mPFC, decreasing glutamate synaptic AMPA receptors. Expression of constitutively active ADF/cofilin in the mPFC rescued the effect of isolation on social dominance. Thus, neonatal isolation affects spines in the mPFC by reducing actin dynamics, leading to altered social behavior later in life.

Comprehensive behavioral study and proteomic analyses of CRMP2-deficient mice.

Collapsin response mediator protein 2 (CRMP2) plays a key role in axon guidance, dendritic morphogenesis and cell polarization. CRMP2 is implicated in various neurological and psychiatric disorders. However, in vivo functions of CRMP2 remain unknown. We generated CRMP2 gene-deficient (crmp2-/- ) mice and examined their behavioral phenotypes. During 24-h home cage monitoring, the activity level during the dark phase of crmp2-/- mice was significantly higher than that of wild-type (WT) mice. Moreover, the time during the open arm of an elevated plus maze was longer for crmp2-/- mice than for WT mice. The duration of social interaction was shorter for crmp2-/- mice than for WT mice. Crmp2-/- mice also showed mild impaired contextual learning. We then examined the methamphetamine-induced behavioral change of crmp2-/- mice. Crmp2-/- mice showed increased methamphetamine-induced ambulatory activity and serotonin release. Crmp2-/- mice also showed altered expression of proteins involved in GABAergic synapse, glutamatergic synapse and neurotrophin signaling pathways. In addition, SNAP25, RAB18, FABP5, ARF5 and LDHA, which are related genes to schizophrenia and methamphetamine sensitization, are also decreased in crmp2-/- mice. Our study implies that dysregulation of CRMP2 may be involved in pathophysiology of neuropsychiatric disorders.

The association between employee lifestyles and the rates of mental health-related absenteeism and turnover in Japanese companies.

We assessed the association of employee lifestyles (e.g., smoking, exercise, drinking, and sleep habits) with mental health-related absenteeism and turnover rates utilizing data from the annual Health and Productivity Management survey by Japan's Ministry of Economy, Trade and Industry. This analysis included data from 1,748 companies, encompassing 4,199,021 employees. The average proportions of mental health-related absenteeism and employee turnover rates were 1.1% (±1.0%) and 5.0% (±5.0%), respectively. In multivariable regression models that incorporated all lifestyle factors and confounders, a 1 percentage point increase in the proportion of employees who slept well was associated with reductions in their turnover rate (mean -0.020%; 95% CI, -0.038% to -0.002%) and in mental health-related absenteeism (mean -0.005%; 95% CI, -0.009% -0.001%). A similar increase in the proportion of employees engaging in regular physical activity corresponded with a 0.005% decrease in the prevalence of mental health-related absenteeism (95% CI, -0.010% to -0.001%). A 1 percentage point increase in the proportion of employees who smoked was associated with a 0.013% reduction in mental health-related absenteeism (95% CI, -0.017% to -0.008%). Nonetheless, the current study's observational and cross-sectional design restricted the ability to establish causality between employee lifestyle factors and mental health issues.

Understanding Sensory-Motor Disorders in Autism Spectrum Disorders by Extending Hebbian Theory: Formation of a Rigid-Autonomous Phase Sequence.

Autism spectrum disorder is a neuropsychiatric disorder characterized by persistent deficits in social communication and social interaction and restricted, repetitive patterns of behavior, interests, or activities. The symptoms invariably appear in early childhood and cause significant impairment in social, occupational, and other important functions. Various abnormalities in the genetic, neurological, and endocrine systems of patients with autism spectrum disorder have been reported as the etiology; however, no clear factor leading to the onset of the disease has been identified. Additionally, higher order cognitive dysfunctions, which are represented by a lack of theory of mind, sensorimotor disorders, and memory-related disorders (e.g., flashbacks), have been reported in recent years, but no theoretical framework has been proposed to explain these behavioral abnormalities. In this study, we extended Hebb's biopsychology theory to provide a theoretical framework that comprehensively explains the various behavioral abnormalities observed in autism spectrum disorder. Specifically, we propose that a wide range of symptoms in autism spectrum disorder may be caused by the formation of a rigid-autonomous phase sequence (RAPS) in the brain. Using the RAPS formation theory, we propose a biopsychological mechanism that could be a target for the treatment of autism spectrum disorders.

Assessments of prolonged effects of desflurane and sevoflurane on motor learning deficits in aged AppNL-G-F/NL-G-F mice.

As the proportion of elderly in society increases, so do the number of older patients undergoing surgical procedures. This is concerning as exposure to anesthesia has been identified as a risk factor for Alzheimer's disease (AD). However, the causal relationship between clinical AD development and anesthesia remains conjectural. Preclinical studies have demonstrated that anesthesia, such as halothane, isoflurane, and sevoflurane, induces AD-like pathophysiological changes and cognitive impairments in transgenic mouse models of AD. Desflurane does not have these effects and is expected to have more potential for use in elderly patients, yet little is known about its effects, especially on non-cognitive functions, such as motor and emotional functions. Thus, we examined the postanesthetic effects of desflurane and sevoflurane on motor and emotional function in aged AppNL-G-F/NL-G-F (App-KI) mice. This is a recently developed transgenic mouse model of AD exhibiting amyloid ß peptide (Aß) amyloidosis and a neuroinflammatory response in an age-dependent manner without non-physiological amyloid precursor protein (APP) overexpression. Mice were subjected to a short behavioral test battery consisting of an elevated plus maze, a balance beam test, and a tail suspension test seven days after exposure to 8.0% desflurane for 6 h or 2.8% sevoflurane for 2 h. App-KI mice showed significant increments in the percentage of entry and time spent in open arms in the elevated plus maze, increments in the number of slips and latency to traverse for the balance beam test, increments in the limb clasping score, increments in immobile duration, and decrements in latency to first immobile episode for the tail suspension test compared to age-matched wild type (WT) controls. Desflurane- and sevoflurane-exposed App-KI mice showed a delayed decrement in the number of slips for each trial in the balance beam test, while air-treated App-KI mice rapidly improved their performance, and increased their clasping behavior in the tail suspension test. Furthermore, App-KI inhibited the change in membrane GluA3 following exposure to anesthetics in the cerebellum. These results suggest high validity of App-KI mice as an animal model of AD.

Fibroblast growth factor 2 induces apoptosis in the early primary culture of rat cortical neurons.

In the central nervous system, fibroblast growth factor 2 (FGF2) is known to have important functions in cell survival and differentiation. In addition to its roles as a neurotrophic factor, we found that FGF2 caused cell death in the early primary culture of cortical neurons. FGF2-induced neuronal cell death showed apoptotic characters, e.g., chromatin condensation and DNA fragmentation. The ultrastructural morphology of FGF2-treated neurons indicated apoptotic features such as progressive cell shrinkage, blebbing of the plasma membrane, loss of cytosolic organelles, clumping of chromatin, and fragmentation of DNA. Tyrosine kinase inhibitors significantly rescued neurons from FGF2-induced apoptosis. FGF2 potentiated a marked influx of Ca(2+) into neurons before apoptosis. Both a calcium chelator and L-type voltage-sensitive Ca(2+) channel (L-VSCC) blockers attenuated FGF2-induced apoptosis, whereas other blockers of VSCCs such as N-type and P/Q-types did not. Blockers of L-VSCCs significantly suppressed FGF2-enhanced Ca(2+) influx into neurons. Moreover, FGF2 also generated reactive oxygen species (ROS) before apoptosis. Radical scavengers reduced not only the FGF2-generated ROS, but also the FGF2-induced Ca(2+) influx and apoptosis. In conclusion, we demonstrated that FGF2 caused apoptosis via L-VSCCs in the early neuronal culture.

Gonadal steroids maintain 24 h acetylcholine release in the hippocampus: organizational and activational effects in behaving rats.

Extracellular acetylcholine (ACh) levels in the dorsal hippocampus increases during learning or exploration, exhibiting a sex-specific 24 h release profile. To examine the activational effect of gonadal steroid hormones on the sex-specific ACh levels and its correlation with spontaneous locomotor activity, we observed these parameters simultaneously for 24 h. Gonadectomy severely attenuated the ACh levels, whereas the testosterone replacement in gonadectomized males or 17beta-estradiol replacement in gonadectomized females successfully restored the levels. 17beta-Estradiol-priming in gonadectomized males could not restore the ACh levels, and testosterone replacement in gonadectomized females failed to raise ACh levels to those seen in testosterone-primed gonadectomized males, revealing a sex-specific activational effect. Spontaneous locomotor activity was not changed in males by gonadectomy or the replacement of gonadal steroids, but 17beta-estradiol enhanced the activity in gonadectomized females. Gonadectomy severely reduced the correlation between ACh release and activity levels, but the testosterone replacement in gonadectomized males or 17beta-estradiol replacement in gonadectomized females successfully restored it. To further analyze the sex-specific effect of gonadal steroids, we examined the organizational effect of gonadal steroids on the ACh release in female rats. Neonatal testosterone or 17beta-estradiol treatment not only increased the ACh levels but also altered them to resemble male-specific ACh release properties without affecting levels of spontaneous locomotor activity. We conclude that the activational effects of gonadal steroids maintaining the ACh levels and the high correlation with spontaneous locomotor activity are sex-specific, and that the organizational effects of gonadal steroids suggest estrogen receptor-mediated masculinization of the septo-hippocampal cholinergic system.

Altered behavior in mice overexpressing soluble ST2.

Psychoneuroimmunological studies have clearly demonstrated that both cellular and humoral immunity are related to major depression. Soluble ST2 is regarded as a key molecule regulating immune system as well as cell proliferation. Indeed, soluble ST2 is reported to reduce IL-33-induced IL-6 and TNF-α production in macrophages and IL-33-induced IL-5 and IL-13 production in type 2 innate lymphoid cells. Elevated serum concentrations of soluble ST2 have been reported in patients with neuropsychiatric disorders, suggesting pathophysiological roles of soluble ST2 in behavioral phenotypes. Nevertheless, the relation between soluble ST2 and depressive behavior remain to be uncovered. To complement this point, we performed broad behavioral phenotyping, utilizing transgenic mice with a high concentration of serum ST2 in the present study. Soluble ST2 overexpression mice (ST2 Tg mice) were generated on a C3H/HeJ background. ST2 Tg mice crossed onto the BALB/c genetic background were used. Before starting tests, each mouse was observed in a clean cage for a general health check and neurological screening tests. In Experiment I, comprehensive behavioral phenotyping was performed to reveal the role of soluble ST2 on sensorimotor functions, anxiety-like behaviors, depression-like behaviors, social behaviors, and learning and memory functions. In Experiment II, to confirm the role of soluble ST2 on depression-like behaviors, a depression test battery (two bottle choice test, forced swimming test, and tail suspension test) was applied. The general health check indicated good general health and normal gross appearance for ST2 Tg mice. Further, the neurological reflexes of all the mice were normal. We found that soluble ST2 overexpression resulted in decreased social interaction. Moreover, depression-like behaviors of ST2 Tg mice were observed in two well-established behavioral paradigms, the forced swimming test and the tail suspension test. Nevertheless, hedonic reaction to sucrose was observed in ST2 Tg mice similar to WT mice. These results suggest the depression in the ST2 Tg mice. In conclusion, through a series of experiments, we established the animal model for assessing role of soluble ST2 in neuropsychiatric disorders, and revealed the possible involvement of soluble ST2 in depressive behavior.

Differential Roles of Each Orexin Receptor Signaling in Obesity.

Orexins are hypothalamic neuropeptides that regulate feeding, energy expenditure, and sleep. Although orexin-deficient mice are susceptible to obesity, little is known about the roles of the orexin receptors in long-term energy metabolism. Here, we performed the metabolic characterization of orexin receptor-deficient mice. Ox1r-deficient mice were resistant to diet-induced obesity, and their food intake was similar between chow and high-fat food. Ox2r-deficient mice exhibited less energy expenditure than wild-type mice when fed a high-fat diet. Neither Ox1r-deficient nor Ox2r-deficient mice showed body weight gain similar to orexin-deficient mice. Although the presence of a running wheel suppressed diet-induced obesity in wild-type mice, the effect was weaker in orexin neuron-ablated mice. Finally, we did not detect abnormalities in brown adipose tissues of orexin-deficient mice. Thus, each orexin receptor signaling has a unique role in energy metabolism, and orexin neurons are involved in the interactive effect of diet and exercise on body weight gain.

Gonadal steroid hormones maintain the stress-induced acetylcholine release in the hippocampus: simultaneous measurements of the extracellular acetylcholine and serum corticosterone levels in the same subjects.

To examine the role of gonadal steroid hormones in the stress responses of acetylcholine (ACh) levels in the hippocampus and serum corticosterone levels, we observed these parameters simultaneously in intact, gonadectomized, or gonadectomized steroid-primed rats. In both sexes of rats, neither gonadectomy nor the replacement of gonadal steroid hormone affected the baseline levels of ACh. However, gonadectomy severely attenuated the stress response of ACh, whereas the replacement of corresponding gonadal hormone successfully restored the response to intact levels. The gonadal hormones affected the serum corticosterone levels in a different manner; the testosterone replacement in orchidectomized rats suppressed the baseline and the stress response of corticosterone levels, whereas the 17beta-estradiol replacement in ovariectomized rats increased the levels. We further found that letrozole or flutamide administration in intact male rats attenuated the stress response of ACh. In addition, flutamide treatment increased the baseline levels of corticosterone, whereas letrozole treatment attenuated the stress response of corticosterone. Moreover, we found a low positive correlation between the ACh levels and corticosterone levels, depending on the presence of gonadal steroid hormone. We conclude that: 1) gonadal steroid hormones maintain the stress response of ACh levels in the hippocampus, 2) the gonadal steroid hormone independently regulates the stress response of ACh in the hippocampus and serum corticosterone, and 3) the sex-specific action of gonadal hormone on the cholinergic stress response may suggest a neonatal sexual differentiation of the septohippocampal cholinergic system in rats.

Postpubertal feeding experience affects sex-specific spatial ability in rats.

We previously reported that feeding with powdered diet after weaning (3 weeks of age) enhanced spatial ability, and increased the amount of acetylcholine (ACh) released in the dorsal hippocampus in female rats. In the present study, to specify the time when feeding conditions caused these effects, a radial 8-arm maze task and an in vivo microdialysis study were performed in both sexes of rats. In rats fed standard laboratory diet (i.e., pelleted diet), males learned the radial 8-arm maze faster than females. Moreover, the ACh release showed a distinct diurnal rhythm which was high during the dark phase and low during the light phase, but males showed a greater amount of ACh released in the dorsal hippocampus than females. However, in rats fed powdered diet after 6 weeks of age, no significant sex differences were observed in the radial 8-arm maze task or in the amount of ACh released, since feeding with powdered diet enhanced spatial ability, and increased the amount of ACh released only in females. These results suggest that feeding conditions after 6 weeks of age may contribute to the sex difference in the spatial ability associated with the changes in the amount of ACh released in the dorsal hippocampus in rats.

Sex difference in the 24-h acetylcholine release profile in the premotor/supplementary motor area of behaving rats.

The sex differences in various motor functions suggest a sex-specific neural basis in the nonprimary or primary motor area. To examine the sex difference in the 24-h profile of acetylcholine (ACh) release in the rostral frontal cortex area 2 (rFr2), which is equivalent to the premotor/supplementary motor area in primates, we performed an in vivo microdialysis study in both sexes of rats fed pelleted or powdered diet. The dialysate was automatically collected from the rFr2 for 24 h under freely moving conditions. Moreover, the number of cholinergic neurons in the nucleus basalis magnocellularis (NBM) was examined. Further, to confirm the relation between ACh release in the rFr2 and motor function, the spontaneous locomotor activity was monitored for 24 h. Both sexes showed a distinct 24-h rhythm of ACh release, which was high during the dark phase and low during the light phase. Female rats, however, showed a greater ACh release and more cholinergic neurons in the NBM than male rats. Similarly, spontaneous locomotor activity also showed a 24-h rhythm, which paralleled the changes in ACh release in both sexes, and these changes were again greater in female rats than in male rats. In addition, feeding with powdered diet significantly increased the ACh release and spontaneous locomotor activity. The present study is the first to report the sex difference in the 24-h profile of ACh release in the rFr2 in rats. The sex specific ACh release in the rFr2 may partly contribute to the sex difference in motor function in rats.

Neurotransmitters and neuropeptides in gonadal steroid receptor-expressing cells in medial preoptic area subregions of the male mouse.

Testosterone is involved in male sexual, parental and aggressive behaviors through the androgen receptor (AR) and estrogen receptor (ER) α expressed in the brain. Although several studies have demonstrated that ERα and AR in the medial preoptic area (MPOA) are required for exhibiting sexual and aggressive behaviors of male mice, the molecular characteristics of ERα- and AR-expressing cells in the mouse MPOA are largely unknown. Here, we performed in situ hybridization for neurotransmitters and neuropeptides, combined with immunohistochemistry for ERα and AR to quantitate and characterize gonadal steroid receptor-expressing cells in the MPOA subregions of male mice. Prodynorphin, preproenkephalin (Penk), cocaine- and amphetamine-related transcript, neurotensin, galanin, tachykinin (Tac)1, Tac2 and thyrotropin releasing hormone (Trh) have distinct expression patterns in the MPOA subregions. Gad67-expressing cells were the most dominant neuronal subtype among the ERα- and AR-expressing cells throughout the MPOA. The percentage of ERα- and AR-immunoreactivities varied depending on the neuronal subtype. A substantial proportion of the neurotensin-, galanin-, Tac2- and Penk-expressing cells in the MPOA were positive for ERα and AR, whereas the vast majority of the Trh-expressing cells were negative. These results suggest that testosterone exerts differential effects depending on both the neuronal subtypes and MPOA subregions.

Moxd1 Is a Marker for Sexual Dimorphism in the Medial Preoptic Area, Bed Nucleus of the Stria Terminalis and Medial Amygdala.

The brain shows various sex differences in its structures. Various mammalian species exhibit sex differences in the sexually dimorphic nucleus of the preoptic area (SDN-POA) and parts of the extended amygdala such as the principal nucleus of the bed nucleus of the stria terminalis (BNSTpr) and posterodorsal part of the medial amygdala (MePD). The SDN-POA and BNSTpr are male-biased sexually dimorphic nuclei, and characterized by the expression of calbindin D-28K (calbindin 1). However, calbindin-immunoreactive cells are not restricted to the SDN-POA, but widely distributed outside of the SDN-POA. To find genes that are more specific to sexually dimorphic nuclei, we selected candidate genes by searching the Allen brain atlas and examined the detailed expressions of the candidate genes using in situ hybridization. We found that the strong expression of monooxygenase DBH-like 1 (Moxd1) was restricted to the SDN-POA, BNSTpr and MePD. The numbers of Moxd1-positive cells in the SDN-POA, BNSTpr and MePD in male mice were larger than those in female mice. Most of the Moxd1-positive cells in the SDN-POA and BNSTpr expressed calbindin. Neonatal castration of male mice reduced the number of Moxd1-positive cells in the SDN-POA, whereas gonadectomy in adulthood did not change the expression of the Moxd1 gene in the SDN-POA in both sexes. These results suggest that the Moxd1 gene is a suitable marker for sexual dimorphic nuclei in the POA, BNST and amygdala, which enables us to manipulate sexually dimorphic neurons to examine their roles in sex-biased physiology and behaviors.

High-fat diet feeding alters olfactory-, social-, and reward-related behaviors of mice independent of obesity.

OBJECTIVE: High-fat diet (HFD) consumption causes obesity, which is associated with well-known increased health risks. Moreover, obesity has been associated with altered sensorimotor and emotional behaviors of humans and mice. This study attempted to dissociate the influence of HFD-induced obesity on behaviors from the influence of HFD consumption itself. METHODS: C57BL male mice were randomly allocated to a low-fat diet (LFD) group, an HFD-induced obesity (DIO) group, or a pair-fed HFD-feeding nonobese (HFD) group. A comprehensive behavioral test battery was performed on all three groups to assess sensorimotor functions, anxiety- and depression-like behaviors, reward-related behaviors, social behaviors, and learning/memory functions. RESULTS: Both the DIO and HFD groups exhibited disturbed olfaction, blunted ethanol preference, and enhanced social interactions. The DIO group exhibited blunted sucrose preference, shorter latency before falling off during the rotarod test, and a lower response to mechanical stimuli. CONCLUSIONS: The HFD-fed nonobese mice showed altered behaviors related to olfaction, social interactions, and rewards that were similar to those of the DIO mice. This finding suggests that HFD consumption alters a variety of behaviors independent of obesity.

Feeding with powdered diet after weaning increases visuospatial ability in association with increases in the expression of N-methyl-D-aspartate receptors in the hippocampus of female rats.

We determined whether feeding with powdered diet improved the visuospatial ability in female rats by checking the expression of N-methyl-D-aspartate receptor (NMDAR) subunit 1 (NR1) mRNA in the hippocampus. In rats fed standard pelleted diet, males performed better than females in a radial 8-arm maze task as we reported previously. We found that the expression of NR1 mRNA, which may be the key mediator in visuospatial ability in the hippocampus, was also higher in males than in females. However, in rats fed powdered diet, no sex difference was seen in the radial 8-arm maze task and the expression of NR1 mRNA in the hippocampus, since feeding with powdered diet improved the visuospatial ability with increases in the expression of NR1 mRNA in the hippocampus in females. We suggest that the sex difference in visuospatial ability is at least in part due to feeding conditions.

Postanesthetic effects of isoflurane on behavioral phenotypes of adult male C57BL/6J mice.

Isoflurane was previously the major clinical anesthetic agent but is now mainly used for veterinary anesthesia. Studies have reported widespread sites of action of isoflurane, suggesting a wide array of side effects besides sedation. In the present study, we phenotyped isoflurane-treated mice to investigate the postanesthetic behavioral effects of isoflurane. We applied comprehensive behavioral test batteries comprising sensory test battery, motor test battery, anxiety test battery, depression test battery, sociability test battery, attention test battery, and learning test battery, which were started 7 days after anesthesia with 1.8% isoflurane. In addition to the control group, we included a yoked control group that was exposed to the same stress of handling as the isoflurane-treated animals before being anesthetized. Our comprehensive behavioral test batteries revealed impaired latent inhibition in the isoflurane-treated group, but the concentration of residual isoflurane in the brain was presumably negligible. The yoked control group and isoflurane-treated group exhibited higher anxiety in the elevated plus-maze test and impaired learning function in the cued fear conditioning test. No influences were observed in sensory functions, motor functions, antidepressant behaviors, and social behaviors. A number of papers have reported an effect of isoflurane on animal behaviors, but no systematic investigation has been performed. To the best of our knowledge, this study is the first to systematically investigate the general health, neurological reflexes, sensory functions, motor functions, and higher behavioral functions of mice exposed to isoflurane as adults. Our results suggest that the postanesthetic effect of isoflurane causes attention deficit in mice. Therefore, isoflurane must be used with great care in the clinical setting and veterinary anesthesia.

Developmental trajectory of contextual learning and 24-h acetylcholine release in the hippocampus.

To determine the developmental trajectory of hippocampal function in rats, we examined 24-h changes in extracellular acetylcholine (ACh) levels and contextual learning performance. Extracellular ACh significantly correlated with spontaneous behavior, exhibiting a 24-h rhythm in juvenile (4-week-old), pubertal (6-week-old), and adult (9- to 12-week-old) rats. Although juveniles of both sexes exhibited low ACh levels, adult males had higher ACh levels than adult females. Moreover, juveniles exhibited much more spontaneous activity than adults when they showed equivalent ACh levels. Similarly, juveniles of both sexes exhibited relatively low contextual learning performance. Because contextual learning performance was significantly increased only in males, adult males exhibited better performance than adult females. We also observed a developmental relationship between contextual learning and ACh levels. Scopolamine pretreatment blocked contextual learning and interrupted the correlation. Since long-term scopolamine treatment after weaning impaired contextual learning in juveniles, the cholinergic input may participate in the development of hippocampus.

A subset of thalamocortical projections to the retrosplenial cortex possesses two vesicular glutamate transporter isoforms, VGluT1 and VGluT2, in axon terminals and somata.

Vesicular glutamate transporter isoforms, VGluT1-VGluT3, accumulate glutamate into synaptic vesicles and are considered to be important molecules in glutamatergic transmission. Among them, VGluT2 mRNA is expressed predominantly throughout the dorsal thalamus, whereas VGluT1 mRNA is expressed in a few thalamic nuclei. In the thalamic nuclei that project to the retrosplenial cortex (RSC), VGluT1 mRNA is expressed strongly in the anterodorsal thalamic nucleus (AD), is expressed moderately in the anteroventral and laterodorsal thalamic nuclei, and is not expressed in the anteromedial thalamic nucleus. Thus, it has been strongly suggested that a subset of thalamocortical projections to RSC possesses both VGluT1 and VGluT2. In this study, double-labeled neuronal somata showing both VGluT1 and VGluT2 immunolabelings were found exclusively in the ventral region of AD (vAD). Many double-labeled axon terminals were also found in two major targets of vAD, the rostral part of the reticular thalamic nucleus and layers Ia and III-IV of the retrosplenial granular b cortex (RSGb). Some were also found in layer Ia of the retrosplenial granular a cortex (RSGa). These axon terminals contain significant amounts of both VGluTs. Because the subset of thalamocortical projections to RSC has a unique molecular basis in the glutamatergic transmission system, it might play an important role in the higher cognitive functions processed in the RSC. Furthermore, double-labeled axon terminals of a different type were distributed in RSGb and RSGa. Because they are small and the immunoreactivity of VGluT2 is significantly weaker than that of VGluT1, they seemed to be a subset of corticocortical terminals.