General-Purpose Ultrasound Neuromodulation System for Chronic, Closed-Loop Preclinical Studies in Freely Behaving Rodents.

Transcranial focused ultrasound stimulation (tFUS) is an effective noninvasive treatment modality for brain disorders with high clinical potential. However, the therapeutic effects of ultrasound neuromodulation are not widely explored due to limitations in preclinical systems. The current preclinical studies are head-fixed, anesthesia-dependent, and acute, limiting clinical translatability. Here, this work reports a general-purpose ultrasound neuromodulation system for chronic, closed-loop preclinical studies in freely behaving rodents. This work uses microelectromechanical systems (MEMS) technology to design and fabricate a small and lightweight transducer capable of artifact-free stimulation and simultaneous neural recording. Using the general-purpose system, it can be observed that state-dependent ultrasound neuromodulation of the prefrontal cortex increases rapid eye movement (REM) sleep and protects spatial working memory to REM sleep deprivation. The system will allow explorative studies in brain disease therapeutics and neuromodulation using ultrasound stimulation for widespread clinical adoption.

Oxytocin modulation in the medial prefrontal cortex of pair-exposed rats during fear conditioning.

INTRODUCTION: Social buffering is the phenomenon, in which stress and fear reactions caused by exposure to stressful stimuli when animals are exposed to homogeneous relationships are attenuated. Social buffering reduces fear memory behavior such as escape, avoidance, and freezing behavior in rodents due to social existence. Here, we aimed to determine alterations of fear behavior and neural activity in the medial prefrontal cortex (mPFC) in response to the presence of another rat in fear-exposed conditions and to confirm the role of oxytocin in mPFC in regulating social buffering. METHODS: We performed a passive avoidance test and determined positive c-Fos expression in single- and pair-exposed rats. Anisomycin (a protein synthesis inhibitor) and oxytocin receptor regulators (carbetocin; agonist and atosiban; antagonist) were microinjected into the mPFC to clarify the role of oxytocin in the mPFC. RESULTS: While single-exposed rats showed a significant increase in both freezing and passive avoidance behaviors compared to control rats, pair-exposed rats showed significantly less fear behavior compared to single-exposed rats. The c-Fos expression in the prelimbic (PL) mPFC was significantly increased in pair-exposed rats compared to that in control and single-exposed rats. The pair-exposed effect was blocked by anisomycin injections into the PL mPFC of pair-exposed rats. Furthermore, when a carbetocin was injected into the PL mPFC in single-exposed rats, fear behavior was decreased, and these changes were blocked by atosiban. DISCUSSION: Our findings suggest that reduction of fear-related behavior induced by acute pair-exposure is mediated by oxytocin receptors in the PL mPFC. Pair exposure with conspecifics during fear-inducing situations helps coping with fear by significantly increasing the role of oxytocin in the PL mPFC.

Alteration of fear behaviors in sleep-deprived adolescent rats: increased fear expression and delayed fear extinction.

Disruption of sleep due to acute or chronic stress can lead to changes in emotional memory processing. Sleep disturbances are highly prevalent in post-traumatic stress disorder (PTSD), but still, the contribution of sleep deprivation on the susceptibility to PTSD has received little attention. To determine whether rapid eye movement sleep deprivation (SD) alters the development of fear expression or fear-associated memory impairment in adolescent rats, we performed animal emotional behavior tests using an SD animal model with the flowerpot technique. SD rats showed an increase in locomotor activity frequency and a decrease in sucrose consumption compared to control rats. An increase in freezing behavior during shock trials was observed in SD rats. Noticeably, it was observed that when applying the SD condition after fear stimuli exposure, fear extinction was delayed more in SD rats than in control rats. Overall, these results indicate that SD in adolescent rats leads to increased locomotor activity and anhedonic behavior, as well as increased fear expression and delayed fear extinction, suggesting that SD would lead to increased severity of PTSD-like phenotype.

Role of Medial Prefrontal Cortical Neurons and Oxytocin Modulation in the Establishment of Social Buffering.

Fear-related behaviors are rigidly controlled by the medial prefrontal cortex (mPFC). The mPFC is activated by the prosocial hormone oxytocin, which plays an important role in social buffering. We used a slice patch current-clamp recording in single- and pair-exposed rats who were subjected to electric shocks, to determine the cellular mechanism of the action of oxytocin in the mPFC under social buffering conditions. Pair-exposed rats showed a significant reduction in both freezing and passive avoidance behaviors compared to single-exposed rats. It was observed that input resistance in pyramidal neurons decreased in both single- and pair-exposed rats than naïve rats, but input resistance in interneurons increased in pair-exposed rats than single-exposed rats. We found that the number of action potential (AP) spikes in the mPFC pyramidal neurons decreased significantly in pair-exposed rats than in single-exposed rats. The pyramidal neurons in the mPFC were similarly regulated by oxytocin in singleand pair-exposed rats, while the number of AP spikes in interneurons by oxytocin decreased in single-exposed rats, but there was no significant change in pair-exposed rats. Therefore, our findings reveal that a decrease in mPFC pyramidal neuronal activity in pair-exposed rats through social interaction induces a reduction in fear-related behavior via obstruction of fear-memory formation; however, no such reduction was observed in single-exposed rats. Moreover, we suggest that the oxytocin-mediated decrease in neuronal activity in the mPFC could facilitate social buffering.

Oxytocin-induced anxiogenic behavior in juvenile male rats.

Oxytocin (OT) is considered beneficial to mental health owing to its anxiolytic, prosocial, and anti-stress effects; however, the adverse effects of OT have been controversial, such as its potentially anxiogenic actions. Although OT influences drug abuse and reciprocally affects vulnerability to drug use, the relationship between OT's anxiogenic working and nicotine preference intake has not been clearly defined. To clarify this issue, the effect of acute peripheral administration of OT on anxiety and nicotine preference was investigated in juvenile male rats. Anxiogenic behaviors were noticeably increased in OT-administrated rats, with an increase in serum corticosterone levels. Moreover, increased anxiety-like behaviors and corticosterone levels were observed in the OT analog carbetocin-injected rats. In the nicotine preference test, the rats' aversive responses to initial nicotine choice and preference were not significantly different between saline-injected and OT-injected rats. However, when administered with OT, there was a significant negative correlation between anxiety-like behavior and low-dose nicotine consumption. Collectively, these results provide evidence that acute OT exposure could induce anxiogenic behavior with corticosterone augmentation, contributing to the attenuation of nicotine preference. This suggests that both aspects of OT, as well as their benefits and drawbacks, should be considered.

Determining nicotine-related behavior changes in juvenile female rats through long-term maternal nicotine exposure.

Nicotine replacement therapy (NRT) has been developed as a drug therapy for smoking cessation and has been considered a safe alternative to smoking during pregnancy. However, the effects of long-term nicotine exposure via NRT on the fetus are still being debated. Here, we determined the effects of long-term maternal nicotine exposure in gestation and lactation on nicotine-related behavior and drug vulnerability in dams and offspring rats. To expose long-term nicotine, on gestation day 14, pregnant rats were implanted with osmotic minipumps releasing nicotine tartrate (6 mg/kg/day, subcutaneously, equivalent to 2 mg nicotine-freebase) for 28 days. The concentration of cotinine in blood was 373.0 ± 109.0 ng/ml in dams and 12.50 ± 1.19 ng/ml in offspring rats. In dams, we found no significant differences in anxiety-like behaviors and various maternal behaviors such as touching, sniffing, pup licking, laying on pups, and retrieval between saline- and nicotine-exposed groups. Adolescent offspring female rats showed no significant differences in anxiety-like behavior and forced alcohol consumption between saline- and nicotine-exposed groups. Nicotine-exposed offspring rats showed more increased nicotine aversion than saline-exposed groups, but the effect was disturbed in the forced alcohol consumption condition on the first day of the nicotine consumption test. Taken together, these results suggest that, in the last gestation and lactation period corresponding to the second and third trimester of human pregnancy, long-term maternal nicotine exposure has a minor effect on dam and female offspring health and does not involve serious pathological changes in rat offspring, despite the presence of nicotine in their blood.

Sex differential effect of dexmedetomidine on fear memory extinction and anxiety behavior in adolescent rats.

Exposure to stressful stimuli, including fear and anxiety, modulates the central noradrenergic system. Dexmedetomidine is a commonly used α2-adrenoreceptor agonist. Because the effect of fear acquisition varies between sexes, the present study was designed to investigate sex-related differences in the effects of dexmedetomidine on fear memory and anxiety-like behavior. We conducted a fear test and an elevated plus maze test in 6-8-week-old male and female rats. Two doses of dexmedetomidine (20 and 40 µg/kg) were injected intraperitoneally three times at 24 h intervals after the tests: after fear expression, extinction 1, and extinction 2. The repeated administration of dexmedetomidine showed significant acceleration of fear memory extinction in female rats but not in male rats; the effect increased proportionally to concentrations of dexmedetomidine. Compared to male rats, female rats treated with both concentrations of dexmedetomidine showed significant anxiolytic behavior after 1 week. Dexmedetomidine accelerated the fear memory extinction and reduced anxiety; it was more effective in female rats than in male rats. Our results suggest that dexmedetomidine may exert protective effects against fear-related and anxiety-like behaviors in rats with fear memory after repeated administration, and the sex-specific effects of dexmedetomidine should be considered.

The link between social context-dependent anxious behavior and habenular mast cells in fear-conditioned rats.

Affiliative social behavior relieves the physiological reactivity to stressors, while social inequity, represented by unfairness in the social environment, causes emotional distress in animals. Mast cells are immune cells found in the brain that affect both the nervous system and emotional behavior. To determine the role of neuro-immunity in the programming of emotional behaviors, we observed brain mast cells and anxiety-like behaviors in female rats exposed to electrical foot shocks in different social environments. The following groups of rats were used in this study: control (unshocked) rats, solitarily shock-exposed rats, and shock-exposed rats in the presence of unshocked (unequal) or shocked (equal) conspecifics. An absence of significant difference in body weight or sucrose preference was seen among the different groups. Additionally, fear memory was augmented in rats shocked in the presence of either unshocked or shocked conspecifics than rats in the solitarily shocked group. Furthermore, rats shocked in the presence of unshocked conspecifics showed intensified anxiety-like behaviors after fear conditioning. Finally, we found an increase in the number of habenular mast cells in the intensified anxiogenic group, which had a significant correlation with the decreasing rate of anxiety-like behaviors. This provides evidence that habenular mast cells might be of importance in relieving the amplified biopsychological responses caused by social stress.

Alteration of adolescent aversive nicotine response and anxiety-like behavior in nicotine-exposed rats during late lactation period.

Early nicotine exposure is an important cause of further habitual tobacco smoking. Although nicotine has not only rewarding but also aversive properties, the effects of early nicotine exposure on the distinct properties of nicotine are not well known. To reveal the effects of early adolescent nicotine exposure on further persistent tobacco smoking, we demonstrated developmental changes in nicotine-related appetitive and aversive behaviors of rats exposed to nicotine during the late lactation period. Sprague-Dawley rats were injected with saline or nicotine (2, 6 and 12mg/kg). We performed a two bottle free-choice test using escalating doses of nicotine (25, 50 and 100µg/ml), saccharin and quinine and the open field test in both adolescent and adult rats. The rats' aversive response to nicotine was increased according to the increase in nicotine concentration. Adolescent rats showed higher nicotine preference and consumption behaviors than did adult rats at an aversive dose of nicotine. Nicotine-exposed rats increased adolescent nicotine consumption when the nicotine concentration was 12mg/kg. We observed significant increases in anxious behaviors in adolescent nicotine-injected rats compared to saline-injected rats, but there were no alterations in adult rats. In both adolescent and adult rats, saccharin and quinine intake were not significantly different between groups. Taken together, it suggests that repeated nicotine exposure in late lactation period affect changes in aversive nicotine responses and anxious behaviors during adolescence but there is no difference in adults.