A neural circuit associated with anxiety-like behaviors induced by chronic inflammatory pain and the anxiolytic effects of electroacupuncture.

AIMS: Negative emotions induced by chronic pain are a serious clinical problem. Electroacupuncture (EA) is a clinically proven safe and effective method to manage pain-related negative emotions. However, the circuit mechanisms underlying the effect of EA treatment on negative emotions remain unclear. METHODS: Plantar injection of complete Freund's adjuvant (CFA) was performed to establish a rat model of chronic inflammatory pain-induced anxiety-like behaviors. Adeno-associated virus (AAV) tracing was used to identify excitatory synaptic transmission from the rostral anterior cingulate cortex (rACC) to the dorsal raphe nucleus (DRN). Employing chemogenetic approaches, we examined the role of the rACC-DRN circuit in chronic pain-induced anxiety-like behaviors and investigated whether EA could reverse chronic pain-induced dysfunctions of the rACC-DRN circuit and anxiety-like behaviors. RESULTS: We found that chemogenetic activation of the rACC-DRN circuit alleviated CFA-induced anxiety-like behaviors, while chemogenetic inhibition of the rACC-DRN circuit resulted in short-term CFA-induced anxiety-like behaviors. Further research revealed that the development of CFA-induced anxiety-like behaviors was attributed to the dysfunction of rACC CaMKII neurons projecting to DRN serotonergic neurons (rACCCaMKII-DRN5-HT neurons) but not rACC CaMKII neurons projecting to DRN GABAergic neurons (rACCCaMKII-DRNGABA neurons). This is supported by the findings that chemogenetic activation of the rACCCaMKII-DRN5-HT circuit alleviates anxiety-like behaviors in rats with chronic pain, whereas neither chemogenetic inhibition nor chemogenetic activation of the rACCCaMKII-DRNGABA circuit altered CFA chronic pain-evoked anxiety-like behaviors in rats. More importantly, we found that EA could reverse chronic pain-induced changes in the activity of rACC CaMKII neurons and DRN 5-HTergic neurons and that chemogenetic inhibition of the rACCCaMKII-DRN5-HT circuit blocked the therapeutic effects of EA on chronic pain-induced anxiety-like behaviors. CONCLUSIONS: Our data suggest that the reversal of rACCCaMKII-DRN5-HT circuit dysfunction may be a mechanism underlying the therapeutic effect of EA on chronic pain-induced anxiety-like behaviors.

Nanoassemblies from the aqueous extract of roasted coffee beans modulate the behavioral and molecular effects of smoking withdrawal-induced anxiety in female rats.

Antioxidant-rich plant extracts have demonstrated tremendous value as inflammatory modulators and as nanomaterial precursors. Chronic cigarette smoking alters neurotransmitter systems, particularly the glutamatergic system, and produces neuroinflammation. This study aimed to investigate the behavioral and molecular correlates of cigarette smoking withdrawal-induced anxiety-like behavior in rats, and whether these effects could be mitigated by the administration of antioxidant nanoassemblies prepared by spontaneous oxidation of dark-roasted Arabica coffee bean aqueous extracts. Four experimental groups of female Sprague-Dawley rats were randomly assigned to: (i) a control group that was only exposed to room air, (ii) a COF group that was administered 20 mg/kg of the coffee nanoassemblies by oral gavage, (iii) a SMOK group that was exposed to cigarette smoke and was given an oral gavage of distilled water, (iv) and a SMOK + COF group that was exposed to cigarette smoke and administered 20 mg/kg of the coffee nanoassemblies. Animals were exposed to cigarette smoke for 2 h per day, five days per week, with a 2-day withdrawal period each week. At the end of the 4th week, rats began receiving either distilled water or the coffee nanoassemblies before being exposed to cigarette smoke for 21 additional days. Weekly behavioral tests revealed that cigarette smoking withdrawal exacerbated anxiety, while the administration of the coffee nanoassemblies reduced this effect. The effect of cigarette smoking on astroglial glutamate transporters and nuclear factor kappa B (NF-κB) expression in brain subregions was also measured. Smoking reduced the relative mRNA and protein levels of the glutamate transporter 1 (GLT-1) and the cystine/glutamate antiporter (xCT), and increased the levels of NF-κB, but these effects were attenuated by the coffee nanoassemblies. Thus, administration of the antioxidant nanoassemblies decreased the negative effects of cigarette smoke, which included neuroinflammation, changes in glutamate transporters' expression, and a rise in anxiety-like behavior.

CaMKIIa Neurons of the Ventromedial Hypothalamus Mediate Wakefulness and Anxiety-like Behavior.

Insomnia and anxiety are two common and closely related clinical problems that pose a threat to individuals' physical and mental well-being. There is a possibility that some nuclei and neural circuits in the brain are shared by both insomnia and anxiety. In the present study, using a combination of chemogenetics, optogenetics, polysomnographic recordings and the classic tests of anxiety-like behaviors, we verified that the calmodulin-dependent protein kinase II alpha (CaMKIIa) neurons of the ventromedial hypothalamus (VMH) are involved in the regulation of both wakefulness and anxiety. Chemogenetic manipulation of the VMH CaMKIIa neurons elicited an apparent increase in wakefulness during activation, whereas inhibition decreased wakefulness mildly. It substantiated that the VMH CaMKIIa neurons contribute to wakefulness. Then in millisecond-scale control of neuronal activity, short-term and long-term optogenetic activation induced the initiation and maintenance of wakefulness, respectively. We also observed that mice reduced exploratory behaviors in classic anxiety tests while activating the VMH CaMKIIa neurons and were anxiolytic while inhibiting. Additionally, photostimulation of the VMH CaMKIIa axons in the paraventricular hypothalamus (PVH) mediated wakefulness and triggered anxiety-like behaviors as well. In conclusion, our results demonstrate that the VMH participates in the control of wakefulness and anxiety, and offer a neurological explanation for insomnia and anxiety, which may be valuable for therapeutic interventions such as medication and transcranial magnetic stimulation.

Effects of endocrine disruptors on the electrical activity of leptin receptor neurons in the dorsomedial hypothalamus and anxiety-like behavior in male mice.

There is increasing concern about the effects of endocrine disrupting chemicals (EDCs) on human health. Recently, some EDCs are suggested to affect energy metabolism leading to increased risk of obesity. Obesogenic effects of some EDCs on adipogenesis have been reported, however, there is no study examining their potential actions on the brain circuits controlling feeding and metabolism. We have investigated effects of tributyltin (TBT) and dichlorodiphenyltrichloroethane (p,p'-DDT) on electrical activity on dorsomedial hypothalamic leptin receptor neurons (DMHLepR), morphological adaptations in neuronal anatomy of DMHLepR, locomotion, and anxiety-like behaviors in mice. Twenty-three Lep-Cre transgenic mice were intracranially injected with GFP virus. Control animals received intraperitoneal corn oil alone while group 2 and 3 received TBT (25 µg/kg) and p,p'-DDT (2 mg/kg) for one month. Locomotor activity and anxiety-like behavior of the animals were determined by open field test. Electrophysiological effects of TBT and p,p'-DDT on DMHLepR neurons were determined by patch clamp method. Neuronal anatomy was determined by confocal microscopy. Spontaneous firing frequency of DMHLepR neurons of TBT group of mice was significantly higher than both p,p'-DDT and control groups (p < 0.01). TBT and p,p'-DDT significantly decreased frequency of the spontaneous inhibitory post-synaptic currents to DMHLepR neurons compared to the control group (p < 0.05). The time spent in the center and the number of entrances to the center by the TBT-administered mice were significantly lower than other groups (p < 0.01). The total distance traveled and mean speed of the control group of mice were significantly higher than the p,p'-DDT- and TBT-administered animals (p < 0.0001). c-Fos activity of the p,p'-DDT- and TBT-administered animals were significantly elevated compared to the control group (p < 0.001), while no change in the number of dendritic spines were observed. In conclusion, this study demonstrates that exposure to TBT and p,p'-DDT alters electrical activity in DMHLepR neurons and behavioral state in mice.

ATF6ß Deficiency Elicits Anxiety-like Behavior and Hyperactivity Under Stress Conditions.

Activating transcription factor 6 (ATF6) is an endoplasmic reticulum (ER) stress-regulated transcription factor that induces expression of major molecular chaperones in the ER. We recently reported that ATF6ß, a subtype of ATF6, promoted survival of hippocampal neurons exposed to ER stress and excitotoxicity, at least in part by inducing expression of calreticulin, an ER molecular chaperone with high Ca2+-binding capacity. In the present study, we demonstrate that ATF6ß deficiency in mice also decreases calreticulin expression and increases expression of glucose-regulated protein 78, another ER molecular chaperone, in emotional brain regions such as the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala. Comprehensive behavioral analyses revealed that Atf6b-/- mice exhibit anxiety-like behavior in the light/dark transition test and hyperactivity in the forced swim test. Consistent with these results, PFC and hypothalamic corticotropin-releasing hormone (CRH) expression was increased in Atf6b-/- mice, as was circulating corticosterone. Moreover, CRH receptor 1 antagonism alleviated anxiety-like behavior in Atf6b-/- mice. These findings suggest that ATF6ß deficiency produces anxiety-like behavior and hyperactivity via a CRH receptor 1-dependent mechanism. ATF6ß could play a role in psychiatric conditions in the emotional centers of the brain.

Clerodendrum viscosum leaves attenuate lead-induced neurotoxicity through upregulation of BDNF-Akt-Nrf2 pathway in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Clerodendrum viscosum is an important medicinal plant in Ayurveda in Bangladesh and its leaves are used as a remedy for various diseases such as anti-inflammatory, antibacterial, hyperglycemic, hepatoprotective effects. AIM OF THE STUDY: The present study aimed to evaluate the protective effect of aqueous extract of C. viscosum leaves against Pb-induced neurobehavioral and biochemical changes in mice. MATERIALS AND METHODS: Swiss albino mice were divided as a) control, b) lead treated (Pb) and c) C. viscosum leaves (Cle) d) Pb plus Cle groups. Pb-acetate (10 mg/kg body weight) was given to Pb and Pb + Cle groups mice, and water extract of leaves (50 mg/kg body weight) was provided as supplementation to Cle and Pb + Cle groups mice for 30 days. Elevated plus maze and Morris water maze tests were used for evaluating anxiety, spatial memory and learning, respectively. Status of cholinesterase, SOD, GSH enzyme activity and neurotoxicity markers such BDNF and Nrf2 levels were analyzed in the brain tissue of experimental mice. RESULTS: Poorer learning, inferior spatial memory, and increased anxiety-like behavior in Pb-exposure mice were noted when compared to control mice in Morris water maze and elevated plus maze test, respectively. In addition, expression of BDNF and Nrf2, cholinesterase activity along with antioxidant activity were significantly reduced compared to control group (p < 0.01). Interestingly, C. viscosum leaves' aqueous extract supplementation in Pb-exposed mice provide a significant improved neurochemical and antioxidant properties through the augmentation of activity of cholinergic enzymes, and upregulation of BDNF and Nrf2 levels in the brain tissue compared to Pb-exposed mice. CONCLUSIONS: This study suggested that C. viscosum leaves restore the cognitive dysfunction and reduce anxiety-like behavior through upregulation of BDNF mediated Akt-Nrf2 pathway in Pb-exposure mice.

Zebrafish Larvae Behavior Models as a Tool for Drug Screenings and Pre-Clinical Trials: A Review.

To discover new molecules or review the biological activity and toxicity of therapeutic substances, drug development, and research relies on robust biological systems to obtain reliable results. Phenotype-based screenings can transpose the organism's compensatory pathways by adopting multi-target strategies for treating complex diseases, and zebrafish emerged as an important model for biomedical research and drug screenings. Zebrafish's clear correlation between neuro-anatomical and physiological features and behavior is very similar to that verified in mammals, enabling the construction of reliable and relevant experimental models for neurological disorders research. Zebrafish presents highly conserved physiological pathways that are found in higher vertebrates, including mammals, along with a robust behavioral repertoire. Moreover, it is very sensitive to pharmacological/environmental manipulations, and these behavioral phenotypes are detected in both larvae and adults. These advantages align with the 3Rs concept and qualify the zebrafish as a powerful tool for drug screenings and pre-clinical trials. This review highlights important behavioral domains studied in zebrafish larvae and their neurotransmitter systems and summarizes currently used techniques to evaluate and quantify zebrafish larvae behavior in laboratory studies.

[Effect of electroacupuncture on mood and estrogen receptor α expression in mice with polycystic ovary syndrome induced by bisphenol A exposure].

OBJECTIVE: To compare the effect of electroacupuncture (EA) on "Guanyuan" (CV4) or sensitization points in mice with polycystic ovary syndrome (PCOS), so as to explore its mechanisms underlying improvement of PCOS. METHODS: In the first part of this study, 26 female ICR mice were randomized into control group (8 mice) and model group (18 mice). The PCOS model was established by gavage of bisphenol A (BPA) at a dose of 100 mg/kg, and the control group were gavage of equal volume of corn oil, once daily, 5 days a week for 4 conse-cutive weeks. Evans blue (EB) dye (0.1 mL/10 g) was injected into the caudal vein after modeling. The size, number and distribution of EB exudation points at the skin were observed. In the second part of this study, 32 mice were randomized into control, model, EA-CV4 and EA-sensitization points groups (8 mice in each group). EA (2 Hz, 2 mA) was applied to the sensitization points or CV4 for 20 min, once daily, 5 days a week for 4 conse-cutive weeks. The body weight was measured once a week for 8 consecutive weeks. The behavior changes were evaluated by open field test and elevated plus maze test. H.E. staining was used to observe the histopathologic changes of the ovary tissue. Serum level of estradiol (E2) was measured by ELISA. The expressions of estrogen receptor α (ER-α) in ovarian and uterine tissues were detected by Western blot and immunofluorescence. RESULTS: (1) In PCOS mice, the EB exudation points were found to overlap the lower abdomen, lumbosacral, chest, back and lower limbs regions, and the number of EB points was significantly more than that of the control group (P<0.01). (2) After the intervention and compared with the control group, the ovaries showed polycystic changes and an increase of atresia follicles with a larger diameter, the activity time in the central area, the total distance of movement, the times of open-arm entries, the duration in open-arm, the serum E2 content and the expression of ER-α in ovarian tissue were significantly decreased (P<0.01, P<0.05), while the mice's body weight and the expression of ER-α in uterine tissue were increased (P<0.05) in the model group. After the intervention and compared with the model group, a small number of normal follicles and corpus luteum were observed under microscope, the activity time in the central area, the total distance of movement, the times of open-arm entries, the duration in open-arm, the serum E2 content and the expression of ER-α in ovarian tissue were significantly increased (P<0.05, P<0.01), while the mice's body weight and the expression of ER-α in uterine tissue was decreased (P<0.05) in both EA-CV4 and EA-sensitization points groups. CONCLUSION: EA at sensitization points and CV4 can regulate the expression of estrogen and ER-α in PCOS mice, and improve the anxiety like behavior. EB exudation points on the body surface can not only reflect the functional changes of organs, but also treat diseases through body surface stimulation, suggesting a dual role in diagnosis and treatment.

Effects of Acute Exposure to 3500 MHz (5G) Radiofrequency Electromagnetic Radiation on Anxiety-Like Behavior and the Auditory Cortex in Guinea Pigs.

Numerous studies have shown that radiofrequency electromagnetic radiation (RF-EMR) may negatively affect human health. We detected the effect of 3500 MHz RF-EMR on anxiety-like behavior and the auditory cortex (ACx) in guinea pigs. Forty male guinea pigs were randomly divided into four groups and exposed to a continuous wave of 3500 MHz RF-EMF at an average specific absorption rate (SAR) of 0, 2, 4, or 10 W/kg for 72 h. After exposure, malondialdehyde (MDA) levels, antioxidant enzyme activity, anxiety-like behavior, hearing thresholds, cell ultrastructure, and apoptosis were detected. Our results revealed that hearing thresholds and basic indexes of animal behavior did not change significantly after exposure (P > 0.05). However, the MDA levels of ACx were increased (P < 0.05), and catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) activities were decreased (P < 0.05) in the exposure groups compared to the sham group. Ultrastructural changes of ACx, including swollen mitochondria and layered myelin sheaths, were observed. Cytochrome-c relocalization, caspase-9, and cleaved caspase-3 activation were detected in the exposure groups. In conclusion, these results suggest that oxidative stress is an important mechanism underlying the biological effects of RF-EMR, which can induce ultrastructural damage to the ACx and cell apoptosis through a mitochondria-dependent mechanism. Moreover, oxidative stress, apoptosis induction and ultrastructural damage increase in a SAR-dependent manner. However, RF-EMR does not increase hearing thresholds or induce anxiety. Bioelectromagnetics. 43:106-118, 2022. © 2021 Bioelectromagnetics Society.

Dietary omega-3 fatty acids prevent neonatal seizure-induced early alterations in the hippocampal glutamatergic system and memory deficits in adulthood.

OBJECTIVE: We investigated the influence of dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) on glutamatergic system modulation after a single episode of neonatal seizures and their possible effects on seizure-induced long-lasting behavioral deficits. METHODS: Male Wistar rats receiving an omega-3 diet (n-3) or an n-3 deficient diet (D) from the prenatal period were subjected to a kainate-induced seizure model at P7. Glutamate transporter activity and immunocontents (GLT-1 and GLAST) were assessed in the hippocampus at 12, 24, and 48 h after the seizure episode. Fluorescence intensity for glial cells (GFAP) and neurons (NeuN) was assessed 24 h after seizure in the hippocampus. Behavioral analysis (elevated-plus maze and inhibitory avoidance memory task) was performed at 60 days of age. RESULTS: The D group showed a decrease in glutamate uptake 24 h after seizure. In this group only, the GLT1 content increased at 12 h, followed by a decrease at 24 h. GLAST increased up to 24 h after seizure. GFAP fluorescence was higher, and NeuN fluorescence decreased, in the D group independent of seizures. In adulthood, the D group presented memory deficits independent of seizures, but short-term memory (1.5 h after a training session) was abolished in the D group treated with kainate. SIGNIFICANCE: N-3 PUFA positively influenced the glutamatergic system during seizure and prevented seizure-related memory deficits in adulthood.

Delivery method matters: omega-3 supplementation by restricted feeding period and oral gavage has a distinct impact on corticosterone secretion and anxious behavior in adolescent rats.

Objectives: Oral gavage and time-restricted feeding are common delivery methods for dietary supplementation to rodents. However, the stress associated with selected feeding regimens could represent a confounding variable. In rodents, the adolescence period is particularly vulnerable to stressful events, in part related to ongoing maturation of the brain. In this context, omega-3 dietary supplementation has shown beneficial effects on neuronal growth, cognitive performance and stress regulation, while high-fat diet (HVF) has been associated with enhanced stress and anxiety. Therefore, this study has two aims: (1) evaluate the influence of 21-day supplementation with soybean oil (control group; CSO), fish oil (FO) or hydrogenated vegetable fat (HVF) fatty acids (FA) during the adolescence period on corticosterone secretion and anxiety-like behavior and, (2) compare the impact of dietary supplementation using oral gavage or time-limited feeding on these measures.Methods: Oral gavage or restricted feeding were used to daily feed adolescent rats (PND28-47; n = 49). On supplementation days 1, 7, 14 and 21, droplets of blood were collected for corticosterone (CORT) assessments. The Open Field (OFT) and the Elevated-Plus Maze (EPM) tests served to assess anxiety-like behavior on PND50.Results: Our findings indicate increased CORT secretion in restricted-(R) compared to gavage-fed animals on DAY7 and DAY14, suggesting heightened HPA-axis reactivity. Notably, CORT secretion diminished in FO-R-rats (DAY21), suggesting improved coping/adjustment. Consistent with CORT assessments, findings in the OFT and EPM supported attenuated anxiety in gavage versus restricted groups. FO and CSO supplementation reduced anxiety compared to HVF intake.Conclusions: Our findings uncover a significant impact of feeding methods on anxiety-like behavior and physiological stress response in rodents, supporting oral gavage as a less stressful option during the adolescent developmental stage. Supplement-specific effects on CORT secretion further indicated an influence of fish oil in regulating the stress response.

Vitamin D3 reduces hippocampal NR2A and anxiety in nicotine withdrawal mice.

Nicotine withdrawal symptoms, mainly anxiety, cause high level of relapse rate after quitting smoking. Vitamin D supplementation has shown its potential for the prevention and treatment of anxiety disorders; however, neurobiological studies about the effect of vitamin D on nicotine withdrawal-induced anxiety are limited. To investigate the effect and molecular mechanism of vitamin D3 supplement by dietary on anxiety-like behavior during nicotine withdrawal, male C57/BL6 mice were divided into four groups: vehicle, nicotine only, vitamin D3 only, and nicotine plus vitamin D3. Mice were administrated with nicotine in drinking water (200 µg/mL), and vitamin D3 in feed for 6 weeks. During nicotine withdrawal, vitamin D3-treated mice showed significantly less anxiety-like behavior by an open-field test and marble buried test that performed an increase in the duration of the central zone and a decrease buried marble, respectively. Moreover, vitamin D3 supplementation attenuated the hippocampal NR2A expression on both protein and mRNA levels in nicotine and vitamin D3-treated mice. Our data showed that dietary supplementation with vitamin D3 ameliorated nicotine withdrawal-induced anxiety, which may be related to downregulation of NR2A expression in hippocampus. Vitamin D3 may provide a new dietary intervention with the easy access for smoking cessation.

Global cerebral ischemia in adolescent male Long Evans rats: Effects of vanillic acid supplementation on stress response, emotionality, and visuospatial memory.

The developmental period is critical in delineating plastic response to internal and external events. However, neurobehavioural effects of global cerebral ischemia (GCI) in the maturing brain remain largely unknown. This study characterised the effects of GCI experienced at puberty on adulthood (1) hippocampus CA1 neuronal damage, (2) cognitive and emotional impairments, and (3) glucocorticoid receptor (GR) expression. Effects of adolescent exposure to the phenol vanillic acid (VA) on post-ischemic outcomes were also determined. Male Long Evans rats (n = 35) were supplemented for 21 consecutive days (postnatal days 33-53) with VA (91 mg/kg) or nut paste vehicle (control) prior to a 10-min GCI or sham surgery. As adults, rats were tested in the Open Field Test (OFT), Elevated-Plus Maze (EPM), and Barnes Maze (BM). GR expression was determined in the basolateral amygdala (BLA), CA1, and paraventricular nucleus (PVN), and brain injury assessed via CA1 neuronal density. Adolescent GCI exposure induced extensive hippocampal CA1 injury, which was not prevented by VA supplementation. Behaviourally, GCI increased EPM exploration while having no impact on spatial memory. VA intake increased OFT peripheral exploration. Notably, while no delayed changes in CA1 and PVN GR immunoreactivity were noted, both treatments separately increased BLA GR expression when compared with sham-nut paste rats. Age at GCI occurrence plays a critical role on post-ischemic impairments. The observation of minimal functional impairments despite important CA1 neuronal damage supports use of compensatory mechanisms. Our findings also show daily VA supplementation during adolescence to have no protective effects on post-ischemic outcomes, contrasting adult intake.

Maternal behavior, novelty confrontation, and subcortical c-Fos expression during lactation period are shaped by gestational environment.

The environmental context during gestation may modulate the postpartum variations in maternal behaviors observed within different animal species. Most of our experimental knowledge on this phenomenon and its physiological effects have been gained by confronting the pregnant mother with stressful situations, with the consensual results indicating a reduced maternal behavior and a hyper reactivity of stress-related neural paths. Here, in contrast, by exposing nulliparous rats strictly during pregnancy to a standard laboratory environment (STD) or a highly stimulating sensory and social environment (EE), we investigated the hypothesis that subjects frequently exposed to social stimuli and novel situations during pregnancy will show postpartum changes in subcortical brain areas' activity related to the processing of social stimuli and novelty, such that there will be modifications in maternal behavior. We found that EE mothers doubled the levels of licking and grooming, and active hovering over pups during the first postpartum week than STD dams, without a difference in the time of contact with the pups. Associated with these behaviors, EE dams showed increased c-Fos immunoreaction in hypothalamic nuclei and distinct responses in amygdalar nuclei, than STD dams. In the maternal defensive test, EE dams tripled the levels of aggressive behaviors of the STD rats. Additionally, in two different tests, EE mothers showed lower levels of postpartum anxiety-like behaviors when confronted with novel situations. Our results demonstrate that the activity of brain areas related to social behavior is adaptable by environmental circumstances experienced during gestation, presumably to prepare the progeny for these particular conditions.

An in vivo explorative study to observe the protective effects of Puerariae flos extract on chronic ethanol exposure and withdrawal male mice.

Protective effects of Puerariae flos extract (PFE) on ethanol (EtOH) exposure have been previously verified. This study attempts to explore the protective effects of PEF on EtOH withdrawal models. Sixty male Kunming mice were involved which were randomly divided into five groups (intact control, EtOH group (35-day EtOH exposure), EtOH withdrawal group (28-day exposure + 7-day withdrawal), EtOH withdrawal group + positive control (Deanxit) group, and EtOH withdrawal group + PFE group). The changes of neuropsychological behaviors; hippocampal BDNF expression and CA1 neuronal density; and plasma corticotropin-releasing hormone (CRH), ACTH, and CORT levels were observed. It was found that depression-like behaviors reduced by EtOH exposure and increased by withdrawal under the 28-day EtOH exposure and 7-day withdrawal conditions. In addition, anxiety-like behaviors worsened by EtOH exposure and unchanged by withdrawal. Deanxit and PEF ameliorated such behaviors (vs. withdrawal group). Hippocampal BDNF expression was significantly downregulated by EtOH exposure and upregulated by withdrawal. Deanxit and PEF significantly upregulated the BDNF expression. The hippocampal CA1 neuronal density significantly decreased by EtOH exposure but unchanged by withdrawal and treatments. The plasma CRH, ACTH, and CORT levels show a significant enhancement by EtOH exposure and reduced by withdrawal. They were further reduced by Deanxit and PEF. The protective effects of PEF on EtOH chronic withdrawal mouse models were verified. The results of this study also indicated a complicated scenario of neuropsychological behaviors, hippocampal BDNF expression, and hypothalamic-pituitary-adrenal axis which are affected by the timing of EtOH exposure and withdrawal.

Effects of ketogenic diet and ketone monoester supplement on acute alcohol withdrawal symptoms in male mice.

RATIONALE: After alcohol ingestion, the brain partly switches from consumption of glucose to consumption of the alcohol metabolite acetate. In heavy drinkers, the switch persists after abrupt abstinence, leading to the hypothesis that the resting brain may be "starved" when acetate levels suddenly drop during abstinence, despite normal blood glucose, contributing to withdrawal symptoms. We hypothesized that ketone bodies, like acetate, could act as alternative fuels in the brain and alleviate withdrawal symptoms. OBJECTIVES: We previously reported that a ketogenic diet during alcohol exposure reduced acute withdrawal symptoms in rats. Here, our goals were to test whether (1) we could reproduce our findings, in mice and with longer alcohol exposure; (2) ketone bodies alone are sufficient to reduce withdrawal symptoms (clarifying mechanism); (3) introduction of ketogenic diets at abstinence (a clinically more practical implementation) would also be effective. METHODS: Male C57BL/6NTac mice had intermittent alcohol exposure for 3 weeks using liquid diet. Somatic alcohol withdrawal symptoms were measured as handling-induced convulsions; anxiety-like behavior was measured using the light-dark transition test. We tested a ketogenic diet, and a ketone monoester supplement with a regular carbohydrate-containing diet. RESULTS: The regular diet with ketone monoester was sufficient to reduce handling-induced convulsions and anxiety-like behaviors in early withdrawal. Only the ketone monoester reduced handling-induced convulsions when given during abstinence, consistent with faster elevation of blood ketones, relative to ketogenic diet. CONCLUSIONS: These findings support the potential utility of therapeutic ketosis as an adjunctive treatment in early detoxification in alcohol-dependent patients seeking to become abstinent. TRIAL REGISTRATION: clinicaltrials.gov NCT03878225, NCT03255031.

The effects of laser stimulation at acupoint ST36 on anxiety-like behaviors and anterior cingulate cortex c-Fos expression in a rat post-traumatic stress disorder model.

Post-traumatic stress disorder (PTSD) is a mental disorder that is linked with the onset of multiple anxiety-like behaviors. This study was designed to assess how these behaviors and anterior cingulate cortex (ACC) c-Fos expression were impacted by 10.6-µm laser stimulation at acupoint ST36 a rat model of PTSD. A rat model of PTSD was prepared via prolonged exposure of animals to a stressor, followed by a 7-day period during which animals were allowed to rest undisturbed in their cages. Rats were randomized into four experimental groups (n = 12/group): the control, PTSD, LS, and sham LS groups. Control group animals were not subjected to SPS procedures prior to behavioral testing. LS and sham LS animals were administered LS treatment at bilateral ST36 acupoints or non-acupoints, respectively, for a 7-day period. Animals were then assessed for performance in elevated plus maze (EPM) tests and open-field tests (OFT), and their plasma corticosterone levels were measured. In addition, c-Fos-positive nuclei in the ACC were detected via immunohistochemical staining. Relative to sham LS treatment and PTSD model control rats, LS was associated with increased time spent in both open EPM test arms and in the central area in the OFT (P < 0.05). The PTSD model group exhibited a significant reduction in ACC c-Fox expression, while LS treatment significantly increased this expression (P < 0.001). In addition, a correlation was detected between anxiety-like behaviors and altered ACC neuronal activation. The results of this study indicate that LS at acupoint ST36 can have a previously unreported effect on anxiety-like behaviors in the context of PTSD, with ACC neuronal activation potentially being implicated as a driver of this effect.

Neonatal pyridoxine administration long lastingly accelerates cortical spreading depression in male rats, without affecting anxiety-like behavior.

Objectives: Pyridoxine plays a key role in the development of the human nervous system. Several reports suggest that administration of high doses of pyridoxine can be helpful in improving disturbances such as anxiety and pyridoxine-dependent epilepsy, although it has also been associated with a proconvulsive action. In this study, we investigated in developing rats the effects of repeated administration of various doses of pyridoxine on anxiety-like behavior and the brain excitability-related phenomenon known as cortical spreading depression (CSD).Methods: From postnatal day (P) 7 to P27, Wistar rat pups received per gavage pyridoxine hydrochloride (1 mg/kg/day, or 5 mg/kg/day, or 10 mg/kg/day). On P60-70, the animals were tested in the elevated plus maze (EPM) to evaluate anxiety-like behavior. On P71-80, we recorded the CSD (4-hour recording session).Results: Compared with naïve (gavage-free) and saline-treated controls, pyridoxine-treated groups displayed a significant (p < 0.001) increase in CSD propagation velocity and amplitude of the CSD negative direct-current (DC)-shift, and a decrease in the CSD DC-shift duration. These effects were long-lasting and dose-dependent. In the EPM, no significant pyridoxine-associated effect was observed.Discussion: Our data demonstrate a novel action of pyridoxine on an electrical activity-related phenomenon (CSD) in the developing brain, confirming the hypothesis that the chronic treatment with pyridoxine early in life modulates CSD. Data on CSD propagation suggest that pyridoxine at a high dose might act as a prooxidant agent in the developing brain, a hypothesis that deserves further testing.

Electroacupuncture suppresses the pain and pain-related anxiety of chronic inflammation in rats by increasing the expression of the NPS/NPSR system in the ACC.

BACKGROUND: The neuropeptide S/Neuropeptide S receptor (NPS/NPSR) system is involved in the regulation of anxiety in rodents. Chronic inflammation can induce anxiety. Our lab has observed that electroacupuncture (EA) has a beneficial effect on chronic inflammatory pain and pain-related anxiety; however, the mechanism should be further clarified. In the present study, we used an inflammatory pain model to investigate the role of the NPS/NPSR system in the anterior cingulate cortex (ACC) in the analgesic and antianxiety effects of EA. RESULTS: In an inflammatory pain model, the paw withdrawal thresholds (PWTs) were decreased, pain-related anxiety-like behaviors were induced, and the ipsilateral protein expression of NPS and NPSR was decreased in the ACC. EA stimulation increased the PWTs, reduced pain-related anxiety-like behavior, and enhanced the ipsilateral protein expression of NPS and NPSR in the ACC. NPS microinjection increased the PWTs and decreased pain-related anxiety-like behaviors. Furthermore, an NPSR inhibitor combined with EA reversed the effect of EA on the PWTs and pain-related anxiety-like behaviors. CONCLUSIONS: Our results suggest that EA suppresses pain and pain-related anxiety-like behavior of chronic inflammation in rats by increasing the expression of the NPS/NPSR system in the ACC.

Ultrasound stress compromises the correlates of emotional-like states and brain AMPAR expression in mice: effects of antioxidant and anti-inflammatory herbal treatment.

The modern lifestyle is associated with exposure to "psychological" or "emotional" stress. A growing portion of the population is exposed to emotional stress that results in a high incidence of anxiety disorders, a serious social problem. With this rise, there is a need for understanding the neurobiological causes of stress-induced anxiety and to offer safe remedies for this condition. Side effects of existing pharmaceuticals necessitate the search for alternatives. Having fewer adverse effects than classic remedies, natural extract-based therapies can be a promising solution. Here, we applied a model of emotional stress in BALB/c mice using ultrasound exposure to evoke the signs of anxiety-like behavior. We examined the behavioral and molecular impact of ultrasound and administration of herbal antioxidant/anti-inflammatory treatment (HAT) on AMPA receptor expression, markers of plasticity, inflammation and oxidative stress. A 3-week ultrasound exposure increased scores of anxiety-like behaviors in the standard tests and altered hippocampal expression as well as internalization of AMPA receptor subunits GluA1-A3. Concomitant treatment with HAT has prevented increases of anxiety-like behaviors and other behavioral changes, normalized hippocampal malondialdehyde content, GSK3ß and pro-inflammatory cytokines Il-1ß and Il-6, and the number of Ki67-positive cells. Levels of malondialdehyde, a common measure of oxidative stress, significantly correlated with the investigated end-points in stressed, but not in non-stressed animals. Our results emphasize the role of oxidative stress in neurobiological abnormalities associated with experimentally induced condition mimicking emotional stress in rodents and highlight the potential therapeutic use of anti-oxidants like herbal compositions for management of stress-related emotional disturbances within the community.