Light-dark Box Research Articles

In silico Screening and Behavioral Validation of a Novel Peptide, LCGA-17, With Anxiolytic-Like Properties.

The aim of the study was to develop better anxiolytics and antidepressants. We focused on GABAA receptors and the α2δ auxiliary subunit of V-gated Ca2+ channels as putative targets because they are established as mediators of efficacious anxiolytics, antidepressants, and anticonvulsants. We further focused on short peptides as candidate ligands because of their high safety and tolerability profiles. We employed a structural bioinformatics approach to develop novel tetrapeptides with predicted affinity to GABAA receptors and α2δ. In silico docking studies of one of these peptides, LCGA-17, showed a high binding score for both GABAA receptors and α2δ, combined with anxiolytic-like properties in a Danio rerio behavioral screen. LCGA-17 showed anxiolytic-like effects in the novel tank test, the light–dark box, and the social preference test, with efficacy comparable to fluvoxamine and diazepam. In binding assays using rat brain membranes, [3H]-LCGA-17 was competed more effectively by gabapentinoid ligands of α2δ than ligands of GABAA receptors, suggesting that α2δ represents a likely target for LCGA-17. [3H]-LCGA-17 binding to brain lysates was unaffected by competition with ligands for GABAB, glutamate, dopamine, serotonin, and other receptors, suggesting specific interaction with α2δ. Dose-finding studies in mice using acute administration of LCGA-17 (i.p.) demonstrated anxiolytic-like effects in the open field test, elevated plus maze, and marble burying tests, as well as antidepressant-like properties in the forced swim test. The anxiolytic effects were effectively blocked by bicuculline. Therefore, LCGA-17 is a novel candidate anxiolytic and antidepressant that may act through α2δ, with possible synergism by GABAA receptors.

Assessment of Antidepressant-like, Anxiolytic Effects and Impact on Memory of Pimpinella anisum L. Total Extract on Swiss Albino Mice.

Mental disorders are psychological symptoms that impact multiple areas of an individual’s life. Depression and anxiety are chronic illnesses described as the most prevalent stress-related mood disorders that cause injury and early death. In Morocco, Anise “Pimpinella anisum L.” is one of the most traditionally used condiment plants, which has long been used to cure various illnesses and in phytotherapy. The present study was designed to investigate the antidepressant, anxiolytic, and memory impact of the total extract of Pimpinella anisum (PATE) at the doses of 100 and 200 mg/kg, using the Forced Swimming Test (FST), Tail Suspension Test (TST), Open Field Test (OFT), and Light–Dark Box Test (LDBT) as an experimental paradigm of anxiety and depression, and Novel Object Recognition Test (NORT) and the Morris Water Maze Test (MWMT) as memory tests on Swiss albino mice. The tests were carried out on the 1st, 7th, 14th, and the 21st days of the study, and the extract groups were compared with normal controls and positive controls (receiving bromazepam and paroxetine at the doses of 1 mg/kg and 11.5 mg/kg for anxiety and depression, respectively). The daily oral gavage of the mice by the PATE induced a significant anxiolytic and antidepressant-like effect by shortening immobility time and decreasing downtime in the different tests. PATE at both doses was shown to have no impact on memory following the NORT and MWM tests. Different compounds, such as gallic acid, catechin, chlorogenic acid, caffeic acid, oleuropein, p-coumaric acid, trans-4-hydroxy-3-methoxycinnamic acid, myricetin, and quercetin, were identified during the phytochemical analysis carried out using HPLC analysis. This research supports and promotes the extract’s traditional use, suggesting its use as a phytomedicine against depression and anxiety, and calls for further research to clarify its mode of action.

Reserpine-induced altered neuro-behavioral, biochemical and histopathological assessments prevent by enhanced antioxidant defence system of thymoquinone in mice.

Thymoquinone (Tq), an active compound of Nigella sativa, has been known for its anti-inflammatory, antioxidant, and neuroprotective characteristics. The present study is aimed to evaluate the effect of Tq on reserpine (Rsp)-induced behavioral (anxiety and/or depression) and, memory deficit; hippocampal inflammatory markers, oxidative markers, antioxidant enzymes, acetylcholinesterase (AChE) activity and histopathology in male mice. Animals were injected with Rsp at a dose of 2mg/ml/kg and doses of Tq (10 and 20mg/ml/kg) for 28days. After the treatment period, behavioral tests [Elevated plus maze (Epm); Light dark box test (Lda); Morris water maze (Mwm); Forced swim test (Fst); Tail suspension test (Tst)] were conducted. After analysis of behaviors, mice were decapitated and brain samples were collected, the hippocampus was removed from the whole-brain sample for biochemical analysis and histology. Administration of Tq at both doses prevent adverse effects of Rsp and increased time spent in open arm and lightbox in Lda and Epm respectively, decreased immobility period in Fst and Tst, decreased latency escape in Mwm, reduced lipid peroxidation (lpo) and inflammatory cytokines, increased defensive enzymes, reduced acetylcholinesterase (AChE) activity and corrected histological lines. It is concluded that Rsp-instigated behavioral and memory deficits were prevented by Tq possibly via its strong antioxidant and anti-inflammatory effects.

Anxiolytic Effect of Two Tobacco Essential Oils (Nicotiana tabacum Linn.) on Mice

Tobacco (Nicotiana tabacum Linn.) is a famous traditional herb used in folk medicine. The essential oils of tobacco have been demonstrated in modern studies to possess antioxidant, anti-inflammatory, and neuroprotective properties, while its anxiolytic effect has not been reported. The purpose of this study was to evaluate the anxiolytic effect of Yunnan tobacco essential oil (YTO) and Zimbabwe tobacco essential oil (ZTO) on mice. The constituents of YTO and ZTO were analyzed by GC/MS. The anxiolytic effect of YTO and ZTO (0.1%, 1%, and 10%, v/v) on male ICR mice was evaluated in the light–dark box test (LDB) and the elevated plus maze test (EPM) test via inhalation and transdermal administration. After the behavioral tests, salivary corticosterone levels in mice were measured. The behavioral analysis showed that the administration of both YTO and ZTO elevated the time that the mice spent in the light chamber in the LDB test compared to the untreated control. In the EPM test, YTO and ZTO increased the time spent in open arms and the number of entries into the open arms. In addition, both YTO and ZTO significantly decreased salivary corticosterone levels in mice (p ≤ 0.001). In summary, our results demonstrated that inhalation and transdermal administration of both YTO and ZTO showed anxiolytic effect on male ICR mice.

BOTOX® counteracts the innate anxiety-related behaviours in correlation with increased activities of key antioxidant enzymes in the hippocampus of ageing experimental mice

Cholinergic crisis and oxidative stress in the hippocampus of the brain have been known to induce anxiety disorders upon ageing. BOTOX® is a widely used therapeutic form of botulinum neurotoxin that acts by inhibiting the release of acetylcholine (ACh) from the nerve terminals at the neuromuscular junction. BOTOX® can migrate from the muscle to the brain through retrograde axonal transport and modulate neuroplasticity. While a mild dose of BOTOX® has been used to manage various neurological deficits and psychiatric complications including depression, the efficacy and experimental evidence for its anxiolytic effects and antioxidant properties remain limited. In this study, we have investigated the effect of BOTOX® on the innate anxiety-like behaviours in ageing mice upon exposure to different behavioural paradigms like open field test, elevated plus maze and light-dark box test, and estimated the enzymatic activities of key antioxidants in the hippocampus. Results revealed that animals injected with a mild intramuscular dosage of BOTOX® showed reduced level of innate anxiety-related symptoms and increased activities of hippocampal antioxidant enzymes compared to the control group. This study strongly supports that BOTOX® could be implemented to prevent or treat anxiety and hippocampal oxidative stress resulting from ageing, emotional and mood disorders.

Early-life experiences altered the maturation of the lateral habenula in mouse models, resulting in behavioural disorders in adulthood.

Background:Abnormally high activity in the lateral habenula causes anxiety- or depression-like behaviours in animal experimental models. It has also been reported in humans that excessive stress in early life is correlated with the onset of psychiatric disorders in adults. These findings raise the question of whether maturation of the lateral habenula is affected under the influence of early-life experiences, which could govern behaviours throughout life.Methods:We examined the maturation of the lateral habenula in mice based on neuronal activity markers and plastic components: Zif268/Egr1, parvalbumin and perineuronal nets. We examined the effect of early-life stress using repeated maternal deprivation.Results:First, we found a transient highly sensitive period of the lateral habenula under stress. The lateral habenula matured through 4 stages: postnatal days 1–9 (P1–9), P10–20, around P35 and after P35. At P10–20, the lateral habenula was highly sensitive to stress. We also observed experience-dependent maturation of the lateral habenula. Only mice exposed to chronic stress from P10–20 exhibited changes specific to the lateral habenula at P60: abnormally high stress reactivity shown by Zif268/Egr1 and fewer parvalbumin neurons. These mice showed anxiety- or depression-like behaviours in the light–dark box test and forced swim test.Limitations:The effect of parvalbumin neurons in the lateral habenula on behavioural alterations remains unknown. It will be important to understand the “sensitive period” of the neuronal circuits in the lateral habenula and how the period P10–20 is different from P9 or earlier, or P35 or later.Conclusion:In mice, early-life stress in the period P10–20 led to late effects in adulthood: hyperactivity in the lateral habenula and anxiety or depression, indicating differences in neuronal plasticity between stages of lateral habenula maturation.

Stigmasterol can be new steroidal drug for neurological disorders: Evidence of the GABAergic mechanism via receptor modulation

BackgroundGamma-aminobutyric acid A (GABAA) receptors have been implicated in anxiety and epileptic disorders. Hypothesis/PurposeThis study aimed to investigate the effects of stigmasterol, a plant sterol (phytosterol) isolated from Artemisia indica Linn on neurological disorders. MethodsStigmasterol was evaluated on various recombinant GABAA receptor subtypes expressed in Xenopus laevis oocytes and its anxiolytic and anticonvulsant potential was assessed using the elevated plus maze (EPM), light-dark box (LDB) test, and pentylenetetrazole- (PTZ-) induced seizure paradigms. Furthermore, computational modeling of α2β2γ2L, α4β3δ, and α4β3 subtypes was performed to gain insights into the GABAergic mechanism of stigmasterol. For the first time, a model of GABAδ subtype was generated. Stigmasterol was targeted to all the binding sites (neurotransmitters, positive and negative modulator binding sites) of GABAA α2β2γ2L, α4β3, and α4β3δ complexes by in silico docking. ResultsStigmasterol enhanced GABA-induced currents at ternary α2β2γ2L, α4β3δ, and binary α4β3 GABAAR subtypes. The potentiation of GABA-induced currents at extrasynaptic α4β3δ was significantly higher compared to the binary α4β3 subtype, indicating that the δ subunit is important for efficacy. Stigmasterol was found to be a potent positive modulator of the extrasynaptic α4β3δ subtype, which was also confirmed by computational analysis. The computational analysis reveals that stigmasterol preferentially binds at the transmembrane region shared by positive modulators or a binding site constituted by the M2-M3 region of α4 and M1-M2 of β3 at α4β3δ complex. In in vivo studies, Stigmasterol (0.5–3.0 mg/kg, i.p.) exerted significant anxiolytic and anticonvulsant effects in an identical manner of allopregnanolone, indicating the involvement of a GABAergic mechanism. ConclusionTo our knowledge, this is the first study reporting the positive modulation of GABAA receptors, anxiolytic and anticonvulsant potential of stigmasterol. Thus, stigmasterol is considered to be a candidate steroidal drug for the treatment of neurological disorders due to its positive modulation of GABA receptors.

Bioactive metabolites of Blumea lacera attenuate anxiety and depression in rodents and computer-aided model.

Blumea lacera is an edible plant with imperative medicinal values. However, the anxiolytic and antidepressant roles of B. lacera have not been well‐explained. Therefore, the current study aims to explore the impending bioactive metabolites and roles of B. lacera methanol leaf extract (Me‐BLL) in attenuating anxiety and depression through several experimental and computer‐aided approaches. The chemical characterization of Me‐BLL was performed through standard phytochemical and GC‐MS analyses. To explore the neuropharmacological insights, Swiss albino mice were treated with Me‐BLL at doses of 200–400 mg/kg, p.o. The anxiolytic effects were observed employing elevated plus maze (EPM), light–dark box (LDB), and hole‐board (HBT) tests, while antidepressant effects were evaluated using forced swimming (FST) and tail suspension tests (TST). Diazepam (1 mg/kg, i.p.) and fluoxetine HCl (20 mg/kg, p.o.) were used as the reference standard. The phytochemical analyses revealed several bioactive metabolites, including higher contents of total phenolics and flavonoids. The EPM and LDB tests demonstrated an increased time spent in open arms and light box, and the HBT showed an increased number of head dipping, indicating the anxiolytic effects of Me‐BLL. The TST and FST revealed a decrease in immobility time, meaning the persuasive antidepressant effects. The antioxidative effects of Me‐BLL have also been observed prominently. Correspondingly, the computer‐aided investigation confirmed several bioactive lead molecules. Specifically, thymol and cuminol revealed potential anxiolytic and antioxidant effects, while stigmast‐5‐en‐3.beta.‐ol and gamma‐sitosterol possessed promising antidepressant effects. Taken these results as a base, the plant has imperative potentials in managing anxiety and depression‐like disorders.

Pharmacological Blockade of PPARα Exacerbates Inflammatory Pain-Related Impairment of Spatial Memory in Rats

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that exist in three isoforms: PPARα, PPARβ/δ and PPARγ. Studies suggest that the PPAR signalling system may modulate pain, anxiety and cognition. The aim of the present study was to investigate whether endogenous signalling via PPARs differentially modulates innate anxiety responses and mnemonic function in the presence and absence of inflammatory pain. We examined the effects of intraperitoneal administration of GW6471 (PPARα antagonist), GSK0660 (PPARβ/δ antagonist), GW9662 (PPARγ antagonist), and N-palmitoylethanolamide (PEA) on rat behaviour in the elevated plus maze (EPM), open field (OF), light-dark box (LDB), and novel object recognition (NOR) tests in the presence or absence of chronic inflammatory pain. Complete Freund’s Adjuvant (CFA)-injected rats exhibited impaired recognition and spatial mnemonic performance in the NOR test and pharmacological blockade of PPARα further impaired spatial memory in CFA-treated rats. N-oleoylethanolamide (OEA) levels were higher in the dorsal hippocampus in CFA-injected animals compared to their counterparts. The results suggest a modulatory effect of CFA-induced chronic inflammatory pain on cognitive processing, but not on innate anxiety-related responses. Increased OEA-PPARα signalling may act as a compensatory mechanism to preserve spatial memory function following CFA injection.

CB1 receptor neutral antagonist treatment epigenetically increases neuropeptide Y expression and decreases alcohol drinking

Alcohol consumption is mediated by several important neuromodulatory systems, including the endocannabinoid and neuropeptide Y (NPY) systems in the limbic brain circuitry. However, molecular mechanisms through which cannabinoid-1 (CB1) receptors regulate alcohol consumption are still unclear. Here, we investigated the role of the CB1 receptor-mediated downstream regulation of NPY via epigenetic mechanisms in the amygdala. Alcohol drinking behavior was measured in adult male C57BL/6J mice treated with a CB1 receptor neutral antagonist AM4113 using a two-bottle choice paradigm while anxiety-like behavior was assessed in the light-dark box (LDB) test. The CB1 receptor-mediated changes in the protein levels of phosphorylated cAMP-responsive element binding protein (pCREB), CREB binding protein (CBP), H3K9ac, H3K14ac and NPY, and the mRNA levels of Creb1, Cbp, and Npy were measured in amygdaloid brain structures. Npy-specific changes in the levels of acetylated histone (H3K9/14ac) and CBP in the amygdala were also measured. We found that the pharmacological blockade of CB1 receptors with AM4113 reduced alcohol consumption and, in an ethanol-naïve cohort, reduced anxiety-like behavior in the LDB test. Treatment with AM4113 also increased the mRNA levels of Creb1 and Cbp in the amygdala as well as the protein levels of pCREB, CBP, H3K9ac and H3K14ac in the central and medial nucleus of amygdala, but not in the basolateral amygdala. Additionally, AM4113 treatment increased occupancy of CBP and H3K9/14ac at the Npy gene promoter, leading to an increase in both mRNA and protein levels of NPY in the amygdala. These novel findings suggest that CB1 receptor-mediated CREB signaling plays an important role in the modulation of NPY function through an epigenetic mechanism and further support the potential use of CB1 receptor neutral antagonists for the treatment of alcohol use disorder.

Tolerance to neurochemical and behavioral effects of the hallucinogen 25I-NBOMe

Rationale4-Iodo-2,5-dimethoxy-N-(2-methoxybenzyl)phenethylamine (25I-NBOMe) is a potent serotonin 5-HT2A/2C receptor agonist with hallucinogenic activity. There is no data on the 25I-NBOMe effect on brain neurotransmission and animal performance after chronic administration.ObjectivesWe examined the effect of a 7-day treatment with 25I-NBOMe (0.3 mg/kg/day) on neurotransmitters’ release and rats’ behavior in comparison to acute dose.MethodsChanges in dopamine (DA), serotonin (5-HT), acetylcholine (ACh), and glutamate release were studied using microdialysis in freely moving rats. The hallucinogenic activity was measured in the wet dog shake (WDS) test. The animal locomotion was examined in the open field (OF) test, short-term memory in the novel object recognition (NOR) test. The anxiogenic/anxiolytic properties of the drug were tested using the light/dark box (LDB) test.ResultsRepeated administration of 25I-NBOMe decreased the response to a challenge dose of DA, 5-HT, and glutamatergic neurons in the frontal cortex as well as weakened the hallucinogenic activity in comparison to acute dose. In contrast, striatal and accumbal DA and 5-HT release and accumbal but not striatal glutamate release in response to the challenge dose of 25I-NBOMe was increased in comparison to acute treatment. The ACh release was increased in all brain regions. Behavioral tests showed a motor activity reduction and memory deficiency in comparison to a single dose and induction of anxiety after the drug’s chronic and acute administration.ConclusionsOur findings suggest that multiple injections of 25I-NBOMe induce tolerance to hallucinogenic activity and produce alterations in neurotransmission. 25I-NBOMe effect on short-term memory, locomotor function, and anxiety seems to be the result of complex interactions between neurotransmitter pathways.

The forced swim test: Giving up on behavioral despair (Commentary on Molendijk & de Kloet, 2021).

The forced swim test: Giving up on behavioral despair (Commentary on Molendijk & de Kloet, 2021).

Anxiety-related defensive behavioral responses in mice selectively bred for High and Low Activity.

High and Low Activity strains of mice (displaying low and high anxiety-like behavior, respectively) with 7.8-20 fold differences in open-field activity were selected and subsequently inbred to use as a genetic model for studying anxiety-like behavior in mice (DeFries et al., 1978, Behavior Genetics, 8:3-13). These strains exhibited differences in other anxiety-related behaviors as assessed using the light-dark box, elevated plus-maze, mirror chamber, and elevated square-maze tests (Henderson et al., 2004, Behavior Genetics, 34: 267-293). The purpose of these experiments was three-fold. First, we repeated a 6-day behavioral battery using updated equipment and software to confirm the extreme differences in anxiety-like behaviors. Second, we tested novel object exploration, a measure of anxiety-like behavior that does not rely heavily on locomotion. Third, we conducted a home cage wheel running experiment to determine whether these strains differ in locomotor activity in a familiar, home cage environment. Our behavioral test battery confirmed extreme differences in multiple measures of anxiety-like behaviors. Furthermore, the novel object test demonstrated that the High Activity mice exhibited decreased anxiety-like behaviors (increased nose pokes) compared to Low Activity mice. Finally, male Low Activity mice ran nearly twice as far each day on running wheels compared to High Activity mice, while female High and Low Activity mice did not differ in wheel running. These results support the idea that the behavioral differences between High and Low Activity mice are likely to be due to anxiety-related factors and not simply generalized differences in locomotor activity.

Enhanced neuroprotective and antidepressant activity of curcumin-loaded nanostructured lipid carriers in lipopolysaccharide-induced depression and anxiety rat model

The present study aims to develop curcumin-loaded nanostructured lipid carriers (CUR-NLCs) and investigate their neuroprotective effects in lipopolysaccharide (LPS)-induced depression and anxiety model. Nanotemplate engineering technique was used to prepare CUR-NLCs with Compritol 888 ATO and oleic acid as solid and liquid lipid, respectively. Poloxamer 188, Tween 80 and Span 80 were used as stabilizing agents for solid–liquid lipid core. The physicochemical parameters of CUR-NLCs were determined followed by in vitro drug release and in vivo neuroprotective activity in rats. The optimized CUR-NLCs demonstrated nanometric particle size of 147.8 nm, surface charge of –32.8 mV and incorporation efficiency of 91.0%. CUR-NLCs showed initial rapid followed by a sustained drug release reaching up to 73% after 24 h. CUR-NLCs significantly elevated struggling time and decreased immobility time in forced swim and tail suspension tests. A substantial increase in time spent and number of entries into the light and open compartments was observed in light–dark box and elevated plus maze models. CUR-NLCs improved the tissue architecture and suppressed the expression of p-NF-κB, TNF-α and COX-2 in brain tissues from histological and immunohistochemical analysis. CUR-NLCs improved the neuroprotective effect of curcumin and can be used as a potential therapeutics for depression and anxiety.

102 Single-Prolonged Stress as a Model of PTSD in Mice: Effects on Sleep and Anxiety

Abstract Introduction Sleep problems are common in humans with post-traumatic stress disorder (PTSD). Rapid eye movement (REM) sleep is involved in processing emotional memories; it is often disrupted in those with PTSD, and may be related to increased anxiety. Single prolonged stress (SPS) is a protocol used to model PTSD in rats, however little is known about how this model impacts sleep in mice. Prior research suggests SPS produces short term disturbances in REM sleep and increases in anxiety-like behavior, but further validation of this model is needed to understand how SPS impacts sleep and anxiety-like behaviors in mice specifically, as they have greater potential for transgenic manipulation Methods C57BL6/J mice underwent a SPS protocol in which they were tube-restrained for 2 hours, followed by a 15 minute forced swim in a group, ether exposure until loss of consciousness, and 10 days of social isolation. Following SPS, mice were tested for anxiety-like behavior in a light-dark box and sleep was measured from surgically implanted EEG and EMG leads. Time spent in wake, REM sleep, and non-REM sleep was quantified for 24 continuous hours in SPS and Control mice. Results There were no significant effects of SPS on the amount of time spent in any vigilance state, or in sleep-wake transitions. However, SPS-exposed mice showed significantly more anxiety-like behavior. EEG power spectra were analyzed in relevant frequency bands during each sleep state, and exploratory analyses were conducted Conclusion Minimal effects on sleep macroarchitecture were seen in mice 10 days after SPS. It is possible that sleep disturbances seen immediately after trauma exposure (such as in prior studies in rats) may have diminished over time. Further studies will need to include additional timepoints and analysis of sleep microarchitecture following SPS, and in other mouse models of PTSD, in order to more comprehensively examine changes in sleep. Support (if any) VA CDA #IK2 BX002712, Portland VA Research Foundation, Medical Research Foundation

Therapeutic Potential of Cannabidiol and WIN 55, 212‐2‐mesylate for Treatment of Anxiety

As the medicalization of marijuana occurs across the United States, the psychoactive component of the cannabis plant, Delta9-Tetrahydrocannabinol (THC), has gained recognition for its therapeutic potential. Meanwhile, its non-psychoactive counterpart, cannabidiol (CBD), has been marketed to treat Parkinson's disease, Chron's disease, dystonia, attention-deficit hyperactivity disorder (ADHD), inflammation, depression, fibromyalgia, epilepsy, and most commonly, anxiety. Given the over the counter accessibility of CBD and the fact that anxiety disorders are the most common type of mental illness in the United States, our objective was to access the therapeutic potential of cannabinoid receptor agonists and antagonists for treatment of anxiety. We hypothesized that CBD might be anxiolytic when chronically administered to a newly found mouse model of anxiety and attention deficit (Kv1.3-/- mice). Similarly to THC, WIN 55, 212-2-mesylate (WIN) is an agonist for the cannabinoid receptors CB1 and CB2, but it is not known to have any psychoactive properties. CBD is an antagonist for CB1 and CB2 receptors, and we used a CBD isolate to ensure purity. Our methods employed two-month-old Kv1.3-/- or wildtype mice that were phenotyped for anxiety behaviors using three assays, namely the elevated plus maze (EPM), the light-dark box (LDB), and the marble burying test. Mice were dosed daily, 30 minutes prior to performing a behavioral test. Mice of both sexes were separated into cohorts in which they were designated to receive an intraperitoneal injection of either drug (WIN55 = 1 mg/kg; CBD = 5 mg/kg) or vehicle solution at equivalent volume. Acute cohorts were defined as daily dosage for 5 days and chronic cohorts were treated with 20 doses over a 30 day interval. Without drug treatment, both genotypes exhibited anxiety-like behaviors in the LDB and EPM tasks, preferring dark side or closed arms of the apparatuses, respectively. For acute WIN treatment, Kv1.3-/- mice showed lessened anxiety in the LDB (increased time in light, decreased time in dark), however, following chronic WIN treatment, they showed no change in LDB behavior. In contrast, in wildtype mice, neither acute nor chronic WIN treatment changed LDB behaviors. Both acute and chronic WIN treatment in Kv1.3-/- mice mitigated anxiety in the EPM test (mice decreased time in the closed arms), whereas the same in wildtype mice showed no effect of anxiety if acutely administered and heightened anxiety following chronic treatment (mice increased time in closed arms). For cohorts treated acutely with CBD, Kv1.3-/- mice showed enhanced anxiety behaviors in the LDB (mice decreased time in light and increased time in dark). Mice of both genotypes showed enhanced anxiety behaviors in the EPM (mice increased time in the closed arms) following acute CBD treatment.In conclusion, our data did not support our hypothesis and showed that CBD may act as an anxiogenic drug in both wildtype and Kv1.3-/- mice.WIN acted as an anxiolytic drug for mice with trait anxiety (Kv1.3-/-), which was ineffective in these mice long-term, and ineffective acutely in wildtype mice exposed to state anxiety.

Swimming exercise attenuates anxiety-like behavior by reducing brain oxidative stress in type 2 diabetic mice

Anxiety-related behaviors are among the most prevalent psychiatric disorders in patients with type 2 diabetes (T2D). The protective effect of exercise on neuropsychiatric disorders has been documented. However, there are no studies that examined whether swimming exercise can decrease anxiety-like symptoms in type 2 diabetes. We investigated the effects of swimming exercise on body weight, anxiety-like behavior, glucose and insulin levels, and brain oxidative stress in male C57BL/6 mice. T2D-induced mice were subjected to swimming exercise, then anxiety-like behaviors were measured by the open field, light-dark box, and elevated plus-maze tests. Glucose and insulin levels were measure in serum, and antioxidant/oxidative markers including glutathione (GSH), malondialdehyde (MDA), and glutathione disulfide (GSSG) were measured in the brain. Our findings showed that T2D increased body weight, anxiety-like symptoms, glucose and insulin resistance, and oxidative stress by increasing MDA and GSSG levels in the brain of mice. Interestingly, swimming exercise reversed these parameters in diabetic mice. Our findings clearly indicate that there is a protective impact of swimming exercise on anxiety-like behavior by reducing insulin resistance and brain oxidative stress in mice with type 2 diabetes. Further studies are needed to validate these findings in humans.

Potentiation of Acetylcholine Does Not Improve Renal Inflammation and Behavior in Female Mice with Late‐Stage Systemic Lupus Erythematosus

Chronic inflammation contributes to the morbidity and mortality in advanced systemic lupus erythematosus (SLE). Endogenous neuroimmune mechanisms like the cholinergic anti-inflammatory pathway can be targeted to modulate inflammation, but the ability to manipulate such pathways and reduce inflammation and end organ damage has not been fully explored in SLE. Positive allosteric modulators (PAM) are pharmacological agents that inhibit desensitization of the nicotinic acetylcholine receptor (α7-nAChR), the main anti-inflammatory feature within the cholinergic anti-inflammatory pathway, and may augment α7-dependent cholinergic tone to generate therapeutic benefits in SLE. In the current study, we hypothesized that activating the cholinergic anti-inflammatory pathway at the level of the α7-nAChR with systemic administration of a partial agonist, GTS-21 or a PAM, PNU-120596, would reduce inflammation, eliminating the associated end organ damage in a mouse model of SLE with advanced disease. Further, we hypothesized that these α7 ligands will have central effects and improve behavioral deficits in SLE mice. Female control (NZW) and SLE mice (NZBWF1) were administered GTS-21 (15 mg/kg/day), PNU-120596 (10 mg/kg/day), or vehicle (DMSO) subcutaneously via minipumps for two weeks starting at 33 weeks of age. A subgroup of these mice were subjected to randomized, blinded behavioral tests for sensorimotor and emotional deficits (i.e., open-field, light-dark box and tail suspension tests) pre- and post-drug administration. SLE mice had higher plasma dsDNA autoantibodies, a hallmark of SLE and marker of disease severity, than control mice (9.2e5 ± 1.1e5 vs. 1.6e5 ± 3.9e4 U/mL; p < 0.001), but this increase was not altered by GTS-21 (1.1e6 ± 2.2e5) or PNU-120596 (1.1e6 ± 2.1e5) in SLE mice. The cholinergic anti-inflammatory pathway directly alters splenic inflammation and indirectly alters renal inflammation, so we measured TNF-α in the spleen and renal cortex of control and SLE mice. Splenic and renal TNF-α was 2- and 4-times higher in SLE mice compared to controls (235.0 ± 28.2 and 417.2 ± 122.7; % difference from controls), but neither was altered by GTS-21 (262.5 ± 33.4 and 320.8 ± 91.4) or PNU-120596 (306.1 ± 34.4 and 343.9 ± 97.7) in SLE mice. The anxiety-like behavior (i.e., decrease in locomotion, increase in sleeping, and decrease distance traveled during open-field test in SLE mice compared to controls (all p<0.001)) presented by SLE mice was also not improved by GTS-21 or PNU-120596. Although no significant beneficial effects of α7 ligands were observed in SLE mice at this advanced stage, we predict that potentiating acetylcholine and targeting this receptor earlier in the pathogenesis of SLE may prove to be efficacious and should be addressed in future studies.

ACE2 Knock‐out Mice Show Decreased Blood Pressure Response and Increased Anxiety‐like Behavior to Acute Stress

Stress increases Ang-II production and elevates sympathetic outflow which results in anxiety-related behavior and hypertension. Here, we investigated a potential contribution of Angiotensin Converting Enzyme-2 (ACE2) in the regulation of stress and anxiety-related behavior, in conscious freely moving mice. Previous studies reported that overexpression of ACE2 attenuates HPA axis and decreases anxiety-related behavior (Wang et al., 2018); however, deletion of ACE2 and its implication for such responses are largely unknow. Male mice (30-33 g), 6 ACE2 knock-outs (KO) and 6 wild type (WT) controls were chronically implanted with blood pressure (BP) transducers for continuous recording of BP, heart rate (HR) and locomotor activity. Following a 1-week recovery, mice were subjected to resident-intruder test for 10 min, for the measurement of stress-induced cardiovascular changes. During the dark/active period, a naïve intruder-mouse was suddenly introduced in the experimental mice's cages and their cardiovascular responses were analyzed using the Ponemah software (DSI). In response to intruder-intrusion for 10 min, ACE2 KO mice showed significant (p<0.05) attenuation in BP increases (+15 ±7 mmHg) compared to their WT controls (+39 ±7 mmHg). However, no significant difference was observed in HR increase in ACE2 KO versus WT mice (+166 ±40 and +173±38 bpm, respectively, p=0.45). In contrast, ACE2 KO mice exhibited a significant increase in locomotor activity compared to controls (179 ±19 and 111 ±14 arbitrary units, respectively, p<0.05). These mice were later subjected to the elevated plus maze (EPM) and light-dark box (LDB) tests for 5 min to evaluate anxiety-related behavior. Interestingly ACE2 KO mice were observed to spend more time in closed arms (244 ±19 s) in EPM compared to WT controls (218 ±22 s, p<0.05). However, in the LDB test, ACE2 KO mice showed no difference (286 ±18 s) compared to WT (279 ±19 s, ns). All mice were then chronically implanted subcutaneously with osmotic minipumps filled with an AT1 receptor antagonist (losartan, 10 mg/kg). A week later, mice were again subjected to the intruder test. ACE2 KO mice showed substantial attenuation in BP increase to intruder-intrusion compared to WT mice (+6±3 and +19±6 mmHg respectively, p<0.05); with no significant difference in HR increase in both the groups. Interestingly, pre-treatment with losartan attenuated the intruder-induced locomotor activity in ACE2 KO mice (149±29, p=0.11). In addition, losartan treatment did not attenuate the time ACE2 KO mice spent in closed arms in EPM; but exhibited a decreased time spent in the dark compartment in the LDB test. Surprisingly, our results did not show exacerbated BP responses to acute stress in the absence of ACE2 but instead produced decreased BP responses suggesting other compensatory mechanisms involved. Losartan-mediated attenuation of the increased locomotor activity and anxiety-related behaviors in ACE2 KO mice strongly suggests an Ang-II mediated anxiety-related behaviors. Our experimental findings further extend the current notion that ACE2 potentially contributes to the regulation of stress and anxiety.

Postnatal Oxytocin Treatment Improves Survival and Improves Behavioral Outcomes in an Animal Model of Neonatal Abstinence Syndrome

Objective To investigate the therapeutic effect of the neuropeptide oxytocin in an animal model of neonatal abstinence syndrome. Hypothesis Prenatal exposure to drugs of abuse (eg. opioids and benzodiazepines) results in a severe withdrawal syndrome known as neonatal abstinence syndrome (NAS). NAS is a serious health problem with increasing prevalence. Standard of care pharmacological intervention consists of morphine, methadone, buprenorphine, clonidine, and phenobarbital. Safer and more effective treatments are urgently needed. The neuropeptide oxytocin has been shown to reverse drug and alcohol withdrawal syndromes in adult animals and humans. Here, we examined the effect of postnatal oxytocin treatment on survival and behavioral outcomes in neonatal rats prenatally exposed to opioids or benzodiazepines. Methods Pregnant Sprague-Dawley rats were injected subcutaneously with escalating doses of morphine (10-50 mg/kg/day) or diazepam (2-15 mg/kg/day) starting at gestational day seven through parturition. Upon delivery, rat pups received daily subcutaneous injections of 2 mg/kg oxytocin or saline for the first 10 postnatal days and survival was measured. A second cohort of morphine and diazepam exposed rat pups received injections of 0.3, 1, or 2 mg/kg oxytocin or saline for the first 10 postnatal days and survival was assessed for 30 days. Surviving animals were subjected to a battery of behavioral assessments between postnatal day 30 and 45 to determine how early life oxytocin treatment impacts neurocognitive outcomes. These assessments included a Locomotor Assay, Light-Dark Box Test, Passive Avoidance Task, and Social Interaction Test. Animals were then sacrificed, blood was collected and processed, and glucose oxidase, coritcosterone, and aldosterone was measured using Enzyme Immunoassay (EIA). Results In the first cohort of animals exposed to morphine prenatally, survival was significantly improved with 2 mg/kg oxytocin treatment compared to saline (100% vs 66.67%; P=0.008). The same pattern was observed with animals exposed to diazepam prenatally (100% vs 66.5%; P=0.0149). In the second cohort of animals exposed to morphine prenatally, all doses of oxytocin (0.3, 1, and 2 mg/kg) improved survival compared to saline (88.89%, 100%, 100% vs 50%, respectively; P<0.0001). A similar pattern of improved survival following the two highest doses of oxytocin, but not 0.3 mg/kg, was observed with animals exposed to diazepam prenatally (68.75%, 100%, 100% vs 62.5%, respectively; P=0.0176). Preliminary findings for improvements in learning and memory and social behavior were also observed. No differences in the measured biological outcomes was observed. Conclusions These findings provide insights in to the potential for oxytocin as a novel treatment option for NAS. Given the high unmet need in NAS and the established safety profile of oxytocin, clinical trials appear warranted.