Open Field Assay Research Articles

Oral fentanyl consumption and withdrawal impairs fear extinction learning and enhances basolateral amygdala principal neuron excitatory-inhibitory balance in male and female mice

The number of opioid overdose deaths has increased over the past several years, mainly driven by an increase in the availability of highly potent synthetic opioids, like fentanyl, in the un-regulated drug supply. Over the last few years, changes in the drug supply, and in particular the availability of counterfeit pills containing fentanyl, have made oral use of opioids a more common route of administration. Here, we used a drinking in the dark (DiD) paradigm to model oral fentanyl self-administration using increasing fentanyl concentrations in male and female mice over 5 weeks. Fentanyl consumption peaked in both female and male mice at the 30 µg/mL dose, with female mice consuming significantly more fentanyl than male mice. Mice consumed sufficient fentanyl such that withdrawal was precipitated with naloxone, with males having increased withdrawal symptoms as compared to females, despite lower pharmacological exposure. We also performed behavioral assays to measure avoidance behavior and reward-seeking during fentanyl abstinence. Female mice displayed reduced avoidance behaviors in the open field assay, whereas male mice showed increased avoidance in the light/dark box assay. Female mice also exhibited increased reward-seeking in the sucrose preference test. Fentanyl-consuming mice of both sexes showed impaired cued fear extinction learning following fear conditioning and increased excitatory synaptic drive and increased excitability of BLA principal neurons. Our experiments demonstrate that long-term oral fentanyl consumption results in wide-ranging physiological and behavioral disruptions. This model could be useful to further study fentanyl withdrawal syndrome and behaviors and neuroplasticity associated with protracted fentanyl withdrawal.

Obesity alters circadian and behavioral responses to constant light in male mice

Exposure to artificial light during the night is known to promote disruption to the biological clock, which can lead to impaired mood and metabolism. Metabolic hormone secretion is modulated by the circadian pacemaker and recent research has shown that hormones such as insulin and leptin can also directly affect behavioral outcomes and the circadian clock. In turn, obesity itself is known to modulate the circadian rhythm and alter emotionality. This study investigated the behavioral and metabolic effects of constant light exposure in two models of obesity – a leptin null mutant (OB) and diet-induced obesity via high-fat diet. For both experiments, mice were placed into either a standard Light:Dark cycle (LD) or constant light (LL) and their circadian locomotor rhythms were continuously monitored. After 10 weeks of exposure to their respective lighting conditions, all mice were subjected to an open field assay to assess their explorative behaviors. Their metabolic hormone levels and inflammation levels were also measured. Behaviorally, exposure to constant light led to increased period lengthening and open field activity in the lean mice compared to both obesity models. Metabolically, LL led to increased cytokine levels and poorer metabolic outcomes in both lean and obese mice, sometimes exacerbating the metabolic issues in the obese mice, independent of weight gain. This study illustrates that LL can produce altered behavioral and physiological outcomes, even in lean mice. These results also indicate that obesity induced by different reasons can lead to shortened circadian rhythmicity and exploratory activity when exposed to chronic light.

The open field assay is influenced by room temperature and by drugs that affect core body temperature

Background The open field assay is used to study anxiety-related traits and anxiolytic drugs in rodents. This assay entails measuring locomotor activity and time spent in the center of a chamber that is maintained at ambient room temperature. However, the ambient temperature in most laboratories varies daily and seasonally and can differ between buildings. We sought to evaluate how varying ambient temperature and core body temperature (CBT) affected open field locomotor activity and center time of male wild-type (WT, C57BL/6) and Transient Receptor Potential Subfamily M Member 8 (Trpm8) knock-out (Trpm8-/- ) mice. TRPM8 is an ion channel that detects cool temperatures and is activated by icilin. Methods Mice were placed in the open field at 4°C and 23°C for 1 hour. Distance traveled and time spent in the center were measured. Mice were injected with icilin, M8-B, diazepam, or saline, and changes in activity level were recorded. Results The cooling agent icilin increased CBT and profoundly reduced distance traveled and center time of WT mice relative to controls. Likewise, cooling the ambient temperature to 4°C reduced distance traveled and center time of WT mice relative to Trpm8-/- mice. Conversely, the TRPM8 antagonist (M8-B) reduced CBT and increased distance traveled and center time of WT mice when tested at 4°C. The TRPM8 antagonist (M8-B) had no effect on CBT or open field behavior of Trpm8-/- mice. The anxiolytic diazepam reduced CBT in WT and Trpm8-/- mice. When tested at 4°C, diazepam increased distance traveled and center time in WT mice but did not alter open field behavior of Trpm8-/- mice. Conclusions Environmental temperature and drugs that affect CBT can influence locomotor behavior and center time in the open field assay, highlighting temperature (ambient and core) as sources of environmental and physiologic variability in this commonly used behavioral assay.

Oral Fentanyl Consumption and Withdrawal Impairs Fear Extinction Learning and Enhances Basolateral Amygdala Principal Neuron Excitatory-Inhibitory Balance in Male and Female Mice.

The number of opioid overdose deaths has increased over the past several years, mainly driven by an increase in the availability of highly potent synthetic opioids, like fentanyl, in the un-regulated drug supply. Over the last few years, changes in the drug supply, and in particular the availability of counterfeit pills containing fentanyl, have made oral use of opioids a more common route of administration. Here, we used a drinking in the dark (DiD) paradigm to model oral fentanyl self-administration using increasing fentanyl concentrations in male and female mice over 5 weeks. Fentanyl consumption peaked in both female and male mice at the 30 µg/mL dose, with female mice consuming significantly more fentanyl than male mice. Mice consumed sufficient fentanyl such that withdrawal was precipitated with naloxone, with males having more withdrawal symptoms, despite lower pharmacological exposure. We also performed behavioral assays to measure avoidance behavior and reward-seeking during fentanyl abstinence. Female mice displayed reduced avoidance behaviors in the open field assay, whereas male mice showed increased avoidance in the light/dark box assay. Female mice also exhibited increased reward-seeking in the sucrose preference test. Fentanyl-consuming mice of both sexes showed impaired cued fear extinction learning following fear conditioning and increased excitatory synaptic drive and increased excitability of BLA principal neurons. Our experiments demonstrate that long-term oral fentanyl consumption results in wide-ranging physiological and behavioral disruptions. This model could be useful to further study fentanyl withdrawal syndrome and behaviors and neuroplasticity associated with protracted fentanyl withdrawal.

Hematopoietic stem cell gene therapy for the treatment of SYNGAP1-related non-specific intellectual disability.

Synaptic Ras GTPase activating protein 1 (SYNGAP1)-related non-specific intellectual disability is a neurodevelopmental disorder caused by an insufficient level of SynGAP1 resulting in a dysfunction of neuronal synapses and presenting with a wide array of clinical phenotypes. Hematopoietic stem cell gene therapy has the potential to deliver therapeutic levels of functional SynGAP1 to affected neurons upon transduction of hematopoietic stem and progenitor cells with a lentiviral vector. As a novel approach toward the treatment of SYNGAP1, we have generated a lentiviral vector expressing a modified form of SynGAP1 for transduction of human CD34+ hematopoietic stem and progenitor cells. The gene-modified cells were then transplanted into adult immunodeficient SYNGAP1+/- heterozygous mice and evaluated for improvement of SYNGAP1-related clinical phenotypes. Expression of SynGAP1 was also evaluated in the brain tissue of transplanted mice. In our proof-of-concept study, we have demonstrated significant improvement of SYNGAP1-related phenotypes including an improvement in motor abilities observed in mice transplanted with the vector transduced cells because they displayed decreased hyperactivity in an open field assay and an increased latency to fall in a rotarod assay. An increased level of SynGAP1 was also detected in the brains of these mice. These early-stage results highlight the potential of this stem cell gene therapy approach as a treatment strategy for SYNGAP1.

Employing a Toxic Aging Coin approach to assess hexavalent chromium (Cr[VI])-induced neurotoxic effects on behavior: Heads for age differences

We are facing a rapidly growing geriatric population (65+) that will live for multiple decades and are challenged with environmental pollution far exceeding that of previous generations. Consequently, we currently have a poor understanding of how environmental pollution will impact geriatric health distinctly from younger populations. Few toxicology studies have considered age differences with geriatric individuals. Critically, all top ten most prevalent age-related diseases are linked to metal exposures. Hexavalent chromium [Cr(VI)] is a metal of major environmental health concern that can induce aging phenotypes and neurotoxicity. However, there are many knowledge gaps for Cr(VI) neurotoxicity, including how Cr(VI) impacts behavior. To address this, we exposed male rats across three ages (3-, 7-, and 18-months old) to Cr(VI) in drinking water (0, 0.05, 0.1 mg/L) for 90 days. These levels reflect the maximum contaminant levels determined by the World Health Organization (WHO) and the U.S. Environmental Protection Agency (US EPA). Here, we report how these Cr(VI) drinking water levels impacted rat behaviors using a battery of behavior tests, including grip strength, open field assay, elevated plus maze, Y-maze, and 3-chamber assay. We observed adult rats were the most affected age group and memory assays (spatial and social) exhibited the most significant effects. Critically, the significant effects were surprising as rats should be particularly resistant to these Cr(VI) drinking water levels due to the adjustments applied in risk assessment from rodent studies to human safety, and because rats endogenously synthesize vitamin C in their livers (vitamin C is a primary reducer of Cr[VI] to Cr[III]). Our results emphasize the need to broaden the scope of toxicology research to consider multiple life stages and suggest the current regulations for Cr(VI) in drinking water need to be revisited.

Relative telencephalon size does not affect collective motion in the guppy (Poecilia reticulata).

Collective motion is common across all animal taxa, from swarming insects to schools of fish. The collective motion requires intricate behavioral integration among individuals, yet little is known about how evolutionary changes in brain morphology influence the ability for individuals to coordinate behavior in groups. In this study, we utilized guppies that were selectively bred for relative telencephalon size, an aspect of brain morphology that is normally associated with advanced cognitive functions, to examine its role in collective motion using an open-field assay. We analyzed high-resolution tracking data of same-sex shoals consisting of 8 individuals to assess different aspects of collective motion, such as alignment, attraction to nearby shoal members, and swimming speed. Our findings indicate that variation in collective motion in guppy shoals might not be strongly affected by variation in relative telencephalon size. Our study suggests that group dynamics in collectively moving animals are likely not driven by advanced cognitive functions but rather by fundamental cognitive processes stemming from relatively simple rules among neighboring individuals.

Immunization with Sez6L2 protein mimics the human condition of late-onset cerebellar ataxia with associated Sez6L2 autoantibodies in mice

Abstract Sez6L2 autoantibodies have been identified in a subset of human patients with progressive cerebellar ataxia. Sez6L2 is a transmembrane protein expressed by most neurons, including Purkinje cells and granular cells of the cerebellum. Patients with Sez6L2 autoantibodies present with gait ataxia, slurred speech, and ocular motor symptoms. Some patients also develop limb ataxia and cognitive deficits. These case studies have led to the hypothesis that autoimmunity against Sez6L2 can directly cause cerebellar damage leading to ataxia. We tested whether C57BL/6J mice immunized against Sez6L2 protein develop ataxia symptoms. We also investigated the systemic immune response against Sez6L2 and whether there was increased immune cell infiltration into the brain. We found that mice immunized with Sez6L2 generated significant levels of anti-Sez6L2 antibodies, in addition to a significant population of Sez6L2-specific CD4 and CD8 T cells. Furthermore, these mice had increased CD4+ T cell infiltration into the brain. The Sez6L2 immunized mice also exhibited mild mobility impairments in open field and wire grid walk assays. Overall, our results demonstrate that an autoimmune response to Sez6L2 in mice can lead to mobility impairments and pathology consistent with human patients who develop cerebellar ataxia associated with Sez6L2 autoantibodies. This new mouse model should be useful for mechanistic studies on a poorly understood autoimmune disease.

Changes in ethanol effects in knock-in mice expressing ethanol insensitive alpha1 and alpha2 glycine receptor subunits

AimsGlycine receptors (GlyRs) are potentiated by physiologically relevant concentrations of ethanol, and mutations in the intracellular loop of α1 and α2 subunits reduced the effect of the drug. Knock-in (KI) mice having these individual mutations revealed that α1 and α2 subunits played a role in ethanol-induced sedation and ethanol intake. In this study, we wanted to examine if the effects of stacking both mutations in a 2xKI mouse model (α1/α2) generated by a selective breeding strategy further impacted cellular and behavioral responses to ethanol. Main methodsWe used electrophysiological recordings to examine ethanol's effect on GlyRs and evaluated ethanol-induced neuronal activation using c-Fos immunoreactivity and the genetically encoded calcium indicator GCaMP6s in the nucleus accumbens (nAc). We also examined ethanol-induced behavior using open field, loss of the righting response, and drinking in the dark (DID) paradigm. Key findingsEthanol did not potentiate GlyRs nor affect neuronal excitability in the nAc from 2xKI. Moreover, ethanol decreased the Ca2+ signal in WT mice, whereas there were no changes in the signal in 2xKI mice. Interestingly, there was an increase in c-Fos baseline in the 2xKI mice in the absence of ethanol. Behavioral assays showed that 2xKI mice recovered faster from a sedative dose of ethanol and had higher ethanol intake on the first test day of the DID test than WT mice. Interestingly, an open-field assay showed that 2xKI mice displayed less anxiety-like behavior than WT mice. SignificanceThe results indicate that α1 and α2 subunits are biologically relevant targets for regulating sedative effects and ethanol consumption.

The open field assay is influenced by room temperature and by drugs that affect core body temperature.

The open field assay is used to study anxiety-related traits and anxiolytic drugs in rodents. This assay entails measuring locomotor activity and time spent in the center of a chamber that is maintained at ambient room temperature. However, the ambient temperature in most laboratories varies daily and seasonally and can differ between buildings. We sought to evaluate how varying ambient temperature and core body temperature (CBT) affected open field locomotor activity and center time of male wild-type (WT, C57BL/6) and Transient Receptor Potential Subfamily M Member 8 ( Trpm8) knock-out ( Trpm8 -/- ) mice. TRPM8 is an ion channel that detects cool temperatures and is activated by icilin. Mice were placed in the open field at 4°C and 23°C for 1 hour. Distance traveled and time spent in the center were measured. Mice were injected with icilin, M8-B, diazepam, or saline, and changes in activity level were recorded. The cooling agent icilin increased CBT and profoundly reduced distance traveled and center time of WT mice relative to controls. Likewise, cooling the ambient temperature to 4°C reduced distance traveled and center time of WT mice relative to Trpm8 -/- mice. Conversely, the TRPM8 antagonist (M8-B) reduced CBT and increased distance traveled and center time of WT mice when tested at 4°C. The TRPM8 antagonist (M8-B) had no effect on CBT or open field behavior of Trpm8 -/- mice. The anxiolytic diazepam reduced CBT in WT and Trpm8 -/- mice. When tested at 4°C, diazepam increased distance traveled and center time in WT mice but did not alter open field behavior of Trpm8 -/- mice. Environmental temperature and drugs that affect CBT can influence locomotor behavior and center time in the open field assay, highlighting temperature (ambient and core) as sources of environmental and physiologic variability in this commonly used behavioral assay.

Studies on neurobehavioural properties of Caralluma dalzielii N.E Br. aqueous aerial parts extract in mice

Ethnopharmacological relevanceCaralluma dalzielii (Asclepiadiaceae) is a shrub used in folkloric medicine to treat epilepsy, pain and infertility in sub-Saharan Africa. Previous studies demonstrated its analgesic, antiulcer, anticonvulsant, and anti-inflammatory activities. AimThis study aimed to determine the neurobehavioural properties of Caralluma dalzielii aqueous aerial parts extract (CDAE) in mice using standard experimental models. Materials and methodsNeurobehavioural activities of CDAE were evaluated (100, 200, and 400 mg/kg) in Swiss Albino mice using the beam walk, staircase, hole board, object recognition, open field assay, Y-maze and forced swimming tests. Phytochemical constituents were analysed using GC-MS. ResultsCDAE significantly increased the mean number of head dips, recognition index and spontaneous alternation in hole board (14.03 at 400 mg/kg and 6.01 in distilled water group; p < 0.05), object recognition (68.16% at 400 mg/kg compared with 51.66% of distilled water group) and Y maze (9.16 at 400 mg/kg as against 4.66 of distilled water group; p < 0.05) tests respectively. It decreased the rearing counts as well as the peripheral and central square crossing in the staircase (4.2 at 400 mg/kg as against 7.87 of the distilled water group; p < 0.05) and open field tests (central, 0.81; peripheral, 1.66 at 400 mg/kg as against central, 5.23; peripheral 11.83 of the distilled water control group; p < 0.05), respectively. There were no significant effects on beam walk assays and forced swim tests. The GC-MS analysis identified a hundred compounds in CDAE. Some compounds which have been reported to possess neurobehavioural activity that were identified include 3,5-Dimethylpyrazole, 2-Amino-5-methylbenzoic acid, Acetophenone, and Tetrahydropyran. ConclusionCDAE demonstrated anxiolytic, anti-hyperactivity, and memory-improving effects in mice. The extract may possess GABAergic and glutamatergic properties. More studies are needed to confirm this. Isolation of the bioactive compounds is currently ongoing to unravel the bioactive constituents present in C. dalzielii extract.

The open field assay is influenced by room temperature and by drugs that affect core body temperature

Background The open field assay is used to study anxiety-related traits and anxiolytic drugs in rodents. This assay entails measuring locomotor activity and time spent in the center of a chamber that is maintained at ambient room temperature. However, the ambient temperature in most laboratories varies daily and seasonally and can differ between buildings. We sought to evaluate how varying ambient temperature and core body temperature (CBT) affected open field locomotor activity and center time of male wild-type (WT, C57BL/6) and Transient Receptor Potential Subfamily M Member 8 (Trpm8) knock-out (Trpm8-/- ) mice. TRPM8 is an ion channel that detects cool temperatures and is activated by icilin. Methods Mice were placed in the open field at 4°C and 23°C for 30 minutes. Distance traveled and time spent in the center were measured. Mice were injected with icilin, M8-B, diazepam, or saline, and changes in activity level were recorded. Results The cooling agent icilin increased CBT and profoundly reduced distance traveled and center time of WT mice relative to controls. Likewise, cooling the ambient temperature to 4°C reduced distance traveled and center time of WT mice relative to Trpm8-/- mice. Conversely, the TRPM8 antagonist (M8-B) reduced CBT and increased distance traveled and center time of WT mice when tested at 4°C. The TRPM8 antagonist (M8-B) had no effect on CBT or open field behavior of Trpm8-/- mice. The anxiolytic diazepam reduced CBT in WT and Trpm8-/- mice. When tested at 4°C, diazepam increased distance traveled and center time in WT mice but did not alter open field behavior of Trpm8-/- mice. Conclusions Environmental temperature and drugs that affect CBT can influence locomotor behavior and center time in the open field assay, highlighting temperature (ambient and core) as sources of environmental and physiologic variability in this commonly used behavioral assay.

Rosemary (Rosmarinus officinalis L.) hydrosol based on serotonergic synapse for insomnia

Ethnopharmacological relevanceRosemary (Rosmarinus officinalis L.) has been widely used as a traditional remedy for insomnia, depression and anxiety in China and Western countries. Modern pharmacological studies have shown that rosemary has important applications in neurological disorders. However, the mechanism of action of rosemary hydrosol in the treatment of insomnia is not known. Aims of the studyInsomnia is closely linked to anxiety and depression, and its pathogenesis is related to biology, psychology, and sociology. Rosemary is a natural plant that has been used to treat insomnia and depression and has good biological activity, but its material basis and mechanism for the treatment of insomnia are not clear. Here, we report on the role of aqueous extracts of rosemary in the treatment of insomnia. Materials and methodsThe study was based on network pharmacology, using a combination of RNA-sequencing, “quantity-effect” weighting coefficients, and pharmacodynamic experiments. DL-4-chlorophenylalanine (PCPA) was intraperitoneally injected into SD rats to replicate the insomnia model with a blank, model, diazepam, and rosemary hydrosol low-, medium-, and high-dose groups were set up for the experiment. The key pathways in the treatment of insomnia with rosemary hydrosol were analyzed by molecular docking, open field assay, ELISA, western-Blot, Rt-PCR, and immunohistochemical assay. ResultsRosemary hydrosol was analyzed by GC-MS to identify 19 components. 1579 differential genes were obtained by RNA-Seq analysis, 533 targets for rosemary hydrosol and 2705 targets for insomnia, and 29 key targets were obtained by intersection. The KEGG results were ranked by “quantity-effect” weighting coefficients, resulting in serotonergic synapse was the key pathway for the treatment of insomnia with rosemary hydrosol. Molecular docking results showed that 1,7,7-trimethylbicyclo[2.2.1] heptan-2-one, 3-methyl-4-isopropylphenol, caryophyllene, and citronellol of rosemary hydrosol acted synergistically to achieve a therapeutic effect on insomnia. Caryophyllene acts on the HTR1A target by upregulating 5-HT1AR, leading to increased 5-HT release, and upregulation of ADCY5, cAMP, PKA and GABAA at serotonergic synapses; citronellol upregulated ADCY5 and 1,7,7-trimethylbicyclo[2.2.1] heptan-2-one, and 3-methyl-4-isopropylphenol up-regulated GABAA to improve insomnia symptoms. In open-field experiments, ELISA kits (5-HT, GABA, and DA), Western-blotting, Rt-PCR and immunohistochemical assay experiments, insomnia rats in the low-, medium- and high-dose groups of rosemary hydrosol showed different degrees of improvement compared with the model group. ConclusionsIt was shown that rosemary hydrosol may exert its therapeutic effects on insomnia through serotonergic synapses by combining RNA-Seq, “quantity-effect” weighting coefficients network pharmacology and pharmacodynamic experiments. We have provided a preliminary theoretical study for the development of rosemary hydrosol additive into a beverage for the treatment of insomnia, but it needs to be studied in depth. This study was conducted in rats and the results have limitations and may not apply to humans.

0074 Investigating the Impacts of Pharmacological Slow-Wave Sleep Enhancement on Cognition in an Alzheimer's Disease Model

Abstract Introduction Alzheimer’s disease (AD) is a debilitating and irreversible neurodegenerative disease associated with major disruptions to sleep. Recent works point to a strong bidirectional relationship between loss of slow-wave sleep (SWS), the deepest sleep stage, and the pathological and cognitive features of AD. Here, we investigate the impacts of chronic pharmacological SWS enhancement with gaboxadol, a delta GABAA receptor agonist, on cognitive performance the APP/PS1 mouse model of AD. Currently, we are assessing the impacts of this intervention on Abeta plaque burden. Methods Using electroencephalography (EEG), we first assessed if gaboxadol is capable of stimulating SWS sleep in 6-month-old AD mice. Next, 6-month-old control (CTRL) or AD mice received gaboxadol 5x/week for 4 weeks at the start of the light cycle. Groups underwent subsequent testing in the open field (OF), Y-maze, and contextual fear conditioning (CFC) assays (n = 8-9 mice per group). Data were analyzed using two- or three-way ANOVAs, with repeated measures when appropriate. Results Our EEG studies demonstrate that gaboxadol (6 mg/kg) significantly enhances SWS/NREM from baseline (p &amp;lt; 0.0308). Gaboxadol did not impact gross locomotor behavior assessed by distance travelled in the in the OF. However, rearing was decreased in AD mice compared to CTRL mice (p &amp;lt; 0.0128). In the Y-maze, gaboxadol significantly increased time spent in the novel arm in CTRL (p &amp;lt; 0.0386) but not AD mice. We did not find significant effects of gaboxadol on memory recall in the CFC assay. Conclusion Chronic pharmacological stimulation of SWS using gaboxadol does not show robust cognition-enhancing effects in APP/PS1 mice, but may be cognitively enhancing in CTRL mice. It is possible that longer treatment duration is necessary to rescue cognitive deficits in AD mice. Support (if any) NIA: 5R21AG064774-02 T32 GM7367-46

Promotion of the growth and yield of Zea mays by synthetic microbial communities from Jala maize.

Plant growth-promoting bacteria (PGPB) are a source of nutrient supply, stimulate plant growth, and even act in the biocontrol of phytopathogens. However, these phenotypic traits have rarely been explored in culturable bacteria from native maize landraces. In this study, synthetic microbial communities (SynCom) were assembled with a set of PGPB isolated from the Jala maize landrace, some of them with additional abilities for the biocontrol of phytopathogenic fungi and the stimulation of plant-induced systemic resistance (ISR). Three SynCom were designed considering the phenotypic traits of bacterial strains, including Achromobacter xylosoxidans Z2K8, Burkholderia sp. Z1AL11, Klebsiella variicola R3J3HD7, Kosakonia pseudosacchari Z2WD1, Pantoea ananatis E2HD8, Pantoea sp. E2AD2, Phytobacter diazotrophicus Z2WL1, Pseudomonas protegens E1BL2, and P. protegens E2HL9. Plant growth promotion in gnotobiotic and greenhouse seedlings assays was performed with Conejo landrace; meanwhile, open field tests were carried out on hybrid CPL9105W maize. In all experimental models, a significant promotion of plant growth was observed. In gnotobiotic assays, the roots and shoot length of the maize seedlings increased 4.2 and 3.0 times, respectively, compared to the untreated control. Similarly, the sizes and weights of the roots and shoots of the plants increased significantly in the greenhouse assays. In the open field assay performed with hybrid CPL9105W maize, the yield increased from 11 tons/ha for the control to 16 tons/ha inoculated with SynCom 3. In addition, the incidence of rust fungal infections decreased significantly from 12.5% in the control to 8% in the treatment with SynCom 3. All SynCom designs promoted the growth of maize in all assays. However, SynCom 3 formulated with A. xylosoxidans Z2K8, Burkholderia sp. Z1AL11, K. variicola R3J3HD7, P. ananatis E2HD8, P. diazotrophicus Z2WL1, and P. protegens E1BL2 displayed the best results for promoting plant growth, their yield, and the inhibition of fungal rust. This study demonstrated the biotechnological eco-friendly plant growth-promoting potential of SynCom assemblies with culturable bacteria from native maize landraces for more sustainable and economic agriculture.

Temporal Silencing of Purkinje Cell Neurotransmission Reveals a Dynamic Role for Cerebellar Function in Neurodevelopment (P10-9.006)

<h3>Objective:</h3> To determine whether differential timing of disrupted cerebellar function influences the nature and severity of associated neurodevelopmental outcomes. <h3>Background:</h3> Disruptions of cerebellar function have been increasingly implicated in the pathogenesis of a wide array of neurodevelopmental disorders. While there has been increasing focus on genetically mediated etiologies of disrupted cerebellar function, less attention has been given to acquired etiologies although they underlie diseases with high incidence and prevalence, such as the neurodevelopmental disability associated with prematurity and autism spectrum disorders. The present study uses mouse transgenics to develop a synthetic model of cerebellar developmental dysfunction and interrogates the role of cerebellar neurotransmission in early brain development. <h3>Design/Methods:</h3> Using inducible tissue specific Cre-mediated recombination the present model allows for spatial and temporal induction of cerebellar dysfunction through timed excision of the vesicular GABA transporter (VGAT) specifically in Purkinje cells. Using this line, Purkinje cell neurotransmission is silenced <i>in utero</i> (EarlyOff) or at time of weaning (LateOff), corresponding developmentally to the immature and mature cerebellum. Mouse behavior and the electrophysiologic profile of cerebellar nuclear neurons was then performed comparing EarlyOff, LateOff, and control animals at 8–10 weeks of life. <h3>Results:</h3> Compared to controls EarlyOFF showed significantly altered gross motor behavior (rotarod assay, footprinting), social behavior (3-chamber assay), and hyperactivity (open field assay). Compared to controls, LateOff had equivalent performance on gross motor assays but showed significant hyperactivity. Social tests are ongoing. Cerebellar nuclear electrophysiology from awake behaving animals revealed significantly different and divergent spike profiles between EarlyOff, LateOff, and controls. <h3>Conclusions:</h3> The developmental timing of cerebellar dysfunction leads to discrepant behavioral and electrophysiologic profiles. These findings underline that the disparate phenotypes of neurodevelopmental disability associated with prematurity and autism may nonetheless have shared cerebellar etiology, emphasize the necessity of understanding how cerebellar nuclear signaling influences broad cortical function, and reveal a potential target for neuromodulatory intervention. <b>Disclosure:</b> Dr. Gill has received personal compensation in the range of $500-$4,999 for serving as an Expert Witness for Elite Medical Experts. The institution of Dr. Gill has received research support from NIH-NINDS. Miss Nguyen has nothing to disclose. Roy Sillitoe has nothing to disclose.

Timing matters: Sex differences in inflammatory and behavioral outcomes following repetitive blast mild traumatic brain injury

BackgroundRepetitive blast-related mild traumatic brain injury (mTBI) caused by exposure to high explosives is increasingly common among warfighters as well as civilians. While women have been serving in military positions with increased risk of blast exposure since 2016, there are few published reports examining sex as a biological variable in models of blast mTBI, greatly limiting diagnosis and treatment capabilities. As such, here we examined outcomes of repetitive blast trauma in female and male mice in relation to potential behavioral, inflammatory, microbiome, and vascular dysfunction at multiple timepoints. MethodsIn this study we utilized a well-established blast overpressure model to induce repetitive (3x) blast-mTBI in both female and male mice. Acutely following repetitive exposure, we measured serum and brain cytokine levels, blood–brain barrier (BBB) disruption, fecal microbial abundance, and locomotion and anxiety-like behavior in the open field assay. At the one-month timepoint, in female and male mice we assessed behavioral correlates of mTBI and PTSD-related symptoms commonly reported by Veterans with a history of blast-mTBI using the elevated zero maze, acoustic startle, and conditioned odorant aversion paradigms. ResultsRepetitive blast exposure resulted in both similar (e.g., increased IL-6), and disparate (e.g., IL-10 increase only in females) patterns of acute serum and brain cytokine as well as gut microbiome changes in female and male mice. Acute BBB disruption following repetitive blast exposure was apparent in both sexes. While female and male blast mice both exhibited acute locomotor and anxiety-like deficits in the open field assay, only male mice exhibited adverse behavioral outcomes that lasted at least one-month. DiscussionRepresenting a novel survey of potential sex differences following repetitive blast trauma, our results demonstrate unique similar yet divergent patterns of blast-induced dysfunction in female vs. male mice and highlight novel targets for future diagnosis and therapeutic development.

The open field assay is influenced by room temperature and by drugs that affect core body temperature

Background: The open field assay is used to study anxiety-related traits and anxiolytic drugs in rodents. This assay entails measuring locomotor activity and time spent in the center of a chamber that is maintained at ambient room temperature. However, the ambient temperature in most laboratories varies daily and seasonally and can differ between buildings. We sought to evaluate how varying ambient temperature and core body temperature (CBT) affected open field locomotor activity and center time of male wild-type (WT, C57BL/6) and Transient Receptor Potential Subfamily M Member 8 (Trpm8) knock-out (Trpm8-/-) mice. TRPM8 is an ion channel that detects cool temperatures and is activated by icilin. Methods: Mice were placed in the open field at 4°C and 23°C for 30 minutes. Distance traveled and time spent in the center were measured. Mice were injected with icilin, M8-B, diazepam, or saline, and changes in activity level were recorded. Results: The cooling agent icilin increased CBT and profoundly reduced distance traveled and center time of WT mice relative to controls. Likewise, cooling the ambient temperature to 4°C reduced distance traveled and center time of WT mice relative to Trpm8-/- mice. Conversely, the TRPM8 antagonist (M8-B) reduced CBT and increased distance traveled and center time of WT mice when tested at 4°C. The TRPM8 antagonist (M8-B) had no effect on CBT or open field behavior of Trpm8-/- mice. The anxiolytic diazepam reduced CBT in WT and Trpm8-/- mice. When tested at 4°C, diazepam increased distance traveled and center time in WT mice but did not alter open field behavior of Trpm8-/- mice. Conclusions: Environmental temperature and drugs that affect CBT can influence locomotor behavior and center time in the open field assay, highlighting temperature (ambient and core) as sources of environmental and physiologic variability in this commonly used behavioral assay.

Partial changes in apoptotic pathways in hippocampus and hypothalamus of Cc2d1a heterozygous.

Alterations in the apoptosis pathway have been linked to changes in serotonin levels seen in autistic patients. Cc2d1a is a repressor of the HTR1A gene involved in the serotonin pathway. The hippocampus and hypothalamus of Cc2d1a ± mice were analyzed for the expression of apoptosis markers (caspase 3, 8 and 9). Gender differences were observed in the expression levels of the three caspases consistent with some altered activity in the open-field assay. The number of apoptotic cells was significantly increased. We concluded that apoptotic pathways are only partially affected in the pathogenesis of the Cc2d1a heterozygous mouse model. A) Apoptosis is suppressed because the cell does not receive a death signal, or the receptor cannot activate the caspase 8 pathway despite the death signal. B) Since Caspase 8 and Caspase 3 expression is downregulated in our mouse model, the mechanism of apoptosis is not activated.

Propranolol Modulates Cerebellar Circuit Activity and Reduces Tremor.

Tremor is the most common movement disorder. Several drugs reduce tremor severity, but no cures are available. Propranolol, a β-adrenergic receptor blocker, is the leading treatment for tremor. However, the in vivo circuit mechanisms by which propranolol decreases tremor remain unclear. Here, we test whether propranolol modulates activity in the cerebellum, a key node in the tremor network. We investigated the effects of propranolol in healthy control mice and Car8wdl/wdl mice, which exhibit pathophysiological tremor and ataxia due to cerebellar dysfunction. Propranolol reduced physiological tremor in control mice and reduced pathophysiological tremor in Car8wdl/wdl mice to control levels. Open field and footprinting assays showed that propranolol did not correct ataxia in Car8wdl/wdl mice. In vivo recordings in awake mice revealed that propranolol modulates the spiking activity of control and Car8wdl/wdl Purkinje cells. Recordings in cerebellar nuclei neurons, the targets of Purkinje cells, also revealed altered activity in propranolol-treated control and Car8wdl/wdl mice. Next, we tested whether propranolol reduces tremor through β1 and β2 adrenergic receptors. Propranolol did not change tremor amplitude or cerebellar nuclei activity in β1 and β2 null mice or Car8wdl/wdl mice lacking β1 and β2 receptor function. These data show that propranolol can modulate cerebellar circuit activity through β-adrenergic receptors and may contribute to tremor therapeutics.