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AimWe examined the anti-acanthamoebic efficacy of solvent extract of C. sinensis) and its chemical constituents against trophozoites and cysts of A. castellanii.Materials and methodsThe effects of C. sinensis solvent extract on A. castellanii was investigated by using anti-trophozoite, anti-encystation, and anti-excystation assays. The solvent extract was also fractionated using Gas Chromatography and the chemical constituents of C. sinensis were tested, individually or combined, against the trophozoites.ResultsTrophozoite replication was inhibited within 24-72 h with exposure to 625-5000 µg/mL of C. sinensis solvent extract. C. sinensis also exhibited a dose-dependent inhibition of encystation, with a marked cysticidal activity at 2500-5000 µg/mL concentrations. Two constituents of C. sinensis, namely epigallocatechin-3-gallate and caffeine, significantly inhibited trophozoite replication and encystation at 100 μM and 200 μM, respectively. Cytotoxicity analysis showed that 156.25-2500 µg/mL of solvent extract was not toxic to human corneal epithelial cells, while up to 625 µg/mL was not toxic to Madin-Darby Canine Kidney cells.ConclusionsThis study shows the anti-acanthamoebic potential of C. sinensis solvent extract against trophozoites and cysts. Further pre-clinical studies are required to elucidate the in vivo efficacy and safety of C. sinensis solvent extract.Graphical abstract

Anxiety and trauma-related disorders, such as post-traumatic stress disorder (PTSD), are debilitating mental illnesses with great personal and socioeconomic costs. Examining memory formation and relevant behavioural responding associated with aversive stimuli may improve our understanding of the neurobiology underlying fear memory processing and PTSD treatment. The neurocircuitry underpinning learned fear and its inhibition through extinction is complex, involving synergistic interactions between different neurotransmitter systems in inter-connected brain areas. Endocannabinoid and noradrenergic transmission have both been implicated separately in fear memory processing and PTSD but potential interactions between these systems in relation to fear extinction have received little attention to date. Their receptors are expressed together in brain areas crucial for fear extinction, which is enhanced by both cannabinoid and noradrenergic receptor activation in these areas. Moreover, cannabinoid signalling modulates the activity of locus coeruleus noradrenaline neurons and the release of noradrenaline in the medial prefrontal cortex, a brain area that is crucial for fear extinction. Interestingly, endocannabinoid-noradrenergic system interactions have been shown to regulate the encoding and retrieval of fear memory. Thus noradrenergic regulation of fear extinction may also be driven indirectly in part via cannabinoid receptor signalling. In this perspective paper, we collate the available relevant literature and propose a synergistic role for the endocannabinoid and noradrenergic systems in regulating fear extinction, the study of which may further our understanding of the neurobiological substrates of PTSD and its treatment.

The endocannabinoid system has been implicated in both social and cognitive processing. The endocannabinoid metabolism inhibitor, URB597, dose-dependently improves non-social memory in adult Wistar and Sprague Dawley rats, whereas its effect on social interaction (SI) is affected by both rat strain and drug dose. Lister Hooded rats consistently respond differently to drug treatment in general compared with albino strains. This study sought to investigate the effects of different doses of URB597 on social and non-social memory in Lister Hooded rats, as well as analysing the behavioural composition of the SI.Males were tested for novel object recognition (NOR), social preference (between an object and an unfamiliar rat), social novelty recognition (for a familiar vs unfamiliar rat) and SI with an unfamiliar rat. URB597 (0.1 or 0.3 mg/kg) or vehicle was given 30 minutes before testing. During SI testing, total interaction time was assessed along with time spent on aggressive and explorative behaviours.Lister Hooded rats displayed expected non-social and social memory and social preference, which was not affected by URB597. During SI, URB597 did not affect total interaction time. However, the high dose increased aggression, compared to vehicle, and decreased anogenital sniffing, compared to the low dose of URB597. In summary, URB597 did not affect NOR, social preference or social recognition memory but did have subtle behavioural effects during SI in Lister hooded rats.These findings highlight the importance of considering strain as well as the composition of behaviour when investigating drug effects on social behaviour.

We examined the anti-acanthamoebic efficacy of green tea Camellia sinensis solvent extract (SE) or its chemical constituents against Acanthamoeba castellanii by using anti-trophozoite, anti-encystation, and anti-excystation assays. C. sinensis SE (625-5000 µg/mL) inhibited trophozoite replication within 24-72 h. C. sinensis SE exhibited a dose-dependent inhibition of encystation, with a marked cysticidal activity at 2500-5000 µg/mL. Two constituents of C. sinensis, namely epigallocatechin-3-gallate and caffeine, at 100 μM and 200 μM respectively, significantly inhibited both trophozoite replication and encystation. Cytotoxicity analysis showed that 156.25-2500 µg/mL of SE was not toxic to human corneal epithelial cells, while up to 625 µg/mL was not toxic to Madin-Darby canine kidney cells. This study shows the anti-acanthamoebic potential of C. sinensis SE against A. castellanii trophozoites and cysts. Pre-clinical studies are required to elucidate the in vivo efficacy and safety of C. sinensis SE.

Toxoplasma gondii (T. gondii) is an opportunistic protozoan that can cause brain infection and other serious health consequences in immuno-compromised individuals. This parasite has a remarkable ability to cross biological barriers and exploit the host cell microenvironment to support its own survival and growth. Recent advances in label-free spectroscopic imaging techniques have made it possible to study biological systems at a high spatial resolution. In this study, we used conventional Fourier-transform infrared (FTIR) microspectroscopy and synchrotron-based FTIR microspectroscopy to analyze the chemical changes that are associated with infection of human brain microvascular endothelial cells (hBMECs) by T. gondii (RH) tachyzoites. Both FTIR microspectroscopic methods showed utility in revealing the chemical alterations in the infected hBMECs. Using a ZnS hemisphere device, to increase the numerical aperture, and the synchrotron source to increase the brightness, we obtained spatially resolved spectra from within a single cell. The spectra extracted from the nucleus and cytosol containing the tachyzoites were clearly distinguished. RNA sequencing analysis of T. gondii-infected and uninfected hBMECs revealed significant changes in the expression of host cell genes and pathways in response to T. gondii infection. These FTIR spectroscopic and transcriptomic findings provide significant insight into the molecular changes that occur in hBMECs during T. gondii infection.

© 2020 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd Learning to associate certain contexts with threat and adapting to changing environmental contingencies by learning that such contexts are no longer associated with threat are both crucial for survival. Research over the last few decades has made considerable progress in determining the brain areas involved in the encoding, retrieval and extinction of contextual fear. These studies have identified the hippocampus and amygdala, along with the prefrontal cortex and other inter-connected brain areas, as key players in contextual fear processing. In contrast to the neural circuit basis of contextual fear, the neurochemical mechanisms involved in its regulation remain poorly understood. Dopamine is well known for its role in appetitive learning but this neurotransmitter is also important for other types of learning, including spatial and aversive memory processing. Dopamine is ideally positioned to regulate contextual fear given that the areas involved receive dopamine input and express dopamine receptors. Moreover, neuronal activity, functional connectivity and synaptic plasticity in this neural circuitry are modulated by dopamine receptor signalling. Here, we review the evidence indicating that dopamine regulates various contextual fear processes, along with the more recent studies that have begun to elucidate the brain areas and neurophysiological mechanisms involved. From a fundamental research perspective, understanding how dopamine regulates contextual fear will lead to novel insights on the neurochemical modulation of neural circuit function underlying memory processing. This research may also have translational relevance given that contextual fear conditioning and extinction also provide useful preclinical models of certain aspects of anxiety-related disorders and their treatment.

Rationale Dopamine D1 receptor (D1R) signalling is involved in contextual fear conditioning. The D1R antagonist SCH23390 impairs the acquisition of contextual fear when administered systemically or infused locally into the dorsal hippocampus or basolateral amygdala. Objectives We determined if state dependency may account for the impairment in contextual fear conditioning caused by systemic SCH23390 administration. We also examined if the dorsomedial prefrontal cortex (dmPFC), nucleus accumbens (NAc), and ventral hippocampus (VH) are involved in mediating the effect of systemic SCH23390 treatment on contextual fear conditioning. Methods In experiment 1, SCH23390 (0.1 mg/kg) or vehicle was given before contextual fear conditioning and/or retrieval. In experiment 2, SCH23390 (2.5 μg/0.5 uL) or vehicle was infused locally into dmPFC, NAc, or VH before contextual fear conditioning, and retrieval was tested drug-free. Freezing was quantified as a measure of contextual fear. Results In experiment 1, SCH23390 given before conditioning or before both conditioning and retrieval decreased freezing at retrieval, whereas SCH23390 given only before retrieval had no effect. In experiment 2, SCH23390 infused into dmPFC before conditioning decreased freezing at retrieval, while infusion of SCH23390 into NAc or VH had no effect. Conclusions The results of experiment 1 confirm those of previous studies indicating that D1Rs are required for the acquisition but not retrieval of contextual fear and rule out state dependency as an explanation for these findings. Moreover, the results of experiment 2 provide evidence that dmPFC is also part of the neural circuitry through which D1R signalling regulates contextual fear conditioning.

Psychiatric diseases like anxiety-related disorders and schizophrenia are characterized by impaired cognition and emotional regulation linked to corticolimbic disinhibition. Restoring the balance between excitation and inhibition in corticolimbic circuits may therefore ameliorate certain features of these disorders, such as inappropriately attributing affective salience to innocuous cues. Corticolimbic activity is tightly controlled by parvalbumin-expressing GABAergic interneurons, which also regulate fear discrimination. The voltage-gated potassium channels Kv3.1 and Kv3.2 are highly expressed in these neurons, therefore Kv3.1/3.2 modulation may have potential for treating disorders associated with cognitive and emotional dysregulation. We determined the effects of the novel Kv3.1/3.2 positive modulator AUT00206 on fear discrimination. Female rats underwent limited or extended auditory fear discrimination training that we previously showed leads to discrimination or generalization, respectively, based on passive fear responding (i.e. freezing). We also assessed darting as an active fear response. We found that limited training resulted in discrimination based on freezing, which was unaffected by AUT00206. In contrast, we found that extended training resulted in generalization based on freezing and the emergence of discrimination based on darting. Importantly, AUT00206 had dissociable effects on fear discrimination and expression with extended training. While AUT00206 mitigated generalization without affecting expression based on freezing, it reduced expression without affecting discrimination based on darting. Our results indicate that Kv3.1/3.2 modulation regulates the attribution of affective significance to threat- and safety-related cues in a response-specific manner. This suggests that targeting Kv3.1 and Kv3.2 channels may provide a promising avenue for treating cognitive and emotional dysregulation in psychiatric disease.

Hippocampal neural disinhibition, i.e. reduced GABAergic inhibition, is a key feature of schizophrenia pathophysiology. The hippocampus is an important part of the neural circuitry that controls fear conditioning and can also modulate prefrontal and striatal mechanisms, including dopamine signalling, which play a role in salience modulation. Therefore, hippocampal neural disinhibition may contribute to impairments in fear conditioning and salience modulation reported in schizophrenia. To test this hypothesis, we examined the effect of ventral hippocampus (VH) disinhibition in male rats on fear conditioning and salience modulation, as reflected by latent inhibition (LI), in a conditioned emotional response procedure (CER). A flashing light was used as the conditioned stimulus (CS) and conditioned suppression was used to index conditioned fear. In Experiment 1, VH disinhibition via infusion of the GABA-A receptor antagonist picrotoxin prior to CS pre-exposure and conditioning markedly reduced fear conditioning to both the CS and context; LI was evident in saline-infused controls, but could not be detected in picrotoxin-infused rats due to the low level of fear conditioning to the CS. In Experiment 2, VH picrotoxin infusions prior to CS pre-exposure only did not affect the acquisition of fear conditioning or LI. Together, these findings indicate that VH neural disinhibition disrupts contextual and elemental fear conditioning, without affecting the acquisition of LI. The disruption of fear conditioning resembles aversive conditioning deficits reported in schizophrenia and may reflect disruption of neural processing within the hippocampus and its projection sites.Significance StatementHippocampal disinhibition, reduced GABAergic inhibition, is a feature of schizophrenia, but how this contributes to psychological deficits remains to be clarified. Here, we focused on impairments patients show on classical-conditioning assays: aberrant salience allocation to stimuli that healthy participants have learnt to ignore and reduced fear conditioning, which have been linked to psychosis and negative symptoms, respectively. These impairments may be related to hippocampal disinhibition because the hippocampus modulates neural substrates of salience allocation and is part of the fear-conditioning neural circuit. Combining selective pharmacological manipulation of the hippocampus with a conditioning assay in rats, we found hippocampal disinhibition disrupted fear conditioning, without evidence for aberrant salience allocation. This suggests hippocampal disinhibition contributes to fear conditioning deficits in schizophrenia.

Coronavirus disease-19 (COVID-19)-related anxiety and post-traumatic stress symptoms (PTSS) or post-traumatic stress disorder (PTSD) are likely to be a significant long-term issue emerging from the current pandemic. We hypothesize that cannabidiol (CBD), a chemical isolated from Cannabis sativa with reported anxiolytic properties, could be a therapeutic option for the treatment of COVID-19-related anxiety disorders. In the global over-the-counter CBD market, anxiety, stress, depression, and sleep disorders are consistently the top reasons people use CBD. In small randomized controlled clinical trials, CBD (300-800 mg) reduces anxiety in healthy volunteers, patients with social anxiety disorder, those at clinical high risk of psychosis, in patients with Parkinson's disease, and in individuals with heroin use disorder. Observational studies and case reports support these findings, extending to patients with anxiety and sleep disorders, Crohn's disease, depression, and in PTSD. Larger ongoing trials in this area continue to add to this evidence base with relevant patient cohorts, sample sizes, and clinical end-points. Pre-clinical studies reveal the molecular targets of CBD in these indications as the cannabinoid receptor type 1 and cannabinoid receptor type 2 (mainly in fear memory processing), serotonin 1A receptor (mainly in anxiolysis) and peroxisome proliferator-activated receptor gamma (mainly in the underpinning anti-inflammatory/antioxidant effects). Observational and pre-clinical data also support CBD's therapeutic value in improving sleep (increased sleep duration/quality and reduction in nightmares) and depression, which are often comorbid with anxiety. Together these features of CBD make it an attractive novel therapeutic option in COVID-related PTSS that merits investigation and testing through appropriately designed randomized controlled trials.

COVID-19-related anxiety and post-traumatic stress symptoms (PTSS) or disorder (PTSD) are likely to be a significant long-term issue emerging from the current pandemic. We hypothesise that cannabidiol (CBD), a chemical isolated from Cannabis Sativa with reported anxiolytic properties, could be a therapeutic option for the treatment of COVID-19-related anxiety disorders. In the global over-the-counter CBD market, anxiety, stress, depression and sleep disorders are consistently the top reasons people use CBD. In small randomised, controlled clinical trials, CBD reduces anxiety in healthy volunteers, patients with social anxiety disorder, those at clinical high risk of psychosis, in patients with Parkinson’s disease, and in individuals with heroin use disorder. Case reports and series support these findings, extending to patients with anxiety and sleep disorders, Crohn’s disease, depression and in PTSD. Preclinical studies reveal the molecular targets of CBD in these indications as the cannabinoid receptors type 1 and 2 (CB1 and CB2) receptors (mainly in fear memory processing), serotonin 5HT1a receptors (mainly in anxiolysis) and peroxisome proliferator-activated receptor gamma (PPARγ) (mainly in the underpinning anti-inflammatory/anti-oxidant effects). Observational and preclinical data also support CBD’s therapeutic value in improving sleep (increased sleep duration/quality and reduction in nightmares) and depression, often comorbid with anxiety. Together these features of CBD to reduce anxiety and depression, and improve sleep disturbances, could be an attractive novel therapeutic option in relieving COVID-related post-traumatic stress symptoms.

Objective. Graphical networks and network metrics are widely used to understand and characterise brain networks and brain function. These methods can be applied to a range of electrophysiological data including electroencephalography, local field potential and single unit recordings. Functional networks are often constructed using pair-wise correlation between variables. The objective of this study is to demonstrate that functional networks can be more accurately estimated using partial correlation than with pair-wise correlation. Approach. We compared network metrics derived from unconditional and conditional graphical networks, obtained using coherence and multivariate partial coherence (MVPC), respectively. Graphical networks were constructed using coherence and MVPC estimates, and binary and weighted network metrics derived from these: node degree, path length, clustering coefficients and small-world index. Main results. Network metrics were applied to simulated and experimental single unit spike train data. Simulated data used a 10x10 grid of simulated cortical neurons with centre-surround connectivity. Conditional network metrics gave a more accurate representation of the known connectivity: Numbers of excitatory connections had range 3–11, unconditional binary node degree had range 6–80, conditional node degree had range 2–13. Experimental data used multi-electrode array recording with 19 single-units from left and right hippocampal brain areas in a rat model for epilepsy. Conditional network analysis showed similar trends to simulated data, with lower binary node degree and longer binary path lengths compared to unconditional networks. Significance. We conclude that conditional networks, where common dependencies are removed through partial coherence analysis, give a more accurate representation of the interactions in a graphical network model. These results have important implications for graphical network analyses of brain networks and suggest that functional networks should be derived using partial correlation, based on MVPC estimates, as opposed to the common approach of pair-wise correlation.

The increased prevalence of post-traumatic stress disorder (PTSD) that is observed in women may involve sex differences in learned fear inhibition and medial prefrontal cortex (mPFC) function. PTSD is characterized by fear overgeneralization involving impaired fear regulation by safety signals. We recently found that males show fear discrimination and females show fear generalization involving reduced safety signalling after extended fear discrimination training. Here we determined if these sex differences involve altered mPFC function. Male and female rats underwent three days of auditory fear discrimination training, where one tone (CS+) was paired with footshock and another tone (CS−) was presented alone. Local field potentials were recorded from prelimbic (PL) and infralimbic (IL) mPFC during retrieval. We found that males discriminated and females generalized based on cue-induced freezing at retrieval. This was accompanied by sex differences in basal theta and gamma oscillations in PL and IL. Importantly, males also showed PL/IL theta activation during safety signalling by the CS− and IL gamma activation in response to the threat-related CS+, both of which were absent in females. These results add to growing evidence indicating that sex differences in learned fear inhibition are associated with altered mPFC function.

Purpose of Review Anxiety- and trauma-related disorders are prevalent and debilitating mental illnesses associated with a significant socioeconomic burden. Current treatment approaches often have inadequate therapeutic responses, leading to symptom relapse. Here we review recent preclinical and clinical findings on the potential of cannabinoids as novel therapeutics for regulating fear and anxiety. Recent Findings Evidence from preclinical studies has shown that the non-psychotropic phytocannabinoid cannabidiol and the endocannabinoid anandamide have acute anxiolytic effects and also regulate learned fear by dampening its expression, enhancing its extinction and disrupting its reconsolidation. The findings from the relevant clinical literature are still very preliminary but are nonetheless encouraging. Summary Based on this preclinical evidence, larger-scale placebo-controlled clinical studies are warranted to investigate the effects of cannabidiol in particular as an adjunct to psychological therapy or medication to determine its potential utility for treating anxiety-related disorders in the future.

BACKGROUND: The ability to infer network structure from multivariate neuronal signals is central to computational neuroscience. Directed network analyses typically use parametric approaches based on auto-regressive (AR) models, where networks are constructed from estimates of AR model parameters. However, the validity of using low order AR models for neurophysiological signals has been questioned. A recent article introduced a non-parametric approach to estimate directionality in bivariate data, non-parametric approaches are free from concerns over model validity. NEW METHOD: We extend the non-parametric framework to include measures of directed conditional independence, using scalar measures that decompose the overall partial correlation coefficient summatively by direction, and a set of functions that decompose the partial coherence summatively by direction. A time domain partial correlation function allows both time and frequency views of the data to be constructed. The conditional independence estimates are conditioned on a single predictor. RESULTS: The framework is applied to simulated cortical neuron networks and mixtures of Gaussian time series data with known interactions. It is applied to experimental data consisting of local field potential recordings from bilateral hippocampus in anaesthetised rats. COMPARISON WITH EXISTING METHOD(S): The framework offers a non-parametric approach to estimation of directed interactions in multivariate neuronal recordings, and increased flexibility in dealing with both spike train and time series data. CONCLUSIONS: The framework offers a novel alternative non-parametric approach to estimate directed interactions in multivariate neuronal recordings, and is applicable to spike train and time series data.

Purpose of project The purpose of this article is to demonstrate the effectiveness of the Cumulative Complexity Model as a framework to build an Excel tool and a Pareto tool that will enable inpatient case managers to predict the increased risk for and prevent repeat falls. The Excel tool is based on work explained in a previous article by and uses a macro to analyze the factors causing the repeat falls and then calculate the probability of it happening again. This enables the case manager to identify trends in how the patient is transitioning toward goals of care and identify problems before they become barriers to the smooth transition to other levels of care. Thus, the case manager will save the facility money by avoiding unneeded days of care and avoiding the costs that result from rendering medical care for the patient who has fallen. Primary practice settings In July 2015, a group of nurses at a small Veterans Health Administration Hospital in the Northwest collaborated to find ways to reverse a trend of increasing falls and repeat falls. Methodology and sample A retrospective chart review of all falls and repeat falls (N = 73) that happened between January 2013 and July 2015 was used to generate a list of top 11 contributing variables that enabled evaluation of the data. A bundle of 3 interventions was instituted in October 2015: (1) development of a dedicated charge nurse/resource nurse, (2) use of a standardized method of rounding, and (3) use of a noncontact patient monitoring system ("virtual nurses"). Falls pre- and postimplementation (N = 109) were analyzed using linear and logistic regression analyses. Data were entered into an Excel sheet and analyzed to identify the major contributing factors to falls and repeat falls and to identify trends. These data were also evaluated to find out whether length of stay and nurse workload contributed to falls. Results Fifteen months after implementation of the aforementioned interventions, falls on the unit went down from 30 aggregate falls in 2015 to 17 aggregate falls in 2016. Repeat falls in 2015 went from 9 repeat falls after admission to the unit down to 2 repeat falls in 2016. Each additional extrinsic variable that was present added an additional 1.43 to the odds ratio (OR) for a fall. Similarly, each additional intrinsic variable present added 2.08 to the OR for a fall. The linear regression of length of stay and falls demonstrated that 17.5% of falls correlated with length of stay, F(1,36) = 7.63, p = .009, R = .175, adjusted R = .152. Workload correlated with work 17% of the time, as measured by using ward days of care, F(1,100) = 20.84, p = .00001, R = .17, adjusted R = .16. Implications for case managers Two examples of the how to use these tools are located in the "Discussion" section of the article.

RATIONALE: Dopamine D1-like receptor signalling is involved in contextual fear conditioning, but the brain regions involved and its role in other contextual fear memory processes remain unclear. OBJECTIVES: The objective of this study was to investigate (1) the effects of SCH 23390, a dopamine D1/D5 receptor antagonist, on contextual fear memory encoding, retrieval and reconsolidation, and (2) if the effects of SCH 23390 on conditioning involve the dorsal hippocampus (DH) and/or basolateral amygdala (BLA). METHODS: Rats were used to examine the effects of systemically administering SCH 23390 on the acquisition, consolidation, retrieval and reconsolidation of contextual fear memory, and on locomotor activity and shock sensitivity. We also determined the effects of MK-801, an NMDA receptor antagonist, on contextual fear memory reconsolidation. The effects of infusing SCH 23390 locally into DH or BLA on contextual fear conditioning and locomotor activity were also examined. RESULTS: Systemic administration of SCH 23390 impaired contextual fear conditioning but had no effects on fear memory consolidation, retrieval or reconsolidation. MK-801 was found to impair reconsolidation, suggesting that the behavioural parameters used allowed for the pharmacological disruption of memory reconsolidation. The effects of SCH 23390 on conditioning were unlikely the result of any lasting drug effects on locomotor activity at memory test or any acute drug effects on shock sensitivity during conditioning. SCH 23390 infused into either DH or BLA impaired contextual fear conditioning and decreased locomotor activity. CONCLUSIONS: These findings suggest that dopamine D1-like receptor signalling in DH and BLA contributes to the acquisition of contextual fear memory.

Objective: A major challenge in non-stationary signal analysis is reliable estimation of correlation. Neurophysiological recordings can be many minutes in duration with data that exhibits correlation which changes over different time scales. Local smoothing can be used to estimate time-dependency, however, an effective framework needs to adjust levels of smoothing in response to changes in correlation. Approach: Here we present a novel data-adaptive algorithm, the z-tracker, for estimating local correlation in segmented data. The algorithm constructs single segment coherence estimates using multi-taper windows. These are subject to adaptive Kalman filtering/smoothing in the z-domain to construct a local coherence estimate for each segment. The error residual for each segment determines the levels of process noise, allowing the filter to adapt rapidly to sudden changes in correlation while applying greater smoothing to data where the correlation is consistent across segments. The method is compared to wavelet coherence, calculated using orthogonal Morse wavelets. Main results: The performance of the z-tracker is quantified against Morse wavelet coherence using a mean square deviation (MSD) metric. The z-tracker has significantly lower MSD than the wavelet estimate for time-varying coherence over long time scales (∼10–20 s), whereas the wavelet has lower MSD for coherence varying over short time scales (∼1–2 s). The z-tracker also has a lower MSD for slowly varying coherence with occasional step changes. The method is applied to detect changes in coherence in paired LFP recordings from rat prefrontal cortex and amygdala in response to a pharmacological challenge. Significance: The z-tracker provides an effective and efficient method to estimate time varying correlation in multivariate data, leading to better characterisation of neurophysiology signals where correlation is subject to slow modulation over time. A number of suggestions are included for future refinements.

Purpose of study The medical record is a sea of information that can reveal what patients are trying to tell us about their health condition. It can reveal hints and trends as to why veterans with congestive heart failure (CHF) are being readmitted within 30 days after hospital discharge. These hints and trends lead caregivers to key contributing variables to veterans' readmission. Furthermore, these variables can be used to predict patient outcomes such as readmission and even prognosis. This article looks at readmissions for CHF from documentation within the medical record to see what was driving the 30-day readmissions. Second, it examines whether the driving forces can be used to predict a veteran's increased risk for readmission or other poor prognosis. Primary practice setting(s) The study was conducted at a rural 84-bed Veterans Health Administration hospital in the Western United States. Methodology and sample A retrospective screen was performed on 1,279 veterans' admissions of which 217 were identified as having CHF as a primary or secondary diagnosis on admission. The descriptive statistics, odds ratio (OR) and multivariate logistic regression were used to examine the data. The multivariate logistic regression equation was p = 1/1 + e, which can be found in the biostatistics textbook by . developed and validated the equation and used it to screen for undiagnosed diabetic patients. The equation was refined by . The variables selected for this study were based on a literature review of 30 articles. Results The probability and OR for 30-day readmissions for all ages increased as the age increased. The ORs for 30-day readmissions for the variables selected were as follows: brain natriuretic peptide 6.21 (95% CI [0.36, 108.24]), ejection fraction 1.298 (95% CI [0.68, 2.49]), hypertension 1.795 (95% CI [0.83, 3.85]), comorbid conditions 1.02 (95% CI [0.04, 25.02]), Stage III and below were protective, Stage IV 2.057 (95% CI [0.63, 9.32]), lack of discharge education 0.446 (95% CI [0.19, 6.45]). The impact of these variables on veterans with more than 3 readmissions (N = 66) was examined. In 32% of these admissions, there was insufficient data to compare the values of the variables between readmissions. In almost 26% (N = 17) of the cases as the number of variables increased, the time between admissions decreased. In 23% of the cases (N = 15), the values did not change; of these, 14 died and the one who survived had assistance with his care in the form of home health and telehealth. Implications for case management practice Use of this evidence-based tool will help case managers to strategically plan care and prioritize interventions to impact the major variables and risk factors that are impacting veterans' health.

Anxiety and trauma-related disorders are psychiatric diseases with a lifetime prevalence of up to 25%. Phobias and post-traumatic stress disorder (PTSD) are characterized by abnormal and persistent memories of fear-related contexts and cues. The effects of psychological treatments such as exposure therapy are often only temporary and medications can be ineffective and have adverse side effects. Growing evidence from human and animal studies indicates that cannabidiol, the main non-psychotomimetic phytocannabinoid present in Cannabis sativa, alleviates anxiety in paradigms assessing innate fear. More recently, the effects of cannabidiol on learned fear have been investigated in preclinical studies with translational relevance for phobias and PTSD. Here we review the findings from these studies, with an emphasis on cannabidiol regulation of contextual fear. The evidence indicates that cannabidiol reduces learned fear in different ways: (1) cannabidiol decreases fear expression acutely, (2) cannabidiol disrupts memory reconsolidation, leading to sustained fear attenuation upon memory retrieval, and (3) cannabidiol enhances extinction, the psychological process by which exposure therapy inhibits learned fear. We also present novel data on cannabidiol regulation of learned fear related to explicit cues, which indicates that auditory fear expression is also reduced acutely by cannabidiol. We conclude by outlining future directions for research to elucidate the neural circuit, psychological, cellular, and molecular mechanisms underlying the regulation of fear memory processing by cannabidiol. This line of investigation may lead to the development of cannabidiol as a novel therapeutic approach for treating anxiety and trauma-related disorders such as phobias and PTSD in the future.

Highlights • We investigated sex differences in auditory fear discrimination in rats. • Males that received extended discrimination training showed fear discrimination. • Females discriminated after limited training and generalized after extended training. • Generalization with extended training in females involved impaired safety signaling., Post-traumatic stress disorder (PTSD) is more prevalent in women than men. PTSD is characterized by overgeneralization of fear to innocuous stimuli and involves impaired inhibition of learned fear by cues that predict safety. While evidence indicates that learned fear inhibition through extinction differs in males and females, less is known about sex differences in fear discrimination and safety learning. Here we examined auditory fear discrimination in male and female rats. In Experiment 1A, rats underwent 1–3 days of discrimination training consisting of one tone predicting threat (CS+; presented with footshock) and another tone predicting safety (CS−; presented alone). Females, but not males, discriminated between the CS+ and CS− after one day of training. After 2–3 days of training, however, males discriminated whereas females generalized between the CS+ and CS−. In Experiment 1B, females showed enhanced anxiety-like behaviour and locomotor activity in the open field, although these results were unlikely to explain the sex differences in fear discrimination. In Experiment 2, we found no differences in shock sensitivity between males and females. In Experiment 3, males and females again discriminated and generalized, respectively, after three days of training. Moreover, fear generalization in females resulted from impaired safety learning, as shown by a retardation test. Whereas subsequent fear conditioning to the previous CS− retarded learning in males, females showed no such retardation. These results suggest that, while females show fear discrimination with limited training, they show fear generalization with extended training due to impaired safety learning.

Sex differences in learned fear expression and extinction involve the medial prefrontal cortex (mPFC). We recently demonstrated that enhanced learned fear expression during auditory fear extinction and its recall is linked to persistent theta activation in the prelimbic (PL) but not infralimbic (IL) cortex of female rats. Emerging evidence indicates that gamma oscillations in mPFC are also implicated in the expression and extinction of learned fear. Therefore we re-examined our in vivo electrophysiology data and found that females showed persistent PL gamma activation during extinction and a failure of IL gamma activation during extinction recall. Altered prefrontal gamma oscillations thus accompany sex differences in learned fear expression and its extinction. These findings are relevant for understanding the neural basis of post-traumatic stress disorder, which is more prevalent in women and involves impaired extinction and mPFC dysfunction.

The basolateral amygdala (BLA) and medial prefrontal cortex (mPFC) are inter-connected regions involved in fear memory expression. The reciprocal nature of projections between these areas differs along the rostrocaudal extent of BLA. This study investigated the role of functional interactions between BLA and the prelimbic (PL) subregion of mPFC in mediating contextual fear memory. Freezing served as the measure of conditioned fear. Experiments 1–3 examined the effects of left, right or bilateral infusion of bupivacaine into anterior BLA (aBLA), posterior BLA (pBLA) or PL on fear memory expression. Reversible inactivation of left, right or bilateral aBLA impaired fear memory expression. Bilateral inactivation of pBLA or PL also disrupted the expression of fear memory, although left or right inactivation alone had no significant effects in either region. Experiment 4 examined the effects of functionally disconnecting pBLA and PL on contextual fear memory by infusing bupivacaine unilaterally into pBLA and PL in the ipsilateral or contralateral hemisphere. Fear memory expression was impaired by asymmetric inactivation of pBLA and PL; however, a similar effect was also observed with symmetric inactivation of these regions. Bupivacaine infusion did not affect behavior in the open field, likely ruling out non-specific effects of inactivation on innate fear and locomotor activity. These results demonstrate different roles for rostral and caudal BLA in mediating the expression of contextual fear memory. They also raise the possibility that pBLA–PL circuitry is involved in subserving fear memory expression via complex processing mechanisms, although further research is needed to confirm this preliminary finding.

The early rearing environment can impact on emotional reactivity and learning later in life. In this study the effects of neonatal maternal separation (MS) on innate fear and fear learning were assessed in the adult female rat. Pups were subjected to MS (360 min), brief handling (H; 15 min), or animal facility rearing (AFR) on post-natal days 2-14. In the first experiment, innate fear was tested in the open field. No differences between the early rearing groups were observed in unconditioned fear. In the second experiment, separate cohorts were used in a 3-day fear learning paradigm which tested the acquisition (Day 1), expression and extinction (both Day 2) of conditioning to an auditory cue; extinction recall was determined as well (Day 3). Contextual fear conditioning was also assessed prior to cue presentations on Days 2 and 3. Whereas MS attenuated the acquisition and expression of fear conditioning to the cue, H potentiated extinction learning. Cue-induced fear was reduced on Day 3, compared to Day 2, indicating that the recall of extinction learning was evident; however, no early rearing group differences in extinction recall were observed. Similarly, while contextual fear was decreased on Day 3, compared to Day 2, there were no differences between the early rearing groups on either day tested. The present findings of altered cue-conditioned fear learning, in the absence of innate fear changes, lend further support for the important role of the early rearing environment in mediating cognition in adulthood.

Neonatal maternal separation (MS) in the rat increases the vulnerability to stressors later in life. In contrast, brief handling (H) in early life confers resilience to stressors in adulthood. Early life programming of stress reactivity may involve the medial prefrontal cortex (mPFC), a region which modulates various stress responses. Moreover, hemispheric specialization in mPFC may mediate adaptive coping responses to stress. In the present study, neuronal activity was examined simultaneously in left and right mPFC in adult rats previously subjected to MS, H or animal facility rearing (AFR). In vivo electrophysiology, under isoflurane anesthesia, was used to conduct acute recordings of unit and local field potential (LFP) activity in response to systemic administration of N-methyl-beta-carboline-3-carboxamide (FG-7142), a benzodiazepine receptor partial inverse agonist which mimics various stress responses. MS decreased basal unit activity selectively in right mPFC. Basal LFP activity was reduced with MS in left and right mPFC, compared to AFR and H, respectively. Hemispheric synchronization of basal LFP activity was also attenuated by MS at lower frequencies. FG-7142 elicited lateralized effects on mPFC activity with different early rearing conditions. Activity in left mPFC was greater with AFR and MS (AFR>MS), whereas activity was predominantly greater with H in right mPFC. Finally, compared to AFR, MS reduced and H enhanced hemispheric synchronization of LFP activity with FG-7142 treatment in a dose-dependent manner. These results indicate that functionally-relevant alterations in mPFC GABA transmission are programmed by the early rearing environment in a hemisphere-dependent manner. These findings may model the hemispheric specialization of mPFC function thought to mediate adaptive coping responses to stressors. They also suggest the possibility that early environmental programming of hemispheric functional coupling in mPFC is involved in conferring vulnerability or resilience to stressors later in life.

Maternal separation (MS) during the neonatal period enhances stress responsivity in adulthood. The medial prefrontal cortex (mPFC) and the basolateral amygdala (BLA) are involved in coordinating various stress responses. Evidence indicates that MS reduces benzodiazepine and GABAA receptor expression in these regions, although their effects on neuronal function in the mPFC and the BLA remain unknown. The present study was conducted to assess the effects of MS on neuronal activity in the mPFC and BLA in response to the benzodiazepine receptor partial inverse agonist N-methyl-β-carboline-3-carboxamide (FG-7142). Rat pups were subjected to MS (360 min), brief handling (H; 15 min) or standard animal facility rearing (AFR) on postnatal days 2–14. In adult males, in vivo electrophysiology under isoflurane anesthesia was used to conduct acute recordings of extracellular unit activity in response to systemic FG-7142 administration. Animals subjected to H showed significantly increased basal mPFC activity compared to MS and AFR animals. There were no differences in basal BLA activity between the early rearing groups. In response to FG-7142, MS animals showed significantly attenuated mPFC activity compared to H animals and a nonsignificant trend towards attenuated mPFC activity compared to AFR animals. In contrast to mPFC, MS animals showed significantly potentiated FG-7142-induced activity in the BLA, compared to both H and AFR animals. These findings indicate that MS induces functionally relevant alterations in corticolimbic GABAA receptor signaling. Given that FG-7142 mimics several behavioral and physiological effects of stress, these results may also model stress-induced corticolimbic dysfunction caused by early life stress.

The medial prefrontal cortex (mPFC) and basolateral amygdala (BLA) coordinate various stress responses. Although the effects of stressors on mPFC and BLA activity have been previously examined, it remains unclear to what extent stressors affect functional interactions between these regions. In vivo electrophysiology in the anesthetized rat was used to examine mPFC and BLA activity simultaneously in response to FG-7142, a benzodiazepine receptor partial inverse agonist that mimics various stress responses, in an attempt to model the effects of stressors on corticolim- bic functional connectivity. Extracellular unit and local field potential (LFP) record- ings, using multielectrode arrays positioned in mPFC and BLA, were conducted under basal conditions and in response to systemic FG-7142 administration. This drug increased mPFC and BLA unit firing at the lowest dose tested, whereas higher doses of FG-7142 decreased various burst firing parameters in both regions. Moreover, LFP power was attenuated at lower (

Excitotoxic neonatal ventral hippocampus (NVH) lesions in rats result in characteristic post-pubertal hyper-responsiveness to stress and cognitive abnormalities analogous to those described in schizophrenia and suggestive of alterations in dopamine (DA) neurotransmission. Converging lines of evidence also point to dysfunctions in the cortical cholinergic system in neuropsychiatric disorders. In previous studies, we observed alterations in dopaminergic modulation of acetylcholine (Ach) release in the prefrontal cortex (PFC) in post-pubertal NVH-lesioned rats. These two neurotransmitter systems are involved in the stress response as PFC release of DA and Ach is enhanced in response to some stressful stimuli. As adult NVH-lesioned rats are behaviorally more reactive to stress, we investigated the effects of NVH lesions on tail-pinch stress-induced Ach and DA release in the PFC. Using in vivo microdialysis, we observed that tail-pinch stress resulted in significantly greater increases in prefrontal cortical Ach release in post-pubertal NVH-lesioned rats (220% baseline) compared with sham-operated controls (135% baseline). Systemic administration of the D1-like receptor antagonist SCH 23390 (0.5 mg/kg i.p.) or the D2-like receptor antagonist haloperidol (0.2 mg/kg i.p.), as well as intra-PFC administration of the D2-like antagonist sulpiride (100 µm), reduced stress-induced Ach release in PFC of adult NVH-lesioned rats. By contrast, intra-PFC administration of SCH 23390 (100 µm) failed to affect stress-induced Ach release in PFC of NVH-lesioned rats. Interestingly, using in vivo voltammetry, stress-induced stimulation of PFC DA release was found to be attenuated in adult NVH-lesioned rats. Taken together, these data suggest developmentally specific reorganization of prefrontal cortical cholinergic innervation notably regarding its regulation by DA neurotransmission.

Although the basolateral amygdala (BLA) plays a role in the habituation to sensory stimuli, the receptor mechanisms mediating this process remain unclear. In the present study, we investigated the role of BLA dopamine (DA) in the habituation of the acoustic startle response (ASR) with intra-BLA infusions of DA receptor antagonists. Male Long Evans rats were subjected to startle pulses over two consecutive once-daily sessions. Prior to testing on Day 1, separate groups of animals received bilateral intra-BLA infusions of a D1 (SCH 23390: 0, 3.2, 6.4 μg per side) or a D2/D3 (raclopride: 0, 2.5, 5.0 μg per side) receptor antagonist. Animals were retested 24 h later (Day 2) without prior drug infusion in order to assess possible treatment effects on within- and between-session habituation of the ASR. As expected, within- and between-session habituation was observed in vehicle-treated controls. Within-session habituation was also seen in SCH 23390- and raclopride-treated animals both on Day 1 as well as 24 h later (Day 2). Evidence of between-session habituation was observed in SCH 23390-treated animals. However, compared to vehicle, intra-BLA SCH 23390 or raclopride attenuated the initial startle response on Day 1, but not Day 2. No evidence of between-session habituation was found in raclopride-treated animals, although this probably reflected the attenuated initial response to the startling stimulus on Day 1 rather than a reduced rate of habituation on Day 2. The present study suggests that while BLA DA is not involved in habituation of the ASR, it may mediate the perceived aversive nature of the initially startling stimuli.

The basolateral amygdala (BLA) is involved in modulating affective responses to stress and, along with the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC), receives a stress-responsive dopamine (DA) projection from the ventral tegmental area. The present study was undertaken to characterize the role of BLA DA D1 and D2/D3 receptor subtypes in modulating the NAc and mPFC DA responses to stress. Voltammetry was used to monitor, in freely behaving rats, stress-induced DA release in NAc or mPFC after injection of D1 (SCH 23390) or D2/D3 (raclopride) receptor antagonist into BLA. Intra-BLA SCH 23390 injection potentiated stress-induced NAc DA release but attenuated the mPFC DA stress response; raclopride had no effect on either the NAc or mPFC DA responses to stress. Based on these results, we also examined the possibility that BLA can indirectly modulate the NAc DA stress response via its projection to mPFC. To do so we studied the effects of intra-mPFC co-administration of D1 (SKF 38393) and D2/D3 (quinpirole) receptor agonists on the potentiated NAc DA stress response resulting from intra-BLA SCH 23390 injection. Alone, mPFC D1 and D2/D3 receptor co-activation had no effect on stress-induced NAc DA release, but did prevent the potentiated NAc DA stress response produced by BLA D1 receptor blockade. These findings indicate that BLA DA modulates the NAc and mPFC DA stress responses via activation of the D1 receptor subtype. They also suggest that BLA DA modulates stress-induced NAc DA release indirectly by modulating the mPFC DA response to stress.

Associative learning is encoded under anesthesia and involves the medial prefrontal cortex (mPFC). Neuronal activity in mPFC increases in response to a conditioned stimulus (CS+) previously paired with an unconditioned stimulus (US) but not during presentation of an unpaired stimulus (CS−) in anesthetized animals. Studies in conscious animals have shown dissociable roles for different mPFC subregions in mediating various memory processes, with the prelimbic (PL) and infralimbic (IL) cortex involved in the retrieval and extinction of conditioned responding, respectively. Therefore PL and IL may also play different roles in mediating the retrieval and extinction of discrimination learning under anesthesia. Here we used in vivo electrophysiology to examine unit and local field potential (LFP) activity in PL and IL before and after auditory discrimination learning and during later retrieval and extinction testing in anesthetized rats. Animals received repeated presentations of two distinct sounds, one of which was paired with footshock (US). In separate control experiments animals received footshocks without sounds. After discrimination learning the paired (CS+) and unpaired (CS−) sounds were repeatedly presented alone. We found increased unit firing and LFP power in PL and, to a lesser extent, IL after discrimination learning but not after footshocks alone. After discrimination learning, unit firing and LFP power increased in PL and IL in response to presentation of the first CS+, compared to the first CS−. However, PL and IL activity increased during the last CS− presentation, such that activity during presentation of the last CS+ and CS− did not differ. These results confirm previous findings and extend them by showing that increased PL and IL activity result from encoding of the CS+/US association rather than US presentation. They also suggest that extinction may occur under anesthesia and might be represented at the neural level in PL and IL.

Anxiety disorders, such as post-traumatic stress, are more prevalent in women and are characterized by impaired inhibition of learned fear and medial prefrontal cortex (mPFC) dysfunction. Here we examined sex differences in fear extinction and mPFC activity in rats. Females showed more learned fear expression during extinction and its recall, but not fear conditioning. They also showed more spontaneous fear recovery and more contextual fear before extinction and its recall. Moreover, enhanced learned fear expression in females was associated with sustained prelimbic (PL) cortex activity. These results suggest that sex differences in learned fear expression may involve persistent PL activation.

Associative learning can occur under anesthesia and its neural correlates have begun to be elucidated. During discrimination learning under anesthesia in rats, lateral amygdala excitability increases in response to a conditioned stimulus (CS+) previously paired with electrical stimulation of the paw but not to another stimulus presented alone (CS−). Similarly, medial prefrontal cortex activity increases selectively during CS+ presentation after discrimination learning but this occurs only in neurons receiving input from the basolateral amygdala (BLA), the main source of amygdaloid projections to this region. However, BLA activity during discrimination learning under anesthesia has not been investigated. Here we used in vivo electrophysiology to examine BLA activity before and after associative learning and during later memory retrieval in anesthetized rats. We examined extracellular unit and local field potential (LFP) activity using an auditory discrimination learning paradigm. Rats were repeatedly presented with two distinct sounds, one of which was paired with electrical stimulation of the paw. One hour later, the paired sound (CS+) was presented alone along with the sound not paired with electrical stimulation (CS−). We found increased unit firing late (1 h) but not early (5 min) after learning. LFP power was increased both early and late after learning. In control experiments we also found increased unit and LFP activity late after electrical stimulation alone. After discrimination learning, unit firing increased in response to CS+, but not CS−, presentation. LFP power also showed a modest increase during CS+, compared to CS−, presentation. These findings suggest that discrimination learning under anesthesia can occur at the neural level in BLA. The potential relevance of these results is discussed in relation to previous studies examining neural activity during fear learning and memory processing in conscious animals.

The early rearing environment programs corticolimbic function and neuroendocrine stress reactivity in adulthood. Although early environmental programming of innate fear has been previously examined, its impact on fear learning and memory later in life remains poorly understood. Here we examined the role of the early rearing environment in programming fear conditioning and extinction in adult male rats. Pups were subjected to maternal separation (MS; 360 min), brief handling (H; 15 min), or animal facility rearing (AFR) on post-natal days 2-14. As adults, animals were tested in a 3-day fear learning and memory paradigm which assessed the acquisition, expression and extinction of fear conditioning to an auditory cue; the recall of extinction was also assessed. In addition, contextual fear was assessed prior to cued extinction and its recall. We found that the acquisition of fear conditioning to the cue was modestly impaired by MS. However, no early rearing group differences were observed in cue-induced fear expression. In contrast, both the rate of extinction and extinction recall were attenuated by H. Finally, although contextual fear was reduced after extinction to the cue, no differences in context-induced fear were observed between the early rearing groups. These results add to a growing body of evidence supporting an important role for early environmental programming of fear conditioning and extinction. They also indicate that different early rearing conditions can program varying effects on distinct fear learning and memory processes in adulthood.

We examined the effects of brief or long durations of repeated maternal separation (MS) on ultrasonic vocalizations (USVs) in rat dams. 50-kHz USVs putatively identified as maternal in origin were emitted only immediately after pups were returned following MS. Maternal USV emission also depended on separation duration and pup age. Given that 50-kHz USVs are emitted in response to rewarding stimuli, MS may induce duration-dependent effects on maternal positive affect.

The dopamine (DA) projection to the basolateral amygdala (BLA) modulates nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) DA transmission. Given the involvement of the BLA, and of NAc and mPFC DA, in select forms of information processing, we sought to determine the role of BLA DA in modulating prepulse inhibition (PPI) and latent inhibition (LI).The effects of BLA D1 (SCH 23390) and D2/D3 (raclopride) receptor blockade on PPI and LI were examined.Separate groups of male Long-Evans rats received bilateral intra-BLA infusions of SCH 23390 (3.2 or 6.4 microg/0.5 microl per side), raclopride (2.5 or 5.0 microg/0.5 microl per side) or saline prior to testing. In two experiments, the effects of BLA DA receptor antagonism on PPI of the acoustic startle response (ASR) and LI of conditioned taste aversion were determined. A control group received bilateral intra-striatal infusions of SCH 23390 or raclopride prior to PPI testing.Intra-BLA SCH 23390 or raclopride had no effect on the ASR. Intra-BLA SCH 23390 enhanced and raclopride disrupted PPI, both in a dose-related manner. Intra-striatal SCH 23390 or raclopride had no effect on PPI or ASR magnitude. Finally, BLA DA receptor blockade had no effect on LI.These results indicate that PPI is modulated by BLA DA and suggest that this modulation occurs independently of changes in NAc and/or mPFC DA transmission. They also suggest that BLA DA is not involved in modulating LI and add to evidence indicating that PPI and LI are mediated by different neural substrates.

In vivo voltammetry was used to study the effects of basolateral amygdala dopamine depletion on stress-induced dopamine release in the nucleus accumbens and medial prefrontal cortex. Male Long–Evans rats received bilateral microinjections of 6-hydroxydopamine or vehicle into the basolateral amygdala. Changes in dopamine signal were monitored in the nucleus accumbens and in the right and left hemispheres of medial prefrontal cortex, in lesioned animals and shams. Animals were subjected to a physical stressor (tail pinch) and a species-typical threat (fox odour); each stressor was presented twice over four consecutive daily sessions. The results indicate that the nucleus accumbens dopamine responses to both stressors are significantly potentiated by dopamine-depleting lesions to basolateral amygdala. In contrast, while the dopamine stress response in the left medial prefrontal cortex did not differ between lesioned animals and shams, the right medial prefrontal cortical dopamine response to tail pinch, but not fox odour stress, was significantly attenuated in lesioned animals. Therefore, basolateral amygdala dopamine depletion had opposite effects on the nucleus accumbens and medial prefrontal cortical dopamine responses to stress, although the effect on the latter is lateralized to the right hemisphere in a stressor-specific manner. These data indicate that stress-induced activation of meso-amygdaloid dopamine exerts an inhibitory influence on the nucleus accumbens dopamine response to stress. They also suggest the possibility that meso-amygdaloid dopamine influences the nucleus accumbens dopamine response to stress indirectly by modulating stress-induced dopamine release in medial prefrontal cortex. These findings add to a growing body of evidence of a preferential involvement of right medial prefrontal cortical dopamine in a wide range of physiological responses to stress.

In this study, we measured the levels of elements in human brain microvascular endothelial cells (ECs) infected with T. gondii. ECs were infected with tachyzoites of the RH strain, and at 6, 24, and 48 hours post infection (hpi), the intracellular concentrations of elements were determined using a synchrotron-microfocus X-ray fluorescence microscopy (μ-XRF) system. This method enabled the quantification of the concentrations of Zn and Ca in infected and uninfected (control) ECs at sub-micron spatial resolution. T. gondii-hosting ECs contained less Zn than uninfected cells only at 48 hpi (p < 0.01). The level of Ca was not significantly different between infected and control cells (p > 0.05). Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis revealed infection-specific metallome profiles characterized by significant increases in the intracellular levels of Zn, Fe, Mn and Cu at 48 hpi (p < 0.01), and significant reductions in the extracellular concentrations of Co, Cu, Mo, V, and Ag at 24 hpi (p < 0.05) compared with control cells. Zn constituted the largest part (74%) of the total metal composition (metallome) of the parasite. Gene expression analysis showed infection-specific upregulation in the expression of five genes, MT1JP, MT1M, MT1E, MT1F, and MT1X, belonging to the metallothionein gene family. These results point to a possible correlation between T. gondii infection and increased expression of MT1 isoforms and altered intracellular levels of elements, especially Zn and Fe. Taken together, a combined μ-XRF and ICP-MS approach is promising for studies of the role of elements in mediating host-parasite interaction.