Anatomical and behavioral impact of a lentiviral tool tapping onto hippocampal serotonin reuptake in rats.

We aimed at the further characterization of rats in which SERT gene silencing was achieved by hippocampal injection of a lentiviral vector, carrying three si-RNA to block SERT mRNA at 66% of normal levels. Improved self-control and reduced restlessness were already demonstrated in these rats. Present further studies consisted of male adult rats, bilaterally inoculated within the hippocampus; control rats received lentivirus particles inactivated with heat. Both groups were maintained in isolation for 5 months, starting from inoculation. Neurochemical changes were studied by proton magnetic resonance spectroscopy (1H-MRS): we found increased hippocampal viability and bioenergetic potential; however, rats showed a behaviorally depressive pattern, also characterized by enhanced affiliation. Based on the extent of such effects, the whole lenti-SERT group was divided into two subgroups, termed intermediate- and extreme- phenotype profiles. While all rats had a widespread modification within dorsal/ventral striatum, amygdala, and hypothalamus, only the former subgroup showed an involvement of Raphé medialis, while, for the latter subgroup, an increase of SERT within hippocampus was unexpectedly caused. Within the less-affected "intermediate" rats, hippocampal 5-HT7 receptors were down-modulated, and also similarly within substantia nigra, septum, and neocortex. This picture demonstrates that additional rather than fewer neurobiological changes accompany a lower phenotypic expression. Overall, tapping hippocampal SERT affected the balance between habits versus strategies of coping by promoting morphogenetic processes indicative of a serotonergic fiber plasticity. Supplementary studies about serotonergic dynamics and neurogenesis within fronto-striatal circuits are needed.

Comparative evaluation of adolescent repeated psychological or physical stress effects on adult cognitive performance, oxidative stress, and heart rate in female rats.

Multiple adult health problems are associated with adolescent stress. As the brain discriminates physical and psychological stressors by activation of different neural networks, we hypothesized that behavioral and physiological performance would be modulated differently based on the nature of the stressors. Thus, we studied the comparative effects of adolescent repeated physical and psychological stresses on adult cognitive performance, pro-oxidant-antioxidant balance (PAB) and heart rate in female rats. The aim was to differentiate disparate potency of chronic psychological and physical stresses leading to long-term behavioral and physiological alterations. Twenty-one female rats were divided randomly into three groups of seven rats each; control, physical, and psychological stress. Experimental rats were exposed to the stressors for five consecutive days (10 min daily) via a two-communication box. After verifying stress induction by serum corticosterone measurement, the rats were returned to their home cage for 6 weeks, until adulthood, elevated plus maze (EPM), forced swimming test (FST), Y-maze, object recognition task (ORT), and passive avoidance test (PAT) were used as five different behavioral tests to evaluate cognitive performance of each group. Serum PAB and heart rate were measured to assess long-term stress-induced physiological disorders. The results showed exposure to adolescent psychological stress resulted in a larger set of significant changes (in behavioral variation, oxidative stress, and elevated heart rate) 6 weeks post-stress compared to adolescent physical stress. Hence, mental health care in adolescence and therapies targeting PAB and heart rate could be prevention and treatment approaches to confront persistent adolescent stress-induced disorders. Lay summaryThe aim of our study on female laboratory rats was to differentiate disparate potency of chronic psychological and physical stresses in adolescence leading to long-term behavioral and physiological alterations. The results suggest that psychological stresses result in a greater extent of changes compared to physical stress. Adolescent chronic psychological stress may reveal itself in the form of certain behavioral and physiological variations in adulthood. Therefore, mental health care in adolescence could be a valuable prevention approach to confront a variety of adult stress-induced disorders.

Constructing an Inexpensive Elevated Plus Maze.

Introducing students to the challenges and rewards of legitimate experimentation has become an essential part of many undergraduate lab courses. However, this objective can be difficult to achieve if the students find the topic uninteresting and therefore do not take ownership of the project. Additionally, the budgets of most undergraduate courses do not allow for the purchase of new equipment for student-generated projects. Here we describe a lab project where students engaged in the process of designing and building their own inexpensive apparatus. Driven by their interest in anxiety research, students in a Neuroscience Methods course developed the following protocol to build an elevated plus maze (EPM) and optional data acquisition module, for less than $100 each. The project engaged students in work that required applied critical thinking and real-world problem solving, and produced a functional EPM that was used in multiple projects beyond this course.

The Behavioral Effects of Oral Psychostimulant Ingestion on a Laboratory Rat Sample: An Undergraduate Research Experience.

Presented is a lab exercise designed to augment an upper-level undergraduate class covering the topics of psychopharmacology, biopsychology, physiological psychology, or introductory neuroscience. The exercise was developed as a tool to allow students to investigate behavioral correlates of oral psychomotor stimulant ingestion and observe firsthand the benefits and challenges of using animal models to study behavior. The purpose of the exercise was to observe the unconditioned, natural behaviors of laboratory rats prior to, and following, the oral administration of commonly used, over-the-counter psychomotor stimulants, and for students to experience the process of neuroscience research. Of specific interest was the comparison of behaviors demonstrated by the animals following ingestion of the nonprescription stimulants caffeine and pseudoephedrine. Students went through the steps of a research project by actively participating in all aspects of experimental design, including construction of testing apparatus, animal care, drug measurement and dosage, data collection, and analyzing behavioral data to determine animal response to psychomotor stimulant exposure. Through repetition of treatment conditions separated by a clearance phase, students observed experiment replication and learned about a research design commonly applied in animal research. Successful replication of treatment effects also served to exemplify the concepts of reliability and validity in behavioral research, while observable responses in animal models provided students with the opportunity to extrapolate important considerations for differential behavioral effects of psychostimulant consumption in humans.

The interplay between 5-HT2C and 5-HT3A receptors in the dorsal periaqueductal gray mediates anxiety-like behavior in mice.

The monoamine neurotransmitter serotonin (5-HT) modulates anxiety by its activity on 5-HT2C receptors (5-HT2CR) expressed in the dorsal periaqueductal gray (dPAG). Here, we investigated the presence of 5-HT3A receptors (5-HT3AR) in the dPAG, and the interplay between 5-HT2CR and 5-HT3AR in the dPAG in mediating anxiety-like behavior in mice. We found that 5-HT3AR is expressed in the dPAG and the blockade of these receptors using intra-dPAG infusion of ondansetron (5-HT3AR antagonist; 3.0 nmol) induced an anxiogenic-like effect. The activation of 5-HT3ABR by the infusion of mCPBG [1-(m-Chlorophenyl)-biguanide; 5-HT3R agonist] did not alter anxiety-like behaviors. In addition, blockade of 5-HT3AR (1.0 nmol) prevented the anxiolytic-like effect induced by the infusion of the 5-HT2CR agonist mCPP (1-(3-chlorophenyl) piperazine; 0.03 nmol). None of the treatment effects on anxiety-like behaviors altered the locomotor activity levels. The present results suggest that the anxiolytic-like effect exerted by serotonin activity on 5-HT2CR in the dPAG is modulated by 5-HT3AR expressed in same region.

The Effect of Earthing Mat on Stress-Induced Anxiety-like Behavior and Neuroendocrine Changes in the Rat.

Grounding is a therapeutic technique that involves doing activities that "ground" or electrically reconnect us to the earth. The physiological effects of grounding have been reported from a variety of perspectives such as sleep or pain. However, its anti-stress efficacy is relatively unknown. The present study investigated the stress-related behavioral effects of earthing mat and its neurohormonal mechanisms in the Sprague−Dawley male rat. Rats were randomly divided into four groups: the naïve normal (Normal), the 21 days immobilization stressed (Control), the 21 days stressed + earthing mat for 7 days (A7) or 21 days (A21) group. The depressive-and anxiety like behaviors were measured by forced swimming test (FST), tail suspension test (TST) and elevated plus maze (EPM). Using immunohistochemistry, the expression of corticotrophin-releasing factor (CRF) and c-Fos immunoreactivity were analyzed in the brain. In the EPM, time spent in the open arm of the earthing mat groups was significantly increased compared to the Control group (p < 0.001), even though there were without effects among groups in the FST and TST. The expression of CRF immunoreactive neurons in the earthing mat group was markedly decreased compared to the Control group. Overall, the earthing mat reduced stress-induced behavioral changes and expression of c-Fos and CRF immunoreactivity in the brain. These results suggest that the earthing mat may have the potential to improve stress-related responses via the regulation of the corticotrophinergic system.

Stress during puberty exerts sex-specific effects on depressive-like behavior and monoamine neurotransmitters in adolescence and adulthood.

Psychiatric disorders including major depression are twice as prevalent in women compared to men. This sex difference in prevalence only emerges after the onset of puberty, suggesting that puberty may be a sensitive period during which sex-associated vulnerability to stress-related depression might become established. Thus, this study investigated whether stress occurring specifically during the pubertal window of adolescence may be responsible for this sex difference in depression vulnerability. Male and female rats were exposed to a three-day stress protocol during puberty (postnatal days 35-37 in females, 45-47 in males) and underwent behavioral tests in adolescence or adulthood measuring anhedonia, anxiety-like behavior, locomotor activity and antidepressant-like behavior. Brainstem and striatum tissue were collected from a separate cohort of behavioral test-naïve rats in adolescence or adulthood to quantify the effect of pubertal stress on monoamine neurotransmitters. Pubertal stress increased immobility behavior in the forced swim test in both sexes in adolescence and adulthood. In adolescence, pubertal stress altered escape-oriented behaviors in a sex-specific manner: decreasing climbing in males but not females and decreasing swimming in females but not males. Pubertal stress decreased adolescent brainstem noradrenaline specifically in females and had opposing effects in adolescent males and females on brainstem serotonin turnover. Pubertal stress induced anhedonia in the saccharin preference test in adult males but not females, an effect paralleled by a male-specific decrease in striatal dopamine turnover. Pubertal stress did not significantly impact anxiety-like behavior or locomotor activity in any sex at either age. Taken together, these data suggest that although pubertal stress did not preferentially increase female vulnerability to depressive-like behaviors compared to males, stress during puberty exerts sex-specific effects on depressive-like behavior and anhedonia, possibly through discrete neurotransmitter systems.

In vivo anxiolytic and in vitro anti-inflammatory activities of water-soluble extract (WSE) of Nigella sativa (L.) seeds.

The WSE is a highly polar, gummy and mucilaginous bioactive content of the Nigella sativa (L.) seeds. This study reports the anxiolytic and anti-inflammatory effects of WSE investigated using Elevated Plus Maze (EPM) and Hole-Board Test (HBT) in adult mice and human RBCs haemolysis inhibition and protein denaturation respectively. The oral WSE treatment (100 & 200 mg/kg b.w/day) for 72 hours has exhibited slightly better anxiolytic effect (p < 0.05) through the time span (92.33 & 93.33 s) spent in the opened arms of EPM vs. diazepam (1 mg/kg b.w i.p/day; 69.33 s). In HBT, only WSE (200 mg/kg b.w/day) has shown a promising number of mean head pokes (13.27 times/min) vs. diazepam (12.87 times/min). The WSE (62.5-500 µg/mL) exposure has exhibited 40.14-72.18% protection against lysis of RBCs vs. aspirin (57.04-71.48%) whilst 62.67-67.66% inhibition of protein denaturation vs. diclofenac sodium (43.11-80.64%). The current findings suggested WSE has promising anxiolytic and anti-inflammatory activities.

To Approach or Avoid: An Introductory Overview of the Study of Anxiety Using Rodent Assays.

Anxiety is a widely studied phenomenon in behavioral neuroscience, but the recent literature lacks an overview of the major conceptual framework underlying anxiety research to introduce young researchers to the field. In this mini-review article, which is aimed toward new undergraduate and graduate students, we discuss how researchers exploit the approach-avoidance conflict, an internal conflict rodents face between exploration of novel environments and avoidance of danger, to inform rodent assays that allow for the measurement of anxiety-related behavior in the laboratory. We review five widely-used rodent anxiety assays, consider the pharmacological validity of these assays, and discuss neural circuits that have recently been shown to modulate anxiety using the assays described. Finally, we offer related lines of inquiry and comment on potential future directions.

Combined treatment of scopolamine and group III mGluR antagonist, CPPG, exerts antidepressant activity without affecting anxiety-related behaviors.

Clinical trials have revealed that the muscarinic receptor antagonist scopolamine produces a fast and potent antidepressant response. However, the anticholinergic adverse effects and the risk of psychosis at higher doses limit the widespread clinical use of scopolamine. Combination therapy of scopolamine and other antidepressants in treating depression has been suggested. Our experimentswere designed to examine the effects of the combining ineffective doses of scopolamine and a group III metabotropic glutamate receptor (mGluR) antagonist, CPPG, on depression- and anxiety-related behaviors in male NMRI mice. The forced swim test (FST) and the elevated plus maze (EPM) were selected to evaluate depression- and anxiety-related behaviors, respectively. Intraperitoneal (i.p.) administration of scopolamine (0.01-0.5 mg/kg) exerted profound antidepressive and anxiogenic effects. Intracerebroventricular (i.c.v.) administration of CPPG (12.5-50 nmol/mouse) elicited significant antidepressive and anxiolytic effects. Moreover, the ineffective dose of CPPG (12.5 nmol/mouse) plus ineffective doses of scopolamine (0.01 or 0.05 mg/kg) showed antidepressant-like effect while these combinations had no effect anxiety-related behaviors. It should be noted that none of the compounds altered locomotor activity. Results identify the combined administration of scopolamine and CPPG as a possible hopeful target in the future treatment of the depressive disorder.

Anxiolytic and antidepressant effects of ACPA and harmaline co-treatment.

Depression and anxiety disorders are among the most common illnesses and a close relationship between them has been found. Because the psychotropic effects and abuse liability of cannabis prevent its therapeutic application in depression and anxiety states, we decided to investigate the effects of the combination of ineffective doses of cannabinoid CB1 receptor agonist arachidonylcyclopropylamide (ACPA) and ß-carbolines on anxiety- and depression-related behaviors in male NMRI mice. Anxiety- and depression-related behaviors were assesses using elevated plus maze (EPM) and forced swim test (FST), respectively. Intraperitoneal administration of ACPA (1 mg/kg) decreased the percentage of time spent in the open-arms (%OAT) and the number of entries to the open-arms (OAE) in the EPM, indicating an anxiogenic-like effect. ACPA also decreased immobility time in the FST compared to the control group, suggesting an antidepressant-like effect. ß-carbolines including harmane (5 and 10 mg/kg), norharmane (5 mg/kg) and harmaline (2.5 and 5 mg/kg) produced an anxiogenic-like response, while the highest dose of harmane or harmaline and the middle dose of norharmane induced an antidepressant-like behavior. Furthermore, co-administration of a subthreshold dose of ACPA (0.5 mg/kg) and harmaline (1.25 mg/kg), but not harmane or norharmane (both at the dose of 2.5 mg/kg), caused anxiolytic- and antidepressant-like behaviors and decreased locomotor activity. Our findings suggest a therapeutic potential for combined ineffective doses of ACPA and harmaline on anxiety- and depression-related processes.

Comparison of the effects of 1MeTIQ and olanzapine on performance in the elevated plus maze test and monoamine metabolism in the brain after ketamine treatment.

Anxiety is a common symptom of schizophrenia. Ketamine, which acts as a noncompetitive antagonist of glutamatergic NMDA receptors by binding to the phencyclidine site, may induce schizophrenia-like symptoms and promote anxiogenic-like behaviour. The symptoms of anxiety in rodents can be measured by the elevated plus maze (EPM) test. 1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), as a neuroprotective and antiaddictive substance, produces pharmacological effects by influencing monoaminergic and glutamatergic activity, as previously demonstrated by us. The aim of the present study was to investigate the anxiolytic-like potential of 1MeTIQ after the administration of ketamine. These results were compared to the effects of olanzapine, an antipsychotic drug commonly used in the treatment of schizophrenia. We conducted the EPM test, during which the percentage of time spent in and the number of entries into the open arms were measured. In addition, locomotor activity was measured. Furthermore, we conducted biochemical analyses to verify changes in the levels of neurotransmitters and their metabolites in selected rat brain structures. Behavioural analyses showed that 1MeTIQ, similar to olanzapine, completely inhibited ketamine-induced anxiogenic effects in the EPM test. On the other hand, neurochemical data indicated that 1MeTIQ, as a reversible inhibitor of MAO, significantly blocked the dopamine MAO-dependent oxidation pathway, whereas olanzapine significantly increased the activity of this pathway. The results above suggest that the anxiolytic-like properties of 1MeTIQ are connected to its influence on the catabolism of dopamine, the elevation of serotonin concentrations and the reduction in the levels of noradrenaline.

Nitric oxide in the prelimbic medial prefrontal cortex is involved in the anxiogenic-like effect induced by acute restraint stress in rats.

Neurons containing the neuronal nitric oxide synthase (nNOS) enzyme are located in brain areas related to defensive behavior, such as the ventromedial prefrontal cortex (vMPFC). Rats exposed to a live predator (a cat) present anxiety-like behavior and an increased number of nNOS-positive neurons in this brain area one-week later. Moreover, stress-related behavioral changes in rodents can be prevented by systemic or local vMPFC nNOS inhibition. In the present study we investigated if acute restraint stress (RS)-induced delayed (one-week) anxiogenic-like effect was associated with increased nNOS expression or activity in the vMPFC. Furthermore, we also tested if local pharmacological nNOS inhibition would prevent stress-induced behavioral changes. Male Wistar rats were submitted to RS for 3h and tested in the elevated plus maze (EPM) 24h or 7 days later. Two hours after the EPM test, their brains were removed, processed and nNOS expression in the vMPFC was evaluated by immunohistochemistry. Another group of animals was used for measuring NO metabolites (NOx; an indirect measure of NOS activity) immediately after the EPM test, 24h after RS. Independent groups had guide cannula implanted bilaterally into the prelimbic (PL) portion of vMPFC. Five to six days after surgery, the animals were submitted to RS and 24h later received local administration of the nNOS inhibitor, N-propyl-l-arginine (NPLA; 0.04 nmol). They were tested in the EPM 10 min later. RS-induced anxiogenic-like effect was accompanied by increased nNOS expression in the PL (p<0.05), but not in the infralimbic (IL) vMPFC, both 24h and 7 days after RS. Moreover, open-arm exploration of the EPM was negatively correlated with nNOS expression (p<0.05) and NOx levels (p<0.05) in the PL. The anxiogenic-like effect observed 24h after RS was prevented by NPLA (p<0.05). Our results suggest that RS-induced anxiogenic-like effect might depend on increased nNOS-mediated signaling in the PL MPFC.

Effects of Glycyrrhizae Radix on Repeated Restraint Stress-induced Neurochemical and Behavioral Responses.

Glycyrrhizae radix (GR) is an herbal medicine that is commonly used in the East Asia for treating a variety of diseases, including stomach disorders. The objective of the present study was to examine the anti-stress effects of GR on repeated stress-induced alterations of anxiety, learning and memory in rats. Restraint stress was administered for 14 days (2 h/day) to the rats in the Control and GR groups (400 mg/kg/day, PO). Starting on the eighth day, the rats were tested for spatial memory on the Morris water maze test (MW) and for anxiety on the elevated plus maze (EPM). We studied the changes of the expressions of cholineacetyl transferase (ChAT) and tyrosine hydroxylase (TH) in the locus coerleus (LC) using immunohistochemistry. The results showed that the rats treated with GR had significantly reduced stress-induced deficits on their learning and memory on the spatial memory tasks. In addition, the ChAT immunoreactivities were increased. Gor the EPM, treatment with GR increased the time spent in the open arms (p<0.001) as compared to that of the control group. Moreover, GR treatment also normalized the increases of the TH expression in the LC (p<0.001). In conclusion, administration of GR improved spatial learning and memory and reduced stress-induced anxiety. Thus, the present results suggest that GR has the potential to attenuate the behavioral and neurochemical impairments caused by stress.

Role of nitric oxide in the periaqueductal gray in defensive behavior in mice: influence of prior local N-methyl-D-aspartate receptor activation and aversive condition

Glutamate N-methyl-D-aspartate (NMDA) receptor activation within the dorsal column of the periaqueductal gray (dPAG) leads to antinociceptive, autonomic, and behavioral responses characterized as the fear reaction. Activation of NMDA receptors in the brain increases nitric oxide (NO) synthesis, and NO has been proposed to be a mediator of the aversive action of glutamate. This paper reviews a series of studies investigating the effects of neuronal NO synthase (nNOS) inhibition in the dPAG of mice in different aversive conditions. nNOS inhibition by infusion of Nω-propyl-L-arginine (NPLA) prevents fear-like reactions (e.g., jumping, running, freezing) induced by NMDA receptor stimulation within the dPAG and produces anti-aversive effects when injected into the same midbrain site in mice confronted with a predator. Interestingly, nNOS inhibition within the dPAG does not change anxiety-like behavior in mice exposed to the elevated plus maze (EPM), but it reverses the effect of an anxiogenic dose of NMDA injected into the same site in animals subjected to the EPM. Altogether, the results support a role for glutamate NMDA receptors and NO in the dPAG in the regulation of defensive behaviors in mice. However, dPAG nitrergic modulation of anxiety-like behavior appears to depend on the magnitude of the aversive stimulus.