Anxiogenic-like effects induced by NMDA receptor activation are prevented by inhibition of neuronal nitric oxide synthase in the periaqueductal gray in mice

Glutamate-NMDA (N-methyl-D-aspartate) receptor activation within the periaqueductal gray (PAG) leads to antinociceptive, autonomic and behavioral responses characterized as the fear reaction. We have recently demonstrated that the vigorous defensive-like behaviors (e.g. jumping and running) and antinociception induced by intra-PAG injection of N-methyl-D-aspartate (NMDA) were completely blocked by prior infusion of N(omega)-propyl-L-arginine (NPLA), a specific neuronal nitric oxide synthesis (nNOS) enzyme inhibitor, into the same midbrain structure. It remains unclear however, whether the inhibition of nNOS within the mouse PAG changes the anxiety-like behavior per se or the effects of the inhibition of nNOS depend on the suppression of downstream of glutamate-NMDA receptor activation. This study investigated whether intra-PAG infusion of NPLA (i) attenuates anxiety in the elevated plus-maze (EPM) and (ii) antagonizes the anxiogenic-like effects induced by intra-PAG injection of NMDA. Test sessions were videotaped and subsequently scored for conventional indices of anxiety (percentage of open arm entries and percentage of open arm time) and locomotor activity (closed arm entries). Results showed that intra-PAG infusions of NPLA (0.2, 0.4 or 0.8 nmol/0.1 mu l) did not alter significantly any behavioral response in the EPM when compared to control group (Experiment 1). Intra-PAG infusion of NMDA (0 and 0.02 nmol/0.1 mu l; a dose that does not provoke vigorous defensive behaviors per se in mice) significantly reduced open arm exploration, confirming an anxiogenic-like effect (Experiment 2). When injected into the PAG 10 min prior local NMDA injection (0.02 nmol/0.1 mu l), NPLA (0.4 nmol/0.1 mu l) was able to revert the anxiogenic-like effect of glutamate-NMDA receptor activation. Neither intra-PAG infusion of NMDA nor NPLA altered closed arm entries, a widely used measure of locomotor activity in the EPM. These results suggest that intra-PAG nitric oxide synthesis does not play a role on anxiety-like behavior elicited during EPM exposure; however its synthesis is important for the proaversive effects produced by activation of glutamate-NMDA receptors located within this limbic midbrain structure. (C) 2008 Elsevier B.V. All rights reserved.

Ventrolateral periaqueductal gray lesion attenuates nociception but does not change anxiety-like indices or fear-induced antinociception in mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Reinforcing properties of fencamfamine: Involvement of dopamine and opioid receptors

Fencamfamine (FCF) is a psychostimulant classified as an indirect dopamine agonist. The conditioning place preference (CPP) paradigm was used to investigate the reinforcing properties of FCF. After initial preferences had been determined, animals were conditioned with FCF (1.75, 3.5, or 7.0 mg/kg; IP). Only at the dose of 3.5 mg/kg FCF produced a significant place preference. Pretreatment with SCH23390 (0.05 mg/kg, SC) or naloxone (1.0 mg/kg SC) 10 min before FCF (3.5 mg/kg; IP) blocked both FCF-induced hyperactivity and CPP. Pretreatment with metoclopramide (10.0 mg/kg; IP) or pimozide (1.0 mg/kg, IP), respectively, 30 min or 4 h before FCF (3.5 mg/kg; IP), which blocked the FCF-induced locomotor activity, failed to influence place conditioning produced by FCF. In conclusion, the present study suggests that dopamine D 1 and opioid receptors are related to FCF reinforcing effect, while dopamine D 2 subtype receptor was ineffective in modifying FCF-induced CPP.

Baseline and stress-induced plasma corticosterone concentrations of mice selectively bred for high voluntary wheel running

The hypothalamic-pituitary-adrenal (HPA) axis is important in regulating energy metabolism and in mediating responses to stressors, including increasing energy availability during physical exercise. In addition, glucocorticoids act directly on the central nervous system and influence behavior, including locomotor activity. To explore potential changes in the HPA axis as animals evolve higher voluntary activity levels, we characterized plasma corticosterone (CORT) concentrations and adrenal mass in four replicate lines of house mice that had been selectively bred for high voluntary wheel running (HR lines) for 34 generations and in four nonselected control (C) lines. We determined CORT concentrations under baseline conditions and immediately after exposure to a novel stressor (40 min of physical restraint) in mice that were housed without access to wheels. Resting daytime CORT concentrations were approximately twice as high in HR as in C mice for both sexes. Physical restraint increased CORT to similar concentrations in HR and C mice; consequently, the proportional response to restraint was smaller in HR than in C animals. Adrenal mass did not significantly differ between HR and C mice. Females had significantly higher baseline and postrestraint CORT concentrations and significantly larger adrenal glands than males in both HR and C lines. Replicate lines showed significant variation in body mass, length, baseline CORT concentrations, and postrestraint CORT concentrations in one or both sexes. Among lines, both body mass and length were significantly negatively correlated with baseline CORT concentrations, suggesting that CORT suppresses growth. Our results suggest that selection for increased locomotor activity has caused correlated changes in the HPA axis, resulting in higher baseline CORT concentrations and, possibly, reduced stress responsiveness and a lower growth rate. © 2007 by The University of Chicago. All rights reserved.

Field and Laboratory Studies Provide Insights into the Meaning of Day-Time Activity in a Subterranean Rodent (Ctenomys aff. knighti), the Tuco-Tuco

South American subterranean rodents (Ctenomys aff. knighti), commonly known as tuco-tucos, display nocturnal, wheel-running behavior under light-dark (LD) conditions, and free-running periods >24 h in constant darkness (DD). However, several reports in the field suggested that a substantial amount of activity occurs during daylight hours, leading us to question whether circadian entrainment in the laboratory accurately reflects behavior in natural conditions. We compared circadian patterns of locomotor activity in DD of animals previously entrained to full laboratory LD cycles (LD12:12) with those of animals that were trapped directly from the field. In both cases, activity onsets in DD immediately reflected the previous dark onset or sundown. Furthermore, freerunning periods upon release into DD were close to 24 h indicating aftereffects of prior entrainment, similarly in both conditions. No difference was detected in the phase of activity measured with and without access to a running wheel. However, when individuals were observed continuously during daylight hours in a semi-natural enclosure, they emerged above-ground on a daily basis. These day-time activities consisted of foraging and burrow maintenance, suggesting that the designation of this species as nocturnal might be inaccurate in the field. Our study of a solitary subterranean species suggests that the circadian clock is entrained similarly under field and laboratory conditions and that day-time activity expressed only in the field is required for foraging and may not be time-dictated by the circadian pacemaker.

Enhancement of behavioral sensitization, anxiety-like behavior, and hippocampal and frontal cortical CREB levels following cocaine abstinence in mice exposed to cocaine during adolescence

Adolescence has been linked to greater risk-taking and novelty-seeking behavior and a higher prevalence of drug abuse and risk of relapse. Decreases in cyclic adenosine monophosphate response element binding protein (CREB) and phosphorylated CREB (pCREB) have been reported after repeated cocaine administration in animal models. We compared the behavioral effects of cocaine and abstinence in adolescent and adult mice and investigated possible age-related differences in CREB and pCREB levels. Adolescent and adult male Swiss mice received one daily injection of saline or cocaine (10 mg/kg, i.p.) for 8 days. On day 9, the mice received a saline injection to evaluate possible environmental conditioning. After 9 days of withdrawal, the mice were tested in the elevated plus maze to evaluate anxiety-like behavior. Twelve days after the last saline/cocaine injection, the mice received a challenge injection of either cocaine or saline, and locomotor activity was assessed. One hour after the last injection, the brains were extracted, and CREB and pCREB levels were evaluated using Western blot in the prefrontal cortex (PFC) and hippocampus. The cocaine-pretreated mice during adolescence exhibited a greater magnitude of the expression of behavioral sensitization and greater cocaine withdrawal-induced anxiety-like behavior compared with the control group. Significant increases in CREB levels in the PFC and hippocampus and pCREB in the hippocampus were observed in cocaine-abstinent animals compared with the animals treated with cocaine in adulthood. Interestingly, significant negative correlations were observed between cocaine sensitization and CREB levels in both regions. These results suggest that the behavioral and neurochemical consequences of psychoactive substances in a still-developing nervous system can be more severe than in an already mature nervous system

Adenosine A2A receptor gene expression in the normal striatum and after 6-OH-dopamine lesion

Adenosine A2A receptors are present on enkephalinergic medium sized striatal neurons in the rat and have an important function in the modulation of striatal output. In order to establish more accurately whether adenosine transmission is a generalized phenomenon in mammalian striatum we compared the A2A R expression in the mouse, rat, cat and human striatum. Secondly we compared the modulation of enkephalin gene expression and A2A receptor gene expression in rat striatal neurons after 6-OH-dopamine lesion of the substantia nigra. Hybridization histochemistry was performed with a 35S-labelled radioactive oligonucleotide probe. The results showed high expression of A2A adenosine receptor genes only in the medium-sized cells of the striatum in all examined species. In the rat striatum, expression of A2A receptors was not significantly altered after lesion of the dopaminergic pathways with 6-OH-dopamine even though enkephalin gene expression was up-regulated. The absence of a change in A2A receptor gene expression after 6-OH-dopamine treatment speaks against a dependency on dopaminergic innervation. The maintained inhibitory function of A2A R on motor activity in spite of dopamine depletion could be partly responsible for the depression of locomotor activity observed in basal ganglia disorders such as Parkinson's disease.

ROLE OF DOPAMINE OF NUCLEUS ACCUMBENS IN BEHAVIORAL SENSITIZATION TO METHYLPHENIDATE

Behavioral sensitization is defined as the subsequent augmentation of the locomotor response to a drug following repeated administrations of the drug. It is believed to occur due to alterations in the motive circuit in the brain by stressors, central nervous system stimulants, and similar stimuli. The motive circuit (or mesocorticolimbic system) consists of several interconnected nuclei that determine the behavioral response to significant biological stimuli. A final target of the mesocorticolimbic system is the nucleus accumbens (NAc), which is a key structure linking motivation and action. In particular, the dopaminergic innervations of the Nac are considered to be essential in regulating motivated states of behavior such as goal-directed actions, stimulus-reward associations and reinforcement by addictive substances. Therefore, the objective of this study was to investigate the role of dopaminergic afferents of the NAc in the behavioral sensitization elicited by chronic treatment with methylphenidate (MPD), a psychostimulant that is widely used to treat attention deficit hyperactivity disorder. The dopaminergic afferents can be selectively destroyed using catecholamine neurotoxin 6-hydroxydopamine (6-OHDA). In order to determine whether destruction of dopaminergic afferents of the NAc prevents sensitization, I compared locomotor activity in rats that had received infusions of 6-hydroxydopamine (6-OHDA) into the NAc with that of control and sham-operated animals. All groups of rats received six days of single daily MPD injections after measuring their pre and post surgery locomotor baseline. Following the consecutive MPD injections, there was a washout period of 4 days, where no injections were given. Then, a rechallenge injection of MPD was given. Behavioral responses after repeated MPD were compared to those after acute MPD to assess behavioral sensitization. Expression of sensitization to MPD was not prevented by 6-OHDA infusion into the NAc. Moreover, two distinct responses were seen to the acute injection of MPD: one group of rats had essentially no response to acute MPD, while the other had an augmented (‘sensitized’-like) acute response. Among rats with 6-OHDA infusions, the animals with diminished acute response to MPD had intact behavioral sensitization to repeated MPD, while the animals with increased acute response to MPD did not exhibit further sensitization to it. This suggests that the acute and chronic effects of MPD have distinct underlying neural circuitries.

Locomotor velocity and striatal adaptive gene expression changes of the direct and indirect pathways in Parkinsonian rats

BACKGROUND In Parkinson's disease (PD), bradykinesia, or slowness of movement, only appears after a large striatal dopamine depletion. Compensatory mechanisms probably play a role in this delayed appearance of symptoms. OBJECTIVE Our hypothesis is that the striatal direct and indirect pathways participate in these compensatory mechanisms. METHODS We used the unilateral 6-hydroxydopamine (6-OHDA) rat model of PD and control animals. Four weeks after the lesion, the spontaneous locomotor activity of the rats was measured and then the animals were killed and their brain extracted. We quantified the mRNA expression of markers of the striatal direct and indirect pathways as well as the nigral expression of dopamine transporter (DAT) and tyrosine hydroxylase (TH) mRNA. We also carried out an immunohistochemistry for the striatal TH protein expression. RESULTS As expected, the unilateral 6-OHDA rats presented a tendency to an ipsilateral head turning and a low locomotor velocity. In 6-OHDA rats only, we observed a significant and positive correlation between locomotor velocity and both D1-class dopamine receptor (D1R) (direct pathway) and enkephalin (ENK) (indirect pathway) mRNA in the lesioned striatum, as well as between D1R and ENK mRNA. CONCLUSIONS Our results demonstrate a strong relationship between both direct and indirect pathways and spontaneous locomotor activity in the parkinsonian rat model. We suggest a synergy between both pathways which could play a role in compensatory mechanisms and may contribute to the delayed appearance of bradykinesia in PD.

Dopamine-dependent responses to morphine depend on glucocorticoid receptors

Previous work has shown that glucocorticoid hormones facilitate the behavioral and dopaminergic effects of morphine. In this study we examined the possible role in these effects of the two central corticosteroid receptor types: mineralocorticoid receptor (MR), and glucocorticoid receptor (GR). To accomplish this, specific antagonists of these receptors were infused intracerebroventricularly and 2 hr later we measured: (i) locomotor activity induced by a systemic injection of morphine (2 mg/kg); (ii) locomotor activity induced by an infusion of morphine (1 μg per side) into the ventral tegmental area, which is a dopamine-dependent behavioral response to morphine; (iii) morphine-induced dopamine release in the nucleus accumbens, a dopaminergic projection site mediating the locomotor and reinforcing effects of drugs of abuse. Blockade of MRs by spironolactone had no significant effects on locomotion induced by systemic morphine. In contrast, blockade of GRs by either RU38486 or RU39305, which is devoid of antiprogesterone effects, reduced the locomotor response to morphine, and this effect was dose dependent. GR antagonists also reduced the locomotor response to intraventral tegmental area morphine as well as the basal and morphine-induced increase in accumbens dopamine, as measured by microdialysis in freely moving rats. In contrast, spironolactone did not modify dopamine release. In conclusion, glucocorticoids, via GRs, facilitate the dopamine-dependent behavioral effects of morphine, probably by facilitating dopamine release. The possibility of decreasing the behavioral and dopaminergic effects of opioids by an acute administration of GR antagonists may open new therapeutic strategies for treatment of drug addiction.

Behavioural characterization of Vitamin D receptor knockout mice

Vitamin D (calcitriol) is a nuclear transcription regulator acting via a nuclear hormone receptor (VDR). In addition to its role in the regulation of calcium and phosphate horneostasis and in bone formation, Vitamin D is also thought to be involved in brain function. The aim of this study was to behaviourally phenotype VDR knockout mice. We characterized the behaviour of VDR null mutant mice and wildtype littermate controls by subjecting them to a range of tests including a primary behavioural screen (using the SHIRPA protocol), rotarod, gait analysis, Y-maze, marble burying test, bedding test, holeboard test, elevated plus maze, open field test and prepulse inhibition of the acoustic startle response. There were no effects of genotype on most of the scores from the SHIRPA protocol except that VDR -/- mice had alopecia, were shorter and weighed less than VDR +/+ mice. VDR -/- mice had a shorter gait as well as impairments on the rotarod, in the bedding test and impaired habituation in both the open field and on the acoustic startle response. The VDR -/- mice had normal acoustic startle responses but had impaired PPI at long (256 ms) but not short (64 ms) prepulse to pulse intervals. The VDR -/- mice were less active in the open field and buried fewer marbles in the marble burying test. However, there were no differences in the time spent on the open arms of the elevated plus maze or in working memory as assessed by repeat arm entries on the Y-maze. Therefore, it appears that VDR -/- mice have muscular and motor impairments that significantly affects locomotor behaviour but seemingly no impairments in cognition as indicated by exploration, working memory or anxiety. (C) 2004 Elsevier B.V. All rights reserved.

Characterization of the behavioural phenotype of calpain-knockout mice

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Anticonvulsant and sedative effect of Fufang Changniu pills and probable mechanism of action in mice

Purpose: To investigate the anticonvulsant and sedative effects of Fufang Changniu Pills (FCP) and its probable mechanism of action in mice. Methods: The water decoction of FCP was prepared and the main constituents were determined by high performance liquid chromatography (HPLC). The anticonvulsant activities of FCP were evaluated by maximal electroshock (MES) and pentylenetetrazole (PTZ)-induced seizures in mice. Pentobarbital sodium-induced sleeping time and locomotor activity measurements were performed to evaluate the sedative effects of FCP in mice. Finally, PTZ-induced chronic seizures were established, and expressions of gamma-aminobutyric acid A receptor (GABA-A) and glutamic acid decarboxylase 65 (GAD65) in the brains of the mice were assayed by western blot in order to explore the probable mechanisms of action of the drug. Results: Gallic acid, liquiritin, cinnamyl alcohol, cinnamic acid and glycyrrhizic acid were detected in FCP decoction. FCP (50, 100 and 200 mg/kg) showed significant anticonvulsant and sedative effects on epileptic mice induced by MES (p < 0.05) and PTZ (p < 0.05). Moreover, pentobarbital sodium-induced sleeping time and locomotor activity tests showed that FCP possesses sedative effect (p < 0.05). Western blot data indicate that FCP significantly up-regulated GABA-A and GAD 65 in the brains of chronic epileptic rats (p < 0.05). Conclusion: FCP has significant anticonvulsant and sedative effects, and the mechanism of its action may be related to the up-regulation of GABA-A and GAD 65 in mice brain.

Activité physique et récompense : impact de la leptine et de la signalisation STAT3 dans les neurones dopaminergiques

La course d’endurance active le système de récompense (SR) et est reliée aux comportements de recherche alimentaire. L’influence de la leptine sur l’activité physique (AP) volontaire est bien documentée d’un point de vue physiologique, mais très peu en termes d’impact hédonique. La leptine inhibe l’effet récompensant lié à la consommation de nourriture et joue un rôle semblable pour d’autres types de stimuli. La leptine s’arrime à la forme longue du récepteur à la leptine (Leprb) situé sur les neurones à dopamine (DA) et GABA de l’aire tegmentale ventrale (ATV) dans le mésencéphale. Signal transducer and Activator of Transcription 3 (STAT3) est un facteur de transcription important de la cascade de signalisation de la leptine. La phosphorylation de STAT3 n’est détectée que dans une parcelle des neurones DA positifs pour le Leprb, conférant aux neurones DA STAT3-spécifiques des caractéristiques uniques. Nous avons généré un modèle murin invalidé pour STAT3 sélectivement dans les neurones DA (STAT3DAT-KO). La première expérience consistait à évaluer les paramètres métaboliques de base de notre modèle en utilisant les chambres métaboliques Comprehensive Lab Animal Monitoring System (CLAMS), incluant l’activité ambulatoire, le ratio d’échanges respiratoires (RER) et la production de chaleur. Les STAT3DAT-KO sont hyperactives, démontré par une activité locomotrice augmentée, mais aucune variation entre les deux groupes n’est observée pour le RER et la production de chaleur, en plus d’un gain de poids identique. Une stratégie de récupération ciblant la réinsertion de STAT3 dans les neurones DA du système mésolimbique normalise l’AP anciennement plus élevée des STAT3DAT-KO à celle des contrôles, suivant l’accès libre à une roue d’exercice (RE) pour une durée de 4 semaines, suivant l’accès libre à une roue d’exercice (RE) pour une durée de 4 semaines. L’injection d’un psychostimulant (agoniste du récepteur DA de type 1 (D1R), le Chloro-APB-Hydrobromide (SKF 82958)) reflète une fonction dopaminergique réduite chez les STAT3DAT-KO. Un test de recherche compulsive de nourriture révèle une suppression de la prise alimentaire chez les deux groupes expérimentaux. Nous démontrons pour la première fois que la motivation alliée à la course d’endurance, indépendamment de la régulation de la prise alimentaire par la leptine, est dépendant d’une signalisation leptine-STAT3 amoindrie dans les neurones DA du système mésolimbique, révélant STAT3 comme élément clé dans la régulation du tonus dopaminergique et des propriétés récompensantes de l’AP.

Activité physique et récompense : impact de la leptine et de la signalisation STAT3 dans les neurones dopaminergiques

La course d’endurance active le système de récompense (SR) et est reliée aux comportements de recherche alimentaire. L’influence de la leptine sur l’activité physique (AP) volontaire est bien documentée d’un point de vue physiologique, mais très peu en termes d’impact hédonique. La leptine inhibe l’effet récompensant lié à la consommation de nourriture et joue un rôle semblable pour d’autres types de stimuli. La leptine s’arrime à la forme longue du récepteur à la leptine (Leprb) situé sur les neurones à dopamine (DA) et GABA de l’aire tegmentale ventrale (ATV) dans le mésencéphale. Signal transducer and Activator of Transcription 3 (STAT3) est un facteur de transcription important de la cascade de signalisation de la leptine. La phosphorylation de STAT3 n’est détectée que dans une parcelle des neurones DA positifs pour le Leprb, conférant aux neurones DA STAT3-spécifiques des caractéristiques uniques. Nous avons généré un modèle murin invalidé pour STAT3 sélectivement dans les neurones DA (STAT3DAT-KO). La première expérience consistait à évaluer les paramètres métaboliques de base de notre modèle en utilisant les chambres métaboliques Comprehensive Lab Animal Monitoring System (CLAMS), incluant l’activité ambulatoire, le ratio d’échanges respiratoires (RER) et la production de chaleur. Les STAT3DAT-KO sont hyperactives, démontré par une activité locomotrice augmentée, mais aucune variation entre les deux groupes n’est observée pour le RER et la production de chaleur, en plus d’un gain de poids identique. Une stratégie de récupération ciblant la réinsertion de STAT3 dans les neurones DA du système mésolimbique normalise l’AP anciennement plus élevée des STAT3DAT-KO à celle des contrôles, suivant l’accès libre à une roue d’exercice (RE) pour une durée de 4 semaines, suivant l’accès libre à une roue d’exercice (RE) pour une durée de 4 semaines. L’injection d’un psychostimulant (agoniste du récepteur DA de type 1 (D1R), le Chloro-APB-Hydrobromide (SKF 82958)) reflète une fonction dopaminergique réduite chez les STAT3DAT-KO. Un test de recherche compulsive de nourriture révèle une suppression de la prise alimentaire chez les deux groupes expérimentaux. Nous démontrons pour la première fois que la motivation alliée à la course d’endurance, indépendamment de la régulation de la prise alimentaire par la leptine, est dépendant d’une signalisation leptine-STAT3 amoindrie dans les neurones DA du système mésolimbique, révélant STAT3 comme élément clé dans la régulation du tonus dopaminergique et des propriétés récompensantes de l’AP.