Behavioural characterisation of young adult transgenic mice overexpressing galanin under the PDGF-B promoter

The behavioural phenotype of transgenic mice (3- to 5-months old) overexpressing galanin (GalOE) under the platelet-derived growth factor B (PDGF-B) promoter was evaluated in a battery of tests, including open field, locomotor cages, light-dark exploration test, elevated plus-maze and the Porsolt forced swim test. Learning and memory were assessed in the passive avoidance and the Morris water maze tasks. No difference between genotypes was found in exploratory activity in the open field. GalOE mice showed a slight increase in spontaneous locomotor activity assessed in the locomotor cages, but the amphetamine-induced increase in locomotor activity was somewhat lower in GalOE mice. Anxiety-like behaviour in the three different tests including open field, light-dark exploration and elevated plus-maze did not differ between genotypes. In the Porsolt forced swim test, GalOE mice displayed an increased time of immobility, indicative of increased learned helplessness possibly reflecting increased stress-susceptibility and/or depression-like behaviour. GalOE mice showed normal learning and memory retention in the passive avoidance and the Morris water maze tasks. These data support the hypothesis that galanin may have a role in functions related to mood states including affective disorders.     

Circuit specific functions of cannabinoid CB1 receptor in the balance of investigatory drive and exploration

Well balanced novelty seeking and exploration are fundamental behaviours for survival and are found to be dysfunctional in several psychiatric disorders. Recent studies suggest that the endocannabinoid (eCB) system is an important control system for investigatory drive. Pharmacological treatment of rodents with cannabinergic drugs results in altered social and object investigation. Interestingly, contradictory results have been obtained, depending on the treatment, drug concentration and experimental conditions. The cannabinoid type 1 (CB1) receptor, a central component of the eCB system, is predominantly found at the synapses of two opposing neuronal populations, i.e. on inhibitory GABAergic and excitatory glutamatergic neurons. In the present study, using different transgenic mouse lines, we aimed at investigating the impact of CB1 receptor inactivation in glutamatergic or GABAergic neurons on investigatory behaviour. We evaluated animate (interaction partner) and inanimate (object) exploratory behaviour in three different paradigms. We show that exploration was increased when CB1 receptor was deleted from cortical and striatal GABAergic neurons. No effect was observed when CB1 receptor was deleted specifically from dopamine receptor D1-expressing striatal GABAergic medium spiny neurons. In contrast, deletion of CB1 receptor from cortical glutamatergic neurons resulted in a decreased exploration. Thus, our results indicate that exploratory behaviour is accurately balanced in both, the social and non-social context, by the eCB system via CB1 receptor activation on cortical glutamatergic and GABAergic neurons. In addition, the results could explain the contradictory findings of previous pharmacological studies and could further suggest a possibility to readjust an imbalance in exploratory behaviour observed in psychiatric disorders.     

Taste aversion induced by confinement in a running wheel

Behavioural lateralisation, which has been postulated to be an individual personality trait, is related to the activity of various physiological systems including the immune system. As lateralisation has been related to anxiety, which is known to influence immune reactivity, it can be hypothesized that the relation between lateralisation and immune reactivity involves individual behavioural patterns as they appear in exploratory-based anxiety models. In order to answer this question, a behavioural investigation focussing on exploratory activity was undertaken in male and female C3H mice previously selected for their paw preference. The observations were performed using two generic paradigms: elevated plus-maze and open field. Exploratory behaviour in the open field, but not in the plus-maze, was influenced by the interactive effect of gender and behavioural lateralisation. A significant difference between male and female mice was found in left-pawed but not in right-pawed nor ambidextrous animals, left-pawed female mice displaying the less exploratory behaviours. These results provide a first evidence of inter-individual variations in exploratory behaviours involving interaction between gender and lateralisation.     

Localization and Function of the Cannabinoid CB1 Receptor in the Anterolateral Bed Nucleus of the Stria Terminalis

Background

The bed nucleus of the stria terminalis (BNST) is involved in behaviors related to natural reward, drug addiction and stress. In spite of the emerging role of the endogenous cannabinoid (eCB) system in these behaviors, little is known about the anatomy and function of this system in the anterolateral BNST (alBNST). The aim of this study was to provide a detailed morphological characterization of the localization of the cannabinoid 1 (CB1) receptor a necessary step toward a better understanding of the physiological roles of the eCB system in this region of the brain.

Methodology/Principal Findings

We have combined anatomical approaches at the confocal and electron microscopy level to ex-vivo electrophysiological techniques. Here, we report that CB1 is localized on presynaptic membranes of about 55% of immunopositive synaptic terminals for the vesicular glutamate transporter 1 (vGluT1), which contain abundant spherical, clear synaptic vesicles and make asymmetrical synapses with alBNST neurons. About 64% of vGluT1 immunonegative synaptic terminals show CB1 immunolabeling. Furthermore, 30% and 35% of presynaptic boutons localize CB1 in alBNST of conditional mutant mice lacking CB1 mainly from GABAergic neurons (GABA-CB1-KO mice) and mainly from cortical glutamatergic neurons (Glu-CB1-KO mice), respectively. Extracellular field recordings and whole cell patch clamp in the alBNST rat brain slice preparation revealed that activation of CB1 strongly inhibits excitatory and inhibitory synaptic transmission.

Conclusions/Significance

This study supports the anterolateral BNST as a potential neuronal substrate of the effects of cannabinoids on stress-related behaviors.     

Adaptive behavior of active and passive rats after intranasal administration of the corticotropin-releasing hormone

Behavioural effect of intranasal application of corticotropin-releasing hormone (CRH) was investigated in rats with high (KHA) and low (KLA) rate of learning in "open field" and plus-maze (PM) active avoidance test. The neurohormone provoked the opposed changes in behaviour of rats of this strain in both tests. The level of locomotion and exploratory activity rose in KLA rats and decreased in KHA rats. After the CRH application, the KLA rats but not KHA rats spent more time in the opened alleys of the PM than the control animals. The same behavioural changes were observed in our previous research when CRH was injected in striatum. We suppose that CRH is an endogenous factor of precise correction of the adaptive behaviour.     

CB(1) Receptor Autoradiographic Characterization of the Individual Differences in Approach and Avoidance Motivation

Typically, approach behaviour is displayed in the context of moving towards a desired goal, while avoidance behaviour is displayed in the context of moving away from threatening or novel stimuli. In the current research, we detected three sub-populations of C57BL/6J mice that spontaneously responded with avoiding, balancing or approaching behaviours in the presence of the same conflicting stimuli. While the balancing animals reacted with balanced responses between approach and avoidance, the avoiding or approaching animals exhibited inhibitory or advance responses towards one of the conflicting inputs, respectively. Individual differences in approach and avoidance motivation might be modulated by the normal variance in the level of functioning of different systems, such as endocannabinoid system (ECS). The present research was aimed at analysing the ECS involvement on approach and avoidance behavioural processes. To this aim, in the three selected sub-populations of mice that exhibited avoiding or balancing or approaching responses in an approach/avoidance Y-maze we analysed density and functionality of CB(1) receptors as well as enzyme fatty acid amide hydrolase activity in different brain regions, including the networks functionally responsible for emotional and motivational control. The main finding of the present study demonstrates that in both approaching and avoiding animals higher CB(1) receptor density in the amygdaloidal centro-medial nuclei and in the hypothalamic ventro-medial nucleus was found when compared with the CB(1) receptor density exhibited by the balancing animals. The characterization of the individual differences to respond in a motivationally based manner is relevant to clarify how the individual differences in ECS activity are associated with differences in motivational and affective functioning.     

Sustained Effects of Developmental Exposure to Ethanol on Zebrafish Anxiety-Like Behaviour

In zebrafish developmentally exposed to ambient ethanol (20mM-50mM) 1–9 days post fertilization (dpf), the cortisol response to stress has been shown to be significantly attenuated in larvae, juveniles and 6 month old adults. These data are somewhat at variance with similar studies in mammals, which often show heightened stress responses. To test whether these cortisol data correlate with behavioural changes in treated animals, anxiety-like behaviour of zebrafish larvae (9dpf and 10dpf) and juveniles (23dpf) was tested in locomotor assays designed to this end. In open field tests treated animals were more exploratory, spending significantly less time at the periphery of the arena. Behavioural effects of developmental exposure to ethanol were sustained in 6-month-old adults, as judged by assessment of thigmotaxis, novel tank diving and scototaxis. Like larvae and juveniles, developmentally treated adults were generally more exploratory, and spent less time at the periphery of the arena in thigmotaxis tests, less time at the bottom of the tank in the novel tank diving tests, and less time in the dark area in scototaxis tests. The conclusion that ethanol-exposed animals showed less anxiety-like behaviour was validated by comparison with the effects of diazepam treatment, which in thigmotaxis and novel tank diving tests had similar effects to ethanol pretreatment. There is thus a possible link between the hypophyseal-pituitary-interrenal axis and the behavioural actions of developmental ethanol exposure. The mechanisms require further elucidation.     

Increased fear- and stress-related anxiety-like behavior in mice lacking tuberoinfundibular peptide of 39 residues

Tuberoinfundibular peptide of 39 residues (TIP39) is synthesized by two groups of neurons, one in the subparafascicular area at the caudal end of the thalamus and the other in the medial paralemniscal nucleus within the lateral brainstem. The subparafascicular TIP39 neurons project to a number of brain regions involved in emotional responses, and these regions contain a matching distribution of a receptor for TIP39, the parathyroid hormone 2 receptor (PTH2-R). We have now evaluated the involvement of TIP39 in anxiety-related behaviors using mice with targeted null mutation of the TIP39 gene (Tifp39). Tifp39(-/-) mice (TIP39-KO) did not significantly differ from wild-type (WT) littermates in the open field, light/dark exploration and elevated plus-maze assays under standard test conditions. However, the TIP39-KO engaged in more active defensive burying in the shock-probe test. In addition, when tested under high illumination or after restraint, TIP39-KO displayed significantly greater anxiety-like behavior in the elevated plus-maze than WT. In a Pavlovian fear-conditioning paradigm, TIP39-KO froze more than WT during training and during tone and context recall but showed normal fear extinction. Disruption of TIP39 projections to the medial prefrontal cortex, lateral septum, bed nucleus of the stria terminalis, hypothalamus and amygdala likely account for the fear- and anxiety-related phenotype of TIP39-KO. Current data support the hypothesis that TIP39 modulates anxiety-related behaviors following environmental provocation.     

Risk taking during exploration of a plus-maze is greater in adolescent than in juvenile or adult mice

We investigated age-related changes in exploratory drive and anxiety in a plus-maze paradigm. We observed the behaviour of outbred CD-1 mice, Mus musculus, of both sexes at three ages: juvenile (35 days), adolescent (48 days) and adult (61 days). Juvenile and adult mice strongly avoided the open arms of the apparatus, suggesting high levels of anxiety. In contrast, adolescents spent similar amounts of time in both open and closed arms. They also entered the open arms more quickly and more often than the other age groups. No age-related differences were found in the frequency of the stretched-attend posture, a behavioural pattern considered to indicate risk assessment. The data can be interpreted in terms of either an increased exploratory drive or reduced environment-related anxiety, or both, during the adolescent period. This is consistent with previous evidence of elevated levels of novelty seeking and reduced behavioural and physiological responses to stressful situations in mice and rats around this age. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

A cautionary note regarding the use of nutritional L-tryptophan to alter aversion-related behaviour in mice

Nutritional L-tryptophan is reported to have positive effects on the behaviour of several farm animals. The present experiment describes some effects of nutritional L-tryptophan on the aversion-related behaviour of male mice selected for high litter size. The behavioural effects of L-tryptophan were observed in the elevated plus-maze, the light/dark test and a resident-intruder test. The only significant effect was observed in the resident-intruder test, where the total occurrence of exploratory behaviour by the intruder was reduced by L-tryptophan treatment. The present experiment provides a cautionary note advising against supplementation with L-tryptophan to improve animal welfare before the consequences of such treatment are fully understood.     

Pharmacological Alterations of Anxious Behaviour in Mice Depending on Both Strain and the Behavioural Situation

A previous study comparing non-emotive mice from the strain C57BL/6/ByJ with ABP/Le mice showed ABP/Le to be more anxious in an open-field situation. In the present study, several compounds affecting anxiety were assayed on ABP/Le and C57BL/6/ByJ mice using three behavioural models of anxiety: the elevated plus-maze, the light-dark discrimination test and the free exploratory paradigm. The compounds used were the full benzodiazepine receptor agonist, chlordiazepoxide, and the antagonist, flumazenil, the GABA_A antagonist, bicuculline, the full 5-HT_1A agonist 8-OH-DPAT, and the mixed 5-HT_1A/5-HT_1B agonist, RU 24969. Results showed the effect of the compounds to be dependent on both the strain and the behavioural task. Several compounds found to be anxiolytic in ABP/Le mice had an anxiogenic effect on C57BL/6/ByJ mice. More behavioural changes were observed for ABP/Le in the elevated plus-maze, but the clearest findings for C57BL/6/ByJ mice were observed in the light-dark discrimination apparatus. These data demonstrate that anxious behaviour is a complex phenomenon which cannot be described by a single behavioural task nor by the action of a single compound.     

Differences in spontaneously avoiding or approaching mice reflect differences in CB1-mediated signaling of dorsal striatal transmission

Approach or avoidance behaviors are accompanied by perceptual vigilance for, affective reactivity to and behavioral predisposition towards rewarding or punitive stimuli, respectively. We detected three subpopulations of C57BL/6J mice that responded with avoiding, balancing or approaching behaviors not induced by any experimental manipulation but spontaneously displayed in an approach/avoidance conflict task. Although the detailed neuronal mechanisms underlying the balancing between approach and avoidance are not fully clarified, there is growing evidence that endocannabinoid system (ECS) plays a critical role in the control of these balancing actions. The sensitivity of dorsal striatal synapses to the activation of cannabinoid CB1 receptors was investigated in the subpopulations of spontaneously avoiding, balancing or approaching mice. Avoiding animals displayed decreased control of CB1 receptors on GABAergic striatal transmission and in parallel increase of behavioral inhibition. Conversely, approaching animals exhibited increased control of CB1 receptors and in parallel increase of explorative behavior. Balancing animals reacted with balanced responses between approach and avoidance patterns. Treating avoiding animals with URB597 (fatty acid amide hydrolase inhibitor) or approaching animals with AM251 (CB1 receptor inverse agonist) reverted their respective behavioral and electrophysiological patterns. Therefore, enhanced or reduced CB1-mediated control on dorsal striatal transmission represents the synaptic hallmark of the approach or avoidance behavior, respectively. Thus, the opposite spontaneous responses to conflicting stimuli are modulated by a different involvement of endocannabinoid signaling of dorsal striatal neurons in the range of temperamental traits related to individual differences.     

The endocannabinoid system controls key epileptogenic circuits in the hippocampus

Balanced control of neuronal activity is central in maintaining function and viability of neuronal circuits. The endocannabinoid system tightly controls neuronal excitability. Here, we show that endocannabinoids directly target hippocampal glutamatergic neurons to provide protection against acute epileptiform seizures in mice. Functional CB1 cannabinoid receptors are present on glutamatergic terminals of the hippocampal formation, colocalizing with vesicular glutamate transporter 1 (VGluT1). Conditional deletion of the CB1 gene either in cortical glutamatergic neurons or in forebrain GABAergic neurons, as well as virally induced deletion of the CB1 gene in the hippocampus, demonstrate that the presence of CB1 receptors in glutamatergic hippocampal neurons is both necessary and sufficient to provide substantial endogenous protection against kainic acid (KA)-induced seizures. The direct endocannabinoid-mediated control of hippocampal glutamatergic neurotransmission may constitute a promising therapeutic target for the treatment of disorders associated with excessive excitatory neuronal activity.     

Effect of phenobarbitone administration to pregnant rats on anxiety in offsprings

Adults Charles-Foster rats were prenatally treated to phenobarbitone (10 mg/kg, i.p.) from day 13 to 21 of gestation, this being the critical period of neural development. Pregnant control rats were similarly treated with equal volume of vehicle. Adult rat offsprings at 8-9 weeks of age were subjected to open-field exploratory behaviour, elevated plus-maze and elevated zero-maze tests. The rat offsprings displayed significantly increased ambulation and rearings in an open-field arena when compared to control offsprings whereas self-grooming and faecal droppings remain unchanged. On elevated plus-maze test these prenatally treated rat offsprings spent significantly less time on open arms and more time and more number of entries in enclosed arms as compared to controls. Prenatally exposed rats also showed significant less time on open arms, less number of head dips and stretched attend postures on elevated zero-maze test indicating increased anxiogenic behavioural pattern in these animals. The results suggest that prenatal exposure to phenobarbitone leaves a lasting effect on the anxiety state of the offsprings.     

The cannabinoid receptor-2 is involved in allergic inflammation

AimTo investigate the role of cannabinoid receptor-2 (CB2) in allergic inflammation in CB2 knockout (CB2-KO) mice.Main methodsThe swelling reaction of the pinna to various stimuli was compared between CB2-KO and wild-type (WT) mice in terms of edema and acanthosis.Key findingsEar swelling induced by repeated application of 2,4-dinitrofluorobenzene in CB2-KO mice was significantly decreased compared with that in WT mice. In an ovalbumin model, pinna edema was significantly suppressed in CB2-KO mice in comparison with that in WT mice. The contribution of CB2 to edema was investigated in a more extreme dermatitis model using oxazolone. Delayed-type hypersensitivity reactions in this model were also suppressed in CB2-KO mice. In each of these three different allergic dermatitis models, there was a significant decrease in edema and acanthosis in CB2-KO mice compared with WT mice.SignificanceThese results clearly demonstrate that CB2 and its endogenous ligands participate not only in the acute, edematous phase of allergic dermatitis, but also in the chronic irreversible acanthosis reaction.     

Long-term isolated housing causes anxiety in C57BL/6J female mice

Behaviour of female C57BL/6J strain mice was studied in the elevated plus-maze and Porsolt's tests after either long-lasting individual housing or keeping with daily shifting group-housed females (social instability). After 2-3 months, an increased level of anxiety in the individually housed females was revealed in the elevated plus-maze. However, in 3 months the least passive behaviour in the Porsolt's was in the individually housed females. No changes were found in behaviour of females individually housed at 3 weeks of age for 4 months. Also, females with preliminary social contacts with males and following individual housing for one month had not any abnormalities in the used behavioural tests. Social instability conditions did not significantly influence the females' plus-maze behaviour, but decreased the passive behaviour in the Porsolt's test.     

Psychotropic effects of glutamic acid diethyl ester in mice

In 1 hour after intraperitoneal injection glutamic acid diethyl ester (GED) in doses 200 and 500 mg/kg decreased locomotor activity and exploratory patterns of mice in "open field" test. GED in doses 100 and 200 mg/kg diminished the immobilization period of animals in forced swimming test, that proves the reversal interaction of glutamatergic and catecholaminergic systems in CNS. Glutamate receptors antagonist--GED in doses 100-200 mg/kg disrupted passive avoidance reaction at 30 min before acquisition and retrieval, therefore glutamate receptors are involved into fixation and retrieval of memory engram.     

Role of learning of open arm avoidance in the phenomenon of one-trial tolerance to the anxiolytic effect of chlordiazepoxide in mice

A single exposure to the elevated plus-maze (EPM) test of anxiety reduces or abolishes the anxiolytic efficacy of benzodiazepines on a second trial. Some possible explanations to the occurrence of this phenomenon (one-trial tolerance-OTT) involve behavioral modifications thought to be consequence of some kind of learning in the first trial. In the present study, the influence of learning-impairing situations on the effects of the benzodiazepine chlordiazepoxide on mice re-tested in the EPM is investigated. The results showed that: (1) as expected, the administration of chlordiazepoxide to mice re-tested in the EPM- under the same conditions of the first trial- failed to induce anxiolysis; (2) a decreased percent time in the open arms was observed on the second trial of mice exposed to both trials under the same experimental conditions; (3) neither the increase in open arm avoidance by mice re-exposed to the EPM nor the OTT to chlordiazepoxide effect were modified by administration of the amnestic agent scopolamine; (4) the decrement of the duration of the first trial to 1 min or the change in light and noise conditions in both trials counteracted the increase in open arm avoidance on trial 2; (5) none of the later procedures modified the phenomenon of OTT. Although not discarding the modulation exerted by other memory processes in the OTT phenomenon, the results indicate that situations that impair the learned avoidance response to the open arms in the EPM do not modify the phenomenon of OTT.     

Omega-3 fatty acids deprivation affects ontogeny of glutamatergic synapses in rats: Relevance for behavior alterations

Essential omega-3 polyunsaturated fatty acids (ω3) are crucial to brain development and function, being relevant for behavioral performance. In the present study we examined the influence of dietary ω3 in the development of the glutamatergic system and on behavior parameters in rats. Female rats received isocaloric diets, either with ω3 (ω3 group) or a ω3 deficient diet (D group). In ontogeny experiments of their litters, hippocampal immunocontent of ionotropic NMDA and AMPA glutamatergic receptors subunits (NR2 A\B and GluR1, respectively) and the alpha isoform of the calcium-calmodulin protein kinase type II (αCaMKII) were evaluated. Additionally, hippocampal [³H]glutamate binding and uptake were assessed. Behavioral performance was evaluated when the litters were adult (60 days old), through the open-field, plus-maze, inhibitory avoidance and flinch-jump tasks. The D group showed decreased immunocontent of all proteins analyzed at 02 days of life (P2) in comparison with the ω3 group, although the difference disappeared at 21 days of life (except for αCaMKII, which content normalized at 60 days old). The same pattern was found for [³H]glutamate binding, whereas [³H]glutamate uptake was not affected. The D group also showed memory deficits in the inhibitory avoidance, increased in the exploratory pattern in open-field, and anxiety-like behavior in plus-maze. Taken together, our results suggest that dietary ω3 content is relevant for glutamatergic system development and for behavioral performance in adulthood. The putative correlation among the neurochemical and behavioral alterations caused by dietary ω3 deficiency is discussed.     

The role of vision and proprioception in the aversion of rats to the open arms of an elevated plus-maze

The elevated plus-maze test is usually run with a short edge surrounding the open arms in order to prevent the rats from falling. The present experiment investigated the role of transparent edges differing in heights: 1 (used as control), 5, 10, 20 and 40 cm, the latter the same height as the closed arm walls. Additionally, this 40-cm high transparent edge was also studied covered by white translucent or black opaque paper. The data show that the time spent in the open arms was significantly greater when the edge height was 5, 10 or 40 cm covered by the white or black paper. However, there were no differences from the 1-cm control edge when the height was 40 cm transparent. A similar effect was observed when entries in the open arms and total entries were analyzed. The facts that there were no differences when the open arms were surrounded by 1- or 40-cm transparent edges (which allow thigmotaxis) and that the same 40-cm edge caused increases in exploratory behavior when covered by papers indicate that vision triggers aversion to open spaces.