Social context increases ultrasonic vocalizations during restraint in adult mice.

Adult mice emit many ultrasonic vocalizations (USVs) during social interaction tasks, but only a few studies have yet reported USVs in stressed adult mice. Our aim was to study which experimental conditions favor USV emission during behaviors associated with different emotional states. As USVs likely mediate social communication, we hypothesized that temporary social isolation followed by exposure to a novel social congener would promote USV emission. USVs were recorded in three different behavioral paradigms: restraint, free moving in a new environment, and during a social interaction task. We compared USV emission, with or without the presence of a social congener, in animals socially isolated during different periods (0, 6 or 21 days). Social isolation decreased the number of USVs during free moving, whereas it increased during restraint. During the social interaction task, animals produced high-frequency USVs (median: 72.6 kHz, 25-75% range: 67.6-78.2 kHz), especially when the social partner was active and social motivation was high. During restraint, presence of a social congener increased the call rate of low-frequency USVs (median: 52.4 kHz, 25-75% range: 44.8-56.5 kHz). USV frequency followed two unimodal distributions that distinguished low-frequency USVs (≤ 60 kHz) mainly emitted during free-moving (90.9% of total USVs) and restraint (93.1%) conditions, from high-frequency USVs (> 60 kHz) mainly emitted during the social interaction task (85.1% of total USVs). The present study confirms that USV call rate and frequency depend on behavioral states, and provides evidence that the presence of a congener promotes ultrasonic vocalizations in restrained adult mice.

Beta2-containing neuronal nicotinic receptors as major actors in the flexible choice between conflicting motivations.

Beside a critical role in nicotine addiction, the role of nicotinic receptors in cognitive or emotional processes remains difficult to elucidate, mostly because of a lack of specificity of compounds and because they up or down regulate easily. Using knockout mice may be one key to elucidate the role of nicotinic receptors stimulated by their endogenous ligand acetylcholine. We and others have previously explored the behaviour of mice knockout for the beta2-subunit containing nicotinic receptor - ß2*nAChRs - ß2(-/-) mice. These mice exhibit a particular kind of hyperactive locomotion, with profound deficits in cognitive and social interaction tasks, only when they have to show flexible choices. We wonder here whether the latter is due to a lack of motor control - i.e. motor impulsivity, a lack of estimation of reward value - i.e. cognitive impulsivity, and/or a lack of appropriate ranking or choice between different motivations. We designed behavioural tasks allowing the study of these distinct processes in mice. Our current results highlight the important role of ß2*nAChRs in flexible behaviours in conflicting situations, such as social contact, spatial exploration and food consumption. They also show that the cognitive deficits exhibited by ß2(-/-) mice cannot be explained by impaired inhibitory behaviours. Although social cognition is considerably enriched in humans as compared to rodents, we provide here novel data for the neurobiology of flexible social behaviours that could ultimately be useful for humans. Indeed, the ability to show flexible behaviours and to display adapted social interactions is profoundly impaired in a myriad of psychiatric disorders.

Aggressive behavior during social interaction in mice is controlled by the modulation of tyrosine hydroxylase expression in the prefrontal cortex.

The Balb/c strain and the C57BL/6 strain show constitutive differences for tyrosine hydroxylase expression, and noradrenaline (NA) prefrontal transmission. Male mice of these strains also show striking differences in social interaction behaviors, with an increased aggressiveness for the Balb/c strain. To test a potential link between these neurobiological and behavioral parameters, we evaluated the behavioral effects of chronic treatment of mice with BC19, a noreburnamine compound previously known as RU24722, found to modify cell organisation, tyrosine hydoxylase (TH) expression, and its activity into the locus coeruleus (LC). We compared the pharmacological effects between the two strains in social behaviors. Our results show that the emergence of additional TH-expressing (TH+) neurons in the rostral part of the LC of Balb/c mice was associated with an increase in the density of TH+ and noradrenergic (NA+) fibers in the molecular layer in the cingular (Cg1) and prelimbic (PrL) parts of the prefrontal cortex (PFC). BC19 treatment resulted in the near-equalization of the LC number of TH+ neurons and of the density of TH+ and NA+ fibers between both strains. The aggressiveness in Balb/c mice was considerably diminished by BC19 treatment, while the originally non aggressive behavior of C57Bl/6 mice was much less affected by BC19 treatment, despite a moderate increase in some offensive behaviors. In additional control experiments, we checked the effect of BC19 on a separate test for anxiety and assessed the effect of noradrenergic N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) mediated lesions in C57BL/6 mice on social behaviors. In the present study we show that the BC19 effect in Balb/c mice was independent of anxiety as measured in the light/dark test and that DSP-4 lesions in C57BL/6 mice produced a robust increase in aggressive social interaction. Altogether, these results show that the noradrenergic system, and particularly its projections to the PFC, strongly modulates aggressive behaviors.

Brain activation by short-term nicotine exposure in anesthetized wild-type and beta2-nicotinic receptors knockout mice: a BOLD fMRI study.

RATIONALE: The behavioral effects of nicotine and the role of the beta2-containing nicotinic receptors in these behaviors are well documented. However, the behaviors altered by nicotine rely on the functioning on multiple brain circuits where the high-affinity beta2-containing nicotinic receptors (beta2*nAChRs) are located. OBJECTIVES: We intend to see which brain circuits are activated when nicotine is given in animals naïve for nicotine and whether the beta2*nAChRs are needed for its activation of the blood oxygen level dependent (BOLD) signal in all brain areas. MATERIALS AND METHODS: We used functional magnetic resonance imaging (fMRI) to measure the brain activation evoked by nicotine (1 mg/kg delivered at a slow rate for 45 min) in anesthetized C57BL/6J mice and beta2 knockout (KO) mice. RESULTS: Acute nicotine injection results in a significant increased activation in anterior frontal, motor, and somatosensory cortices and in the ventral tegmental area and the substantia nigra. Anesthetized mice receiving no nicotine injection exhibited a major decreased activation in all cortical and subcortical structures, likely due to prolonged anesthesia. At a global level, beta2 KO mice were not rescued from the globally declining BOLD signal. However, nicotine still activated regions of a meso-cortico-limbic circuit likely via alpha7 nicotinic receptors. CONCLUSIONS: Acute nicotine exposure compensates for the drop in brain activation due to anesthesia through the meso-cortico-limbic network via the action of nicotine on beta2*nAChRs. The developed fMRI method is suitable for comparing responses in wild-type and mutant mice.

Targeted in vivo expression of nicotinic acetylcholine receptors in mouse brain using lentiviral expression vectors.

Nicotinic acetylcholine receptors (nAChRs) in the brain exhibit diverse functional properties and ubiquitous distribution. Yet, except for providing a receptor for the exogenously applied nicotine of tobacco products, their role in the normal functioning of the brain has remained elusive. We have used a lentiviral expression vector to re-express the beta2 subunit specifically in the ventral tegmental area (VTA) of beta2-/- mice. The viral vector efficiently expresses beta2- subunit protein leading to new nAChR-binding sites. VTA neurons transduced by the lentiviral vector are responsive to intravenous nicotine when analyzed using in vivo electrophysiology. Nicotine-induced dopamine release from the nucleus accumbens (NuAcc) was also restored in re-expressing beta2-/- mice. Intra-VTA injection of nicotine was found to be reinforcing in both wild-type and beta2-subunit re-expressing beta2-/- mice, but not in beta2-/- mice. Furthermore, in the absence of applied nicotine, the spontaneous slow exploratory behavior of the mice was restored, whereas fast navigation did not change. This latter behavioral analysis suggests a role for beta2* nAChR, specifically expressed in the VTA, in mammalian cognitive function.

Genetic dissociation of two behaviors associated with nicotine addiction: beta-2 containing nicotinic receptors are involved in nicotine reinforcement but not in withdrawal syndrome.

RATIONALE: Nicotine addiction is characterized by two distinct behaviors, chronic compulsive self-administration and the induction of a withdrawal syndrome upon cessation of nicotine consumption. OBJECTIVE: To examine if these two processes rely on beta2-containing nicotinic receptors--beta2*nAChRs--we analyzed the behavior of mice lacking these receptors in the two situations. RESULTS: First, we showed that, in contrast to wild-type (WT) mice, beta2-knockout (beta2-/-) mice exhibit no intra-ventral tegmental area (VTA) nicotine self-administration, whereas their ability to self-administer morphine is intact. However, beta2-/- mice showed some sensitivity to locomotor effects of nicotine, implying an effect of the drug on other nicotinic subtypes. Then, we observed that beta2-/- mice exhibited a normal nicotine withdrawal syndrome, i.e., increased levels of rearing and jumping upon precipitated withdrawal. Thus, the beta2*nAChRs are not involved in the behaviors induced by cessation of nicotine consumption. CONCLUSION: Taken together, the present data demonstrated a genetic dissociation of two distinct behavioral patterns associated with nicotine addiction. They further suggested that independent molecular mechanisms underlie these two aspects, offering the possibility of controlling them separately.

Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors.

Worldwide, 100 million people are expected to die this century from the consequences of nicotine addiction, but nicotine is also known to enhance cognitive performance. Identifying the molecular mechanisms involved in nicotine reinforcement and cognition is a priority and requires the development of new in vivo experimental paradigms. The ventral tegmental area (VTA) of the midbrain is thought to mediate the reinforcement properties of many drugs of abuse. Here we specifically re-expressed the beta2-subunit of the nicotinic acetylcholine receptor (nAChR) by stereotaxically injecting a lentiviral vector into the VTA of mice carrying beta2-subunit deletions. We demonstrate the efficient re-expression of electrophysiologically responsive, ligand-binding nicotinic acetylcholine receptors in dopamine-containing neurons of the VTA, together with the recovery of nicotine-elicited dopamine release and nicotine self-administration. We also quantified exploratory behaviours of the mice, and showed that beta2-subunit re-expression restored slow exploratory behaviour (a measure of cognitive function) to wild-type levels, but did not affect fast navigation behaviour. We thus demonstrate the sufficient role of the VTA in both nicotine reinforcement and endogenous cholinergic regulation of cognitive functions.

Individual differences in prefrontal cortical activation on the Tower of London planning task: implication for effortful processing.

Solving challenging ('effortful') problems is known to involve the dorsal and dorsolateral prefrontal cortex in normal volunteers, although there is considerable individual variation. In this functional magnetic resonance imaging study, we show that healthy subjects with different levels of performance in the Tower of London planning task exhibit different patterns of brain activation. All subjects exhibited significant bilateral activation in the dorsolateral prefrontal cortex, the anterior and posterior cingulate areas and the parietal cortex. However, 'standard performers' (performance < 70% correct) and 'superior performers' (performance >70% correct) differed in the patterns of activation exhibited. Superior performers showed a significantly more spatially extended activation in the left dorsolateral prefrontal cortex than did standard performers, whereas the latter group tended to show increased activation of the anterior cingulate region.

The lipase C-terminal domain. A novel unusual inhibitor of pancreatic lipase activity.

In vertebrates, dietary fat digestion mainly results from the combined effect of pancreatic lipase, colipase, and bile. It has been proposed that in vivo lipase adsorption on oil-water emulsion is mediated by a preformed lipase-colipase-mixed micelle complex. The main lipase-colipase binding site is located on the C-terminal domain of the enzyme. We report here that in vitro the isolated C-terminal domain behaves as a potent noncovalent inhibitor of lipase and that the inhibitory effect is triggered by the presence of micelles. Lipase inhibition results from the formation of a nonproductive C-terminal domain-colipase-micelle ternary complex, which competes for colipase with the active lipase-colipase-micelle ternary complex, thus diverting colipase from its lipase-anchoring function. The formation of such a complex has been evidenced by molecular sieving experiments. This nonproductive complex lowers the amount of active lipase thus reducing lipolysis. Preliminary experiments performed in rats show that the C-terminal domain also behaves as an inhibitor in vivo and thus could be considered a potential new tool for specifically reducing intestinal lipolysis.

Enhanced and impaired attentional performance after infusion of D1 dopaminergic receptor agents into rat prefrontal cortex.

The role in spatial divided and sustained attention of D1 and D2-like dopamine (DA) receptors in the rat prelimbic medial prefrontal cortex (mPFC) was investigated in a five-choice serial reaction time task. Rats were trained to detect brief flashes of light (0.5-0.25 sec) presented randomly in a spatial array of five apertures. When performance stabilized, animals received bilateral microinfusions of either the D1 DA receptor antagonist SCH 23390, the D1 DA receptor agonist SKF 38393, or the D2 DA antagonist sulpiride into the mPFC. Rats were divided into two groups, with low (<75% correct) and high (>75%) baseline levels of accuracy. Infusions of the D2 receptor antagonist sulpiride had no significant effect on any task variable. SCH 23390 (0.3 microg) selectively impaired the accuracy of attentional performance in rats in the high baseline condition. By contrast, SKF 38393 (0.06 microg) enhanced the accuracy of attentional performance in the low baseline condition, a lower dose (0.03 microg) also increasing the speed of making correct responses. Finally, the beneficial effects of SKF-383893 on choice accuracy were antagonized by SCH 23390 (1.0 microg). The results provide apparently the first demonstration of enhanced cognitive function after local administration of a D1 receptor agonist to the mPFC and suggest dissociable roles of D1 and D2 DA receptors of the mPFC in modulating attentional function.

Evidence for the involvement of the rat prefrontal cortex in sustained attention.

Previous studies suggest that, in both humans and rats, the prefrontal cortex (PFC) is involved in both selective and divided attention. We have also shown that the PFC is involved in response selection and that its involvement is modulated by the cognitive effort required by the task. However, the role of the PFC is much less clear when no response selection is required. The purpose of the present experiments was to assess the role of the PFC in attentional functions with a low response-selection demand. We used two tasks in which information processing was effortful but where the demand on a response selection process is low. Moreover, we assessed two different types of visual attentional functions: selective attention (Experiment 1) and sustained attention (Experiment 2). The results showed a differential involvement for the PFC in the two tasks. Selective attention was not impaired by prefrontal lesions when the number of possible positions for the stimulus on which the subjects must focus was restricted to two (Experiment 1). In contrast, prefrontal rats were unable to sustain their attention long enough to detect, and react to, subtle variations in brightness (Experiment 2). This results suggests a dissociation between different types of attentional functions depending upon the integrity of the PFC. More specifically, results in Experiment 2 suggest an involvement of the PFC in sustained attention. Finally, the overall results show that even in tasks involving low demands on response selection the PFC is involved in attentional functions.

Neural systems underlying arousal and attention. Implications for drug abuse.

The monoaminergic and cholinergic systems are implicated in different forms of behavioral arousal that can be dissected in terms of their forebrain targets and the nature of the behavioral processes they modulate in distinct regions. Thus, evidence in rats with selective neurochemical manipulations tested behaviorally using an analog of an attentional task developed for human subjects indicates that the coeruleo-cortical noradrenergic system is implicated in divided and selective attention, the basal forebrain cholinergic system in stimulus detection, the mesostriatal and mesolimbic dopaminergic systems in response speed and vigor, and the mesencephalic serotoninergic or 5-HT systems in response inhibition. Our recent studies have focused on fractionating, in the same task, the differential contributions of the dorsal and median raphé 5-HT systems as well as elucidating the functions of the mesocortical dopaminergic system, each of which may be relevant to understanding the behavioral and cognitive sequelae of cocaine administration in human subjects as well as in experimental animals.

Neural Systems Underlying Arousal and Attention: Implications for Drug Abusea.

The monoaminergic and cholinergic systems are implicated in different forms of behavioral arousal that can be dissected in terms of their forebrain targets and the nature of the behavioral processes they modulate in distinct regions. Thus, evidence in rats with selective neurochemical manipulations tested behaviorally using an analog of an attentional task developed for human subjects indicates that the coeruleo-cortical noradrenergic system is implicated in divided and selective attention, the basal forebrain cholinergic system in stimulus detection, the mesostriatal and mesolimbic dopaminergic systems in response speed and vigor, and the mesencephalic serotoninergic or 5-HT systems in response inhibition. Our recent studies have focused on fractionating, in the same task, the differential contributions of the dorsal and median raphé 5-HT systems as well as elucidating the functions of the mesocortical dopaminergic system, each of which may be relevant to understanding the behavioral and cognitive sequelae of cocaine administration in human subjects as well as in experimental animals.

Effortful information processing in a spontaneous spatial situation by rats with medial prefrontal lesions.

Previous research has suggested that the rat prefrontal cortex might play a role in spatial information processing and in divided attention. More recent work showed that the effect of prefrontal lesions is more important when the task involves response selection in complex situations. The first aim of the present study was to test the effect of lesions of the prelimbic area of the rat prefrontal cortex in spatial exploration, a situation involving the processing of spatial and non-spatial information, but requiring no response selection. The second aim was to manipulate the degree of cognitive effort required by the task. The latter effect was tested by manipulating the number of items to explore. Rats explored either a simple (3 objects) or a complex (6 objects) situation. We reasoned that acquiring spatial information so as to react adequately to spatial or non spatial changes involved more effortful processing in the complex situation than in the simpler one. The results suggest that the medial prefrontal cortex is not crucially involved in effortful processing when the task requires no response selection.

Recombinant C-terminal domain of pancreatic lipase retains full ability to bind colipase.

The organization of the pancreatic lipase in two well defined domains has been correlated to a specific function for each domain, catalytic activity for the N-terminal domain and colipase binding for the C-terminal domain. In order to see if such an organization implies that the two domains can behave as separate entities, we expressed the N- and C-terminal domains in insect cells. The recombinant proteins secreted in the cell supernatants present the expected molecular properties. However, whereas the C-terminal domain retains its function of colipase binding, the N-terminal domain appears to be unable to ensure catalysis. The lack of activity of the recombinant N-terminal domain could result either from a (partially) incorrect folding or from an incapacity to function by itself. These results suggest that, although both are structurally well defined, the two domains of the pancreatic lipase behave differently when they are expressed as separate entities.

Effects of limitations on the use of some visual and kinaesthetic information in spatial mapping during exploration in the rat.

The purpose of this experiment was to study the effects of limiting visual and/or locomotor access to a part of the environment in the building up of a spatial representation of the whole space. During five sessions, rats were allowed to explore separately and successively the two halves (subspaces) of a circular open field containing four objects. During exploration of each half, continuous or discontinuous locomotor and/or visual access to the other half was provided by using opaque or transparent partitions, with or without doors. Once habituation was complete, the partition was removed for some subjects but remained for others. The locomotor and exploratory reactions to this removal were recorded. Whatever their locomotor experience (continuous or discontinuous), rats that had a discontinuous visual experience between the subspaces displayed a renewal of exploratory activity, whereas the rats that had received a continuous visual experience did not re-explore the objects. This result suggests that continuous visual access to the whole space is necessary for the construction of an overall representation. Furthermore, continuous locomotor activity does not seem to compensate for the discontinuity of visual information.

Crystallographic study of a cleaved, non-activatable form of porcine zymogen E.

The crystal structure of a cleaved form of porcine zymogen E has been solved by molecular replacement using the bovine procarboxypeptidase A-S6 subunit III structure as search model. Crystallographic refinement using simulated annealing and energy minimization techniques resulted in a final R-factor of 0.189 for all data between 8 and 2.3 A resolution. The zymogen E three-dimensional model is very close to that of bovine subunit III and represents the second member of the zymogen E family for which the crystal structure is known. The two structures indicate that, in contrast to trypsinogen and chymotrypsinogen, zymogens of this family are highly organized molecules. The amino acid sequence of zymogen E has only been determined for the first 40 residues. Based on the electron density map, we have introduced six sequence changes relative to subunit III. Out of the 11 residues in the activation peptide, only the first six present well matching electron density; they are connected to the rest of the zymogen by an unexpected Cys1-Cys122 disulphide bridge (according to the bovine chymotrypsinogen A numbering system). Amino acid sequencing of protein solutions both from dissolved crystals and from the initial stock clearly indicated that the Val17-Asn18 bond had been cleaved during the crystallization process. This result adds weight to the assumption that the autolysis of the bovine zymogen E gives rise to subunit III and that this maybe a regulatory mechanism for protease E activity.

Medial prefrontal lesions in the rat and spatial navigation: evidence for impaired planning.

Rats with medial prefrontal cortical lesions were tested in a modified water maze navigation task. In Stage 1, the rats were trained to locate a hidden platform from a single start location. They were then subjected to a series of trials during which a second start position was used (Stage 2). In Stage 3, the rats had to navigate to a new goal location from the 2 experienced start positions. Stage 4 required the rats to navigate to the same goal as in Stage 3, starting from 4 distinct positions. Finally, a single probe trial with no platform was conducted. Rats with prefrontal lesions were impaired only during Stage 4. This deficit was specific to the 2 start positions newly introduced during this stage, suggesting a dysfunction of planning processes. This impairment might result from a working memory deficit, precluding the animal from forming an adequate representation of the whole course of movements required to reach the platform.

Nicotinic and muscarinic receptors in the rat prefrontal cortex: differential roles in working memory, response selection and effortful processing.

The aim of the present study was to evaluate the effects of cholinergic receptor blockade in the rat prefrontal cortex on cognitive processes. The nicotinic antagonists neuronal bungarotoxin and dihydro-beta-erythroidine and the muscarinic antagonist scopolamine were injected into the prelimbic area of the prefrontal cortex. Their behavioural effects were assessed in a T-maze to test reference memory (visual discrimination task) and working memory in delayed matching (MTS) and non-matching to sample (NMTS) tasks. Neuronal bungarotoxin produced a significant decrease in working memory performance in the MTS task but not in the NMTS task. In contrast, scopolamine impaired working memory in both MTS and NMTS tasks. Reference memory was not altered by any of the cholinergic antagonists. These results demonstrate a differential role of nicotinic and muscarinic receptors in the rat prefrontal cortex. Nicotinic transmission appears to be important in delayed response tasks requiring effortful processing for response selection, while the muscarinic system is involved in general working memory processes.

Working memory, response selection, and effortful processing in rats with medial prefrontal lesions.

This study examined the effects of lesions of the prelimbic area of the rat prefrontal cortex on acquisition and retention of nonmatching (NMTS) and matching-to-sample (MTS) tasks. Both tasks involved a reference and a working memory component, but only working memory was impaired by the lesions. A comparison of the 2 tasks revealed quantitatively similar deficits in postoperatively trained rats. In preoperatively trained rats, however, the deficits were more important in the MTS task than in the NMTS task. In addition, an effect of interference between successive trials was observed in the NMTS task but not in the MTS task. Perseverative tendencies were observed in the MTS task only. These results suggest that prefrontal lesions induce working memory deficits as a result of poor temporal encoding and increased susceptibility to interference and impair effortful processing, such as that engaged in response selection mechanisms.