Monogenic mouse models of social dysfunction: implications for autism.

Autism is a pervasive disorder characterized by a complex symptomatology, based principally on social dysfunction. The disorder has a highly complex, largely genetic etiology, involving an impressive variety of genes, the precise contributions of which still remain to be determined. For this reason, a reductionist approach to the study of autism has been proposed, employing monogenic animal models of social dysfunction, either by targeting a candidate gene, or by mimicking a single-gene disorder characterized by autistic symptoms. In the present review, we discuss this monogenic approach by comparing examples of each strategy: the mu opioid receptor knock-out (KO) mouse line, which targets the opioid system (known to be involved in the control of social behaviors); and the Fmr1-KO mouse, a model for Fragile X syndrome (a neurodevelopmental syndrome that includes autistic symptoms). The autistic-relevant behavioral phenotypes of the mu-opioid and Fmr1-KO mouse lines are described here, summarizing previous work by our research group and others, but also providing novel experimental evidence. Relevant factors influencing the validity of the two models, such as sex differences and age at testing, are also addressed, permitting an extensive evaluation of the advantages and limits of monogenic mouse models for autism.

Modeling socially anhedonic syndromes: genetic and pharmacological manipulation of opioid neurotransmission in mice.

Social anhedonia, or the diminished capacity to experience pleasure and reward from social affiliation, is a major symptom of different psychiatric disorders, including some forms of infantile autism and schizophrenia spectrum disorders. The brain opioid hypothesis of social attachment is a promising model for achieving insights into how neurobiological and developmental factors contribute to the regulation of social reward. In this study, genetic knocking-out and naltrexone (NTRX) treatment during the first 4 days of life were used to disrupt opioid neurotransmission in mouse pups and their attachment relationships with the mother. Both permanent (genetic) and transient (pharmacological) manipulations of opioid neurotransmission exerted long-term effects on social affiliation. When juveniles, both µ-opioid receptor knockout mice and NTRX-treated pups showed reduced interest in peers and no preference for socially rewarding environment. These results demonstrate that sociability in juvenile mice is highly dependent on the establishment during infancy of a positive affective relationship with their mothers and that opioid neurotransmission has a major role in the regulation of social hedonic capacity. If the validity of this animal model will be confirmed by future research, translational studies focusing on the interaction between early experience and opioid neurotransmission could provide useful insights for identifying endophenotypes of human psychiatric disorders associated with social anhedonia.

Implication of the VGF-derived peptide TLQP-21 in mouse acute and chronic stress responses.

The impact of stress is widely recognized in the etiology of multiple disorders. In particular, psychological stress may increase the risk of cardiovascular, metabolic, immune, and mood disorders. Several genes are considered potential candidates to account for the deleterious consequences of stress and recent data point to role of Vgf. VGF mRNA is abundantly expressed in the hypothalamus, where it has been involved in metabolism and energy homeostasis; more recently a link between VGF-derived peptides and mood disorders has been highlighted. The following experiments were performed to address the contribution of the VGF-system to stress induced changes in mice: the distribution of VGF immuno-reactivity in hypothalamic nuclei and its modulation by social stress; the role of VGF-derived peptide TLQP-21 in plasma catecholamine release induced by acute restraint stress (RS); the efficacy of chronic TLQP-21 in a mouse model of chronic subordination stress (CSS). VGF fibers were found in high density in arcuate, dorsomedial, and suprachiasmatic and, at lower density, in lateral, paraventricular, and ventromedial hypothalamic nuclei. Central administration of either 2 or 4 mM TLQP-21 acutely altered the biphasic serum epinephrine release and decreased norepinephrine serum levels in response to RS. Finally, 28-day of 40 µg/day TLQP-21 treatment increased CSS-induced social avoidance of an unfamiliar conspecific. Overall these data support a role for TLQP-21 in stress responses providing a promising starting point to further elucidate its role as a player in stress-related human pathologies.

Postnatal handling reverses social anxiety in serotonin receptor 1A knockout mice.

Mice lacking the serotonin receptor 1A (Htr1a knockout, Htr1a(KO)) show increased innate and conditioned anxiety. This phenotype depends on functional receptor activity during the third through fifth weeks of life and thus appears to be the result of long-term changes in brain function as a consequence of an early deficit in serotonin signaling. To evaluate whether this phenotype can be influenced by early environmental factors, we subjected Htr1a knockout mice to postnatal handling, a procedure known to reduce anxiety-like behavior and stress responses in adulthood. Offspring of heterozygous Htr1a knockout mice were separated from their mother and exposed 15 min each day from postnatal day 1 (PD1) to PD14 to clean bedding. Control animals were left undisturbed. Maternal behavior was observed during the first 13 days of life. Adult male offspring were tested in the open field, social approach and resident-intruder tests and assessed for corticosterone response to restraint stress. Knockout mice showed increased anxiety in the open field and in the social approach test as well as an enhanced corticosterone response to stress. However, while no effect of postnatal handling was seen in wild-type mice, handling reduced anxiety-like behavior in the social interaction test and the corticosterone response to stress in knockout mice. These findings extend the anxiety phenotype of Htr1a(KO) mice to include social anxiety and demonstrate that this phenotype can be moderated by early environmental factors.

Chronic administration of olanzapine affects Behavioral Satiety Sequence and feeding behavior in female mice.

The aim of the study was to assess the effects of chronic olanzapine (Ola) administration on feeding behavior. Although atypical antipsychotics (AAPs) have greatly improved the management of schizophrenia and extrapyramidal symptoms, substantial bodies of literature point out that most of these agents are highly related to a major risk of metabolic drawbacks, leading to dyslipidemia and obesity. Among these compounds, Ola is one of the more weight gain-inducing AAPs. In the present study, we analyzed the Behavioral Satiety Sequence (BSS) in female mice given a palatable diet (wet mash) and chronically administered Ola (0.75, 1.5, 3 mg/kg per os) for 36 days. The results showed that administration of the highest dose of Ola postponed the onset of satiation, as suggested by the rightward shift of the BSS. This effect was confirmed by an increase in the actual food intake by the Ola (3 mg/kg) mice. These results suggest that one of the possible mechanisms involved in AAPinduced weight gain is alteration of the hunger-satiety regulation in female mice. These findings are consistent with the hypothesis that enhanced food intake and diminished central sensitivity to satiation signaling may cooperate in promoting weight gain and metabolic dysregulation in rodents and patients taking antipsychotic medications.

Interaction with stressed mothers affects corticosterone levels in pups after reunion and impairs the response to dexamethasone in adult mice.

BACKGROUND: Early adverse experiences are preeminent factors for the development of affective disorders. In the present study, we analyzed the effects of different postnatal manipulations applied either on the mother or on the offspring in mice. Maternal behavior and adrenocortical activity of both mothers and offspring at the end of postnatal stress and at adulthood were considered. METHODS: From postnatal day (PND) 1 to 14 mice underwent 15min of: (a) brief (15min) pups' exposure to clean bedding (CB: clean bedding), (b) mothers' exposure to the odor of a novel male (SM: stressed mother) or (c) mothers' exposure to a clean cage (CSM: control stressed mother), and (d) standard rearing (N-H: non-handled). The behavior of mouse dams during and after stress sessions was analyzed. Serum corticosterone of mothers and pups at the end of the stress session and 30min after reunion was assessed on PND 14. Moreover, anxiety levels and HPA-axis inhibitory feedback in response to dexamethasone administration were evaluated in adult male offspring. RESULTS: Overall, during the 14 days of treatment CB mothers when reunited with their pups showed higher maternal behavior than other dams. After the last stress (PND 14) SM and CSM maternal corticosterone levels increased as well as those of CB pups. While 30min of mother-infant interaction restored baseline corticosterone levels in SM and CSM mothers and in CB pups, SM and CSM offspring showed a decrease of corticosterone under baseline levels. At adulthood, SM and CSM males did not show the suppressive hormonal response to dexamethasone treatment. Moreover, adult CB and SM male mice displayed decreased anxiety in the open field. CONCLUSIONS: Maternal psychosocial stress during lactation seems to permanently affect the offspring's HPA functioning. These effects may be dissociated from the behavioral response as suggested by the decrease of anxiety in SM and CB adult mice.

TLQP-21, a VGF-derived peptide, increases energy expenditure and prevents the early phase of diet-induced obesity.

The vgf gene has been identified as an energy homeostasis regulator. Vgf encodes a 617-aa precursor protein that is processed to yield an incompletely characterized panel of neuropeptides. Until now, it was an unproved assumption that VGF-derived peptides could regulate metabolism. Here, a VGF peptide designated TLQP-21 was identified in rat brain extracts by means of immunoprecipitation, microcapillary liquid chromatography-tandem MS, and database searching algorithms. Chronic intracerebroventricular (i.c.v.) injection of TLQP-21 (15 mug/day for 14 days) increased resting energy expenditure (EE) and rectal temperature in mice. These effects were paralleled by increased epinephrine and up-regulation of brown adipose tissue beta2-AR (beta2 adrenergic receptor) and white adipose tissue (WAT) PPAR-delta (peroxisome proliferator-activated receptor delta), beta3-AR, and UCP1 (uncoupling protein 1) mRNAs and were independent of locomotor activity and thyroid hormones. Hypothalamic gene expression of orexigenic and anorexigenic neuropeptides was unchanged. Furthermore, in mice that were fed a high-fat diet for 14 days, TLQP-21 prevented the increase in body and WAT weight as well as hormonal changes that are associated with a high-fat regimen. Biochemical and molecular analyses suggest that TLQP-21 exerts its effects by stimulating autonomic activation of adrenal medulla and adipose tissues. In conclusion, we present here the identification in the CNS of a previously uncharacterized VGF-derived peptide and prove that its chronic i.c.v. infusion effected an increase in EE and limited the early phase of diet-induced obesity.

Pain sensitivity in mice lacking the Ca(v)2.1alpha1 subunit of P/Q-type Ca2+ channels.

The role of voltage-gated Ca(2+) (Ca(V)) channels in pain mechanisms has been the object of intense investigation using pharmacological approaches and, more recently, using mutant mouse models lacking the Ca(V)alpha(l) pore-forming subunit of N-, R- and T-type channels. The role of P/Q-type channels in nociception and pain transmission has been investigated by pharmacological approaches but remains to be fully elucidated. To address this issue, we have analyzed pain-related behavioral responses of null mutant mice for the Ca(V)2.1alpha(1) subunit of P/Q-type channels. Homozygous null mutant Ca(V)2.1alpha(1)-/- mice developed dystonia at 10-12 days after birth and did not survive past weaning. Tested at ages where motor deficit was either absent or very mild, Ca(V)2.1alpha(1)-/- mice showed reduced tail withdrawal latencies in the tail-flick test and reduced abdominal writhes in the acetic acid writhing test. Adult heterozygous Ca(V)2.1alpha(1)+/- mice did not show motor deficits in the rotarod and activity cage tests and did not show alterations in pain responses in the tail-flick test and the acetic acid writhing test. Strikingly, they showed a reduced licking response during the second phase of formalin-induced inflammatory pain and a reduced mechanical allodynia in the chronic constriction injury model of neuropathic pain. Our findings show that P/Q-type channels play an antinociceptive role in sensitivity to non-injurious noxious thermal stimuli and a pronociceptive role in inflammatory and neuropathic pain states, pointing to an important role of Ca(V)2.1 channels in central sensitization.

Psychosocial stress affects energy balance in mice: modulation by social status.

Stress has been associated with changes in eating behaviour and food preferences. Moreover, psychosocial and socio-economical challenges have been related with neuroendocrine-autonomic dysregulation followed by visceral obesity and associated risk factors for disease. In the current study, we provide a model of body weight development, food intake, energy expenditure of subordinate and dominant mice under psychosocial stress either in the presence of a standard diet or of a high palatable diet. When only standard chow was available stressed animals consumed more food in comparison to the control counterpart. Moreover, subordinate mice, at the end of the stress period were heavier in comparison to dominant animals. This last result was due to a decrease in the caloric efficiency of dominant animals in comparison to subordinates. Confirming this, the results of the experiment 2 showed that dominant mice significantly increase their energy expenditure at the end of the chronic psychosocial stress procedure in comparison to subordinate mice, as measured by indirect calorimetry. When a palatable high fat diet was available subordinate animals became heavier in comparison with both dominant and control animals. No differences in the caloric intake were found between groups. Subordinate mice ingested more calories from fat than controls, while dominant animals ingested more calories from carbohydrates. These results suggest that psychosocial stress can be a risk factor for overeating and weight gain in mice. However, social status influences the extent to which an individual keeps up with adverse environment, influencing the vulnerability toward stress related disorders.

Postnatal stress in mice: does "stressing" the mother have the same effect as "stressing" the pups?

Short- and long-term effects of brief maternal separation, maternal exposure to novel male odor, and standard rearing were compared in NMRI mice. The first condition consisted of 15 min of daily exposure of pups to clean bedding (CB), and the second condition consisted of 15 min of mothers' exposure to the odor of strange males (SM), for 14 days after birth starting from postnatal Day 1. Thus, both conditions entailed the same period of maternal separation. A control mother-offspring group was left undisturbed (nonhandled, N-H). Corticosterone levels of mothers and pups were measured at the end of the last manipulation session. Corticosterone levels were higher in SM mothers, differing from both those of CB and of control dams; CB pups showed the highest corticosterone levels in comparison with the pups belonging to the other groups. Maternal behavior observed as furthest as possible from the daily separation session did not differ among the three groups. The behavioral response to 0.5 mg/kg of apomorphine in 15-day-old pups was enhanced in both CB and SM animals, which suggests an alteration of dopaminergic functioning. Finally, adult CB and SM male mice showed an increase in the percentage of time and entries into the open arms of the plus-maze in comparison to nonhandled males. This study indicates that exposure to ecologically relevant stimuli elicited a stress response in lactating dams. This "social stress" brings about short- and long-term effects in the offspring, even in the absence of any direct manipulation of the pups.

Ultrasonic vocalizations as an index of social memory in female mice.

Ultrasonic vocalization (UV) as a measure of social memory was investigated in female mice. UVs emitted by a resident female in the presence of a same-sex partner were measured during a 3-min, pretest social interaction. In a second 3-min test session, mice were reexposed to the familiar partner or presented with a novel partner. In the first case, there was a decline in UVs emitted by resident mice when the intervals between the 2 sessions were 15, 30, or 60 min. After 24 hr, this effect disappeared. In contrast, with a novel female partner, the number of UVs remained unchanged. Scopolamine (0.05 mg/kg ip) disrupted this memory process: Drug-treated females did not show the expected decrease in UVs when reexposed to the familiar female after 30 min. This study provides behavioral and pharmacological evidence that ultrasonic calls can be used as a measure of social memory in female mice.

A model of social stress in dominant mice: effects on sociosexual behaviour.

The possibility of socially stressing the dominant/aggressive member of a pair of male mice is tested. Male mice (NMRI outbreed strain) were housed in pairs to assess dominant and subordinate roles by agonistic interactions and urine-marking test. Social stress for dominant males consisted in 30 min/day of exposure to their subordinate partner interacting with a female in the adjacent compartment of the cage, for 9 days. Results showed that dominance status was maintained. Behavioural observations indicated that neither the subordinates nor the dominant males habituated to this experimental procedure. At the end of the chronic stress, dominant animals were given the opportunity to interact for 30 min with a female in their compartment. Results indicated that stressed dominants showed impairment in their sexual behaviour and were more oriented towards the physical environment in comparison with control dominants. The behavioural response to apomorphine (0.25 mg/kg) indicated an alteration of the dopaminergic functioning in socially stressed dominant mice. This study suggests that the characteristics of the stressor and the effects of the chronic social stress could be different, according to male social status.

Behavioral and mesocorticolimbic dopamine responses to non aggressive social interactions depend on previous social experiences and on the opponent's sex.

In these experiments we evaluated the relationship between behavioral and brain dopamine (DA) responses to social interactions. Subjects were group housed male mice confronted with a non aggressive male or female conspecific following either repeated defeat (defeated) or repeated non aggressive experiences (social). Defeated mice showed more defensive/submissive reactions then mice of the social group regardless of the opponent sex. However, mice defeated by females showed reduced social exploration without significant differences in non social exploration whilst the opposite was true for mice defeated by male opponents. Non aggressive social interactions enhanced dopamine metabolism in the prefrontal cortex (pFC) of DEFEATED mice regardless of opponent sex. However, only mice defeated by females showed enhanced dopamine metabolism and release in the nucleus accumbens septi (NAS) and olfactory tubercle (OT) following interaction with the non aggressive opponent. Finally, correlation between central and behavioral responses evidenced that 3,4-dihydroxiphenilacetic acid levels in the pFC were positively correlated with defensive behaviors and negatively correlated with non social exploration in mice confronted with male opponents but not in those confronted with females. The latter, showed a significant positive correlation between 3-methoxytyramine (3-MT) levels in the OT and defensive responses and significant negative correlation between social investigation and 3-MT levels in the OT and in the NAS. These results indicate a strict relationship between mesocorticolimbic dopamine transmission and behavior responses to social cues. Moreover, they strongly support the view that mesocorticolimbic DA modulates social behavior by affecting perceptive processing.

Male olfactory cues affect mothers' behavior in mice: effects of benzodiazepines.

RATIONALE: Threatening social stimuli were used in this study as aversive conditions to test anxiety in lactating female mice. The odors of potential infanticidal males or the "stress odor" left by restrained mice represented two aversive conditions that have been suggested to modulate the time spent by the mothers to reach their pups after 30 min of separation. OBJECTIVES: The effects of drugs acting at the benzodiazepine receptors were evaluated on the behavior of mothers exposed to different threatening social cues. METHODS: Lactating mice of the NMRI outbred strain with 8-day old pups were treated with (1) chlordiazepoxide (CDP) 2.5, 5.0 and 10 mg/kg i.p.; (2) flumazenil 10 mg/kg i.p. and (3) methyl beta-carboline-3-carboxylate (beta-CCM) 3.0 mg/kg i.p. RESULTS: The odors left by stressed females changed the mothers' exploratory behavior, but not the latency to reach pups. The latency was higher in the presence of cues from potentially infanticidal males. CDP (5.0 mg/kg) reduced the time spent to contact pups, whereas the other CDP doses did not modify the dam's behavior. Flumazenil, given in combination with CDP (5.0 mg/kg) antagonized the latter anxiolytic effect. In addition, in the presence of cues from potentially infanticidal males beta-CCM had anxiogenic activity, increasing latency to reach pups. The same CDP and beta-CCM doses were ineffective in the presence of cues from stressed females and in the absence of olfactory cues from conspecifics. CONCLUSIONS: This study provides behavioral and pharmacological validation of a new model of anxiety specifically designed for lactating females.

Effects of postnatal manipulation on nociception and morphine sensitivity in adult mice.

The long-term effects of postnatal manipulation on nociception were studied in NMRI albino male mice. During the first two weeks of life, pups were removed from their cage and deprived of maternal/nest odour for 15 min/day. To evaluate pain sensitivity, adult mice exposed to this postnatal manipulation (CB group) were tail flick and formalin tested for acute and tonic pain, respectively. CB mice showed a reduced pain sensitivity both in tail-flick and in formalin tests in comparison with control animals. Moreover, responsiveness to morphine (MO 1.0, 2.5, and 5.0 mg/kg, i.p.) in young (35 days old) and adult (90 days old) postnatally manipulated animals was evaluated with the tail-flick test: a decrease of the antinociceptive effects induced by morphine both in young and adult males was observed in postnatally manipulated animals. Morphine induced significant analgesic effects in control mice at doses lower than those affecting nociceptive thresholds both in young and adult CB mice. In addition, young animals showed a higher sensitivity to morphine than adults, independently of postnatal manipulation. The long-term effects of postnatal manipulation on nociception are discussed in terms of involvement of the opioid system and of the characteristics of pup manipulation.

Long-term effects of postnatal manipulation on emotionality are prevented by maternal anxiolytic treatment in mice.

This study investigated the role of maternal behavior on the long-term effects of postnatal manipulation (15 min of daily separation from the dam and exposure to clean bedding from Day 1 to Day 14 of postnatal life) on emotionality in the mouse. Mothers were treated with an antianxiety agent (Chlordiazepoxide: 5 mg/kg), daily upon removal of the litter from the nest. Emotionality in adult offspring was tested in the elevated plus maze. Mice manipulated during postnatal development were more explorative and less anxious than unhandled mice, but this effect was not observable in the offspring of Chlordiazepoxide-treated dams. No effect of maternal Chlordiazepoxide was observed in unhandled offspring. The pharmacological treatment of the mother did not affect either pups' ultrasonic calling during separation, or maternal behavior far apart from the daily manipulation sessions. By contrast, Chlordiazepoxide-treated dams were less responsive toward pups upon reunion following daily separation. This alteration of dams' behavior was not related to alterations in the amount of ultrasonic calls emitted by pups during reunion. Finally, when dams were daily injected with Chlordiazepoxide far apart from pups' removal, the pharmacological treatment was devoid of effects. These results support the view that the mother-infant interaction which follows separation plays a major role in determining the effects of postnatal manipulations on adult emotionality.

Kin interaction enhances morphine analgesia in male mice.

The additive effect of social and pharmacological treatments was evaluated in pairs of male mice. Ineffective and effective doses of morphine (2.5 and 5.0 mg/kg, i.p.) were tested on pain threshold in dyads of males at different times after pair formation and drug treatment. During the second hour of social interaction after reunion, saline-injected adult sibling male mice showed a decrease in nociception as measured by the tail-flick test. Pairs of unrelated, unfamiliar control mice showed no changes in pain sensitivity during a 2-h social session. An ineffective dose of 2.5 mg/kg of morphine in non-sibling males, significantly increased tail-flick latencies in sibling pairs, before the effect of the social environment (sibling) reached statistical significance. The higher dose of morphine (5.0 mg/kg) produced analgesia in sibling as well as in non-sibling males, but the effect in the latter disappeared 60 min after drug treatment, whereas siblings were still analgesic. These results indicate that an ineffective dose of morphine, combined with the activation of the endogenous opioid system by social factors, can affect nociception.

Neurobiological and behavioral aspects of recognition in female mice.

Female mice were tested for their capability to recognize female littermates in adulthood. Sibling females did not show any of the behavioral indices of recognition reported in the literature for sibling males during a 2-h social interaction. Conversely, adult females that experienced 7 days of separation from unrelated cagemates were able to recognize each other: during reunion, they showed reduced olfactory exploration, higher levels of affiliative behavior, and changes in nociception that were opioid dependent. Familiar females treated with naloxone (5.0 mg/kg, i.p.) did not show the increase in tail-flick latency that characterized saline-treated familiar pairs. Unfamiliar females did not show any behavioral modifications during social interaction. These data stress the role of familiarity in female-female social interaction and suggest that more recent experiences conceal earlier information gained before weaning. The importance of familiarity in females was found to be in contrast with the relevance of genetic recognition in male mice, but was well adapted to male and female roles within the social structure of mouse populations.

Time-related effects of stress on cholinergic sensitivity.

The effect of the administration of the muscarinic cholinergic agonist oxotremorine on locomotor activity was investigated in DBA/2 mice subjected to chronic restraint stress of different durations (120 min daily for 10, 14 or 18 days). Oxotremorine induced a depressant effect on locomotion, which was reduced after 10 and 14 days of restraint, but not after a 18-day restraint stress. Acetylcholine (ACh) content was significantly reduced in prefrontal cortex after 10 and 14 days of stress but returned to control values after 18 days of restraint. No changes in ACh content were observed in nucleus accumbens and striatum. These results are discussed in terms of possible changes in muscarinic receptor sensitivity.

Reunion of separated sibling mice: neurobiological and behavioral aspects.

Outbred albino NMRI male mice encountering a brother in adulthood, after a long period of separation, show an opioid-dependent increase in pain threshold. Unrelated and unfamiliar males show no similar changes in pain sensitivity. This study investigates which kind of stimuli from the partner may be responsible for such a modification at the neural level. The tail-flick test is used as a measure of pain sensitivity. Exposure to the scent of the brother's home cage, as well as exposure to visual, olfactory and auditory stimuli and partial physical contact with the related male, are not sufficient to induce changes in nociception. Physical affiliative contact between males is higher in sib than in nonsib pairs, and a positive correlation exists in sib pairs between huddling behavior and pain sensitivity at the end of a 2-h social session. Siblings injected with naloxone, an opioid receptor blocker, show a decrease in social behaviors involving physical contact. These results suggest that physical affiliative contact between sibling mice may be responsible for the enhancement of nociceptive threshold.