Oestrogen-deficient female aromatase knockout (ArKO) mice exhibit depressive-like symptomatology

We recently found that female aromatase knockout (ArKO) mice that are deficient in oestradiol due to a targeted mutation in the aromatase gene show deficits in sexual behaviour that cannot be corrected by adult treatment with oestrogens. We determined here whether these impairments are associated with changes in general levels of activity, anxiety or 'depressive-like' symptomatology due to chronic oestrogen deficiency. We also compared the neurochemical profile of ArKO and wild-type (WT) females, as oestrogens have been shown to modulate dopaminergic, serotonergic and noradrenergic brain activities. ArKO females did not differ from WT in spontaneous motor activity, exploration or anxiety. These findings are in line with the absence of major neurochemical alterations in hypothalamus, prefrontal cortex or striatum, which are involved in the expression of these behaviours. By contrast, ArKO females displayed decreased active behaviours, such as struggling and swimming, and increased passive behaviours, such as floating, in repeated sessions of the forced swim test, indicating that these females exhibit 'depressive-like' symptoms. Adult treatment with oestradiol did not reverse the behavioural deficits observed in the forced swim test, suggesting that they may be due to the absence of oestradiol during development. Accordingly, an increased serotonergic activity was observed in the hippocampus of ArKO females compared with WT, which was also not reversed by adult oestradiol treatment. The possible organizational role of oestradiol on the hippocampal serotonergic system and the 'depressive-like' profile of ArKO females provide new insights into the pathophysiology of depression and the increased vulnerability of women to depression.     

Behavioral and biochemical responses to d-amphetamine in MCH1 receptor knockout mice

The melanin-concentrating hormone (MCH) system is anatomically and functionally interlaced with the mesocorticolimbic dopamine system. Therefore, we investigated whether MCH(1) receptor knockout (KO) mice are more susceptible than wild-type (WT) mice to psychostimulant-induced locomotor stimulation and sensitization, dopamine receptor-mediated phosphorylation events and c-fos expression within the frontal cortex and ventral striatum. MCH(1) receptor KO mice have 20% higher basal locomotor activity, are hypersensitive to the locomotor activating effects of d-amphetamine (1 mg/kg), and develop behavioral sensitization to a regimen of repeated d-amphetamine administration that does not induce sensitization in WT mice. In addition, d-amphetamine-mediated regulation of p44-mitogen activated protein kinase (MAPK) phosphorylation within the frontal cortex was significantly enhanced in MCH(1) receptor KO mice, when compared with WT mice. No significant genotype difference in the effects of d-amphetamine on MAPK phosphorylation events within the ventral striatum, phosphorylation at Ser(897) of the NR1 subunit of the NMDA receptor or Ca(2+) and cyclic AMP response-element binding-protein (CREB) at Ser(133) in the frontal cortex was detected. d-Amphetamine (3 mg/kg) increased c-fos expression within the frontal cortex in MCH(1) receptor KO mice, but not WT mice. There were no d-amphetamine-induced changes in c-fos expression within the ventromedial striatum in KO or WT mice. Overall, MCH(1) receptor KO mice are hypersensitive to the behavioral and molecular effects of the dopaminergic psychostimulant d-amphetamine. Increased frontal cortical MAPK phosphorylation and c-fos expression in MCH(1) receptor KO mice indicates that the MCH(1) receptor may be an important target for treating neuropsychiatric disorders characterized by frontal cortex dysfunction, including depression, attention deficit hyperactivity disorder (ADHD) and schizophrenia.     

Relationships between aromatase activity in the brain and gonads and behavioural deficits in homozygous and heterozygous aromatase knockout mice

The present study was carried out to determine whether aromatase knockout (ArKO) mice are completely devoid of aromatase activity in their brain and gonads and to compare aromatase activity in wild-type and ArKO mice, as well as in heterozygous (HET) mice of both sexes that were previously shown to display a variety of reproductive behaviours at levels intermediate between wild-type and ArKO mice. Aromatase activity was extremely low, and undetectable by the tritiated water assay, in homogenates of the preoptic area-hypothalamus of adult wild-type mice, but was induced following a 12-day treatment with testosterone. The induction of aromatase activity by testosterone was significantly larger in males than in females. Even after 12 days exposure to testosterone, no aromatase activity was detected in the brain of ArKO mice of either sex whereas HET mice showed intermediate levels of activity between ArKO and wild-type. Aromatase activity was also undetectable in the ovary of adult ArKO females but was very high in the wild-type ovary and intermediate in the HET ovary. In wild-type mice, a high level of aromatase activity was detected on the day of birth even without pretreatment with testosterone. This neonatal activity was higher in males than in females, but females nevertheless appear to display a substantial level of oestrogen production in their brain. Aromatase activity was undetectable in the brain of newborn ArKO males and females and was intermediate between wild-type and ArKO in HET mice. In conclusion, the present study confirms that ArKO mice are unable to synthesize any oestrogens, thereby validating the ArKO mouse as a valuable tool in the study of the physiological roles of oestradiol. In addition, it demonstrates that the intermediate behaviour of HET mice presumably reflects the effect of gene dosage on aromatase expression and activity, that aromatase activity is sexually differentiated in mice during the neonatal period as well as in adulthood and, finally, that the neonatal female brain produces substantial amounts of oestrogens that could play a significant role in the sexual differentiation of the female brain early in life.     

Central forebrain Fos responses to familiar male odours are attenuated in recently mated female mice

Exposure of recently mated female mice to the urinary odours of an unfamiliar male blocks pregnancy (the Bruce effect). The absence of a pregnancy block in response to the stud male's familiar odours depends on an olfactory memory that is formed in the accessory olfactory bulb (AOB) in response to vomeronasal organ (VNO) inputs during mating. Sexually naive Balb/c female mice in pro-oestrus/oestrus were either placed onto soiled bedding ('bedding-only' females) from, or allowed to mate with, a Balb/c male ('recently mated' females). After 42 h, females were placed for 90 min onto clean bedding (controls) or onto soiled bedding from either a C57BL/6 male (unfamiliar bedding) or a Balb/c male (familiar bedding). Significant increases in Fos-immunoreactivity (Fos-IR, a marker of neuronal activation) occurred in the medial amygdala and the medial preoptic area (MPA) of 'bedding only' females exposed to either unfamiliar or familiar bedding and in 'recently mated' females exposed to unfamiliar bedding but not to familiar bedding. This suggests that a mating-induced memory prevents the later activation by the familiar stud male's odours of neurons in forebrain regions that receive inputs from the VNO--AOB. 'Bedding-only' females later exposed to either familiar or unfamiliar bedding had increased Fos-IR in the G alpha(o) protein-expressing basal zone of the VNO whereas no such effect occurred in 'recently mated' females. Familiar, as well as unfamiliar, male odours augmented Fos-IR in significantly more rostral than caudal AOB granule cells in all groups, with the effect being strongest in 'recently mated' females exposed to familiar male bedding. This outcome is consistent with the absence of odour-induced Fos-IR in forebrain regions of these females and, presumably, the absence of a pregnancy block.     

Aberrant responses in social interaction of dopamine transporter knockout mice

The dopamine (DA) transporter (DAT) controls the temporal and spatial resolution of dopaminergic neurotransmission. Disruption of the Dat1 gene in mice leads to increased extracellular DA concentrations and reduced expression of D1- and D2-like receptors in striatum. The mutants are hyperactive in the open field and they display deficits in learning and memory. In humans, dopaminergic dysfunction has been associated with a number of different psychiatric disorders and some of these conditions are accompanied by abnormal social responses. To determine whether social responses were also impaired in DAT knockout (KO) mice, behaviors of group- and isolation-housed animals were compared. All group-housed animals readily established hierarchies. However, the social organizations of the mutants were changed over time. Under both group- and isolation-housed conditions, mutants exhibited increased rates of reactivity and aggression following mild social contact. In isolation, exposure to a novel environment exacerbated these abnormal responses. Regardless of housing context, stereotyped and perseverative patterns of social responses were a common feature of the KO repertoire. In fact, many abnormal behaviors were due to the emergence and predominance of these inflexible behaviors. These data suggest that KO mice may serve as a useful animal model for understanding not only how DA dysfunction contributes to social abnormalities, but also how behavioral inflexibility distorts their social responses.     

Androgen receptor is essential for sexual differentiation of responses to olfactory cues in mice

During sexual differentiation males and females are exposed to different levels of testosterone, which promotes sex differences in the adult brain and in behavior. Testosterone can act after aromatization or reduction via a number of steroid hormone receptors. Here we provide new evidence that the androgen receptor (AR) is essential for sexual differentiation in mice. We used mice carrying the testicular feminization (Tfm) mutation of the AR. Adult Tfm males, wild-type male and female littermates were gonadectomized and given subcutaneous estradiol implants. In all sexually dimorphic traits, Tfm males had responses equivalent to females and different from males. In simultaneous choice tests, males spent significantly more time investigating female-soiled bedding, whereas females and Tfm males preferred to investigate male-soiled bedding. Tfm males and females did not have a partner preference in tests with awake stimulus animals, whereas males showed a preference for females over males. Exposure to male-soiled, but not clean, bedding produced a significant increase in c-Fos-immunoreactive cells in the medial preoptic area and bed nucleus of the stria terminalis in Tfm males and females, no increase was noted in males. Masculine sexual behavior (mounting and thrusting) was not sexually dimorphic, and all groups displayed these behaviors. Our results support data collected in humans suggesting a role for the androgen receptor in sexual differentiation of social preferences and neural responses to pheromones.     

Pituitary-adrenal responses to oxotremorine and acute stress in male and female M1 muscarinic receptor knockout mice: comparisons to M2 muscarinic receptor knockout mice

Both within the brain and in the periphery, M(1) muscarinic receptors function primarily as postsynaptic receptors and M(2) muscarinic receptors function primarily as presynaptic autoreceptors. In addition to classical parasympathetic effectors, cholinergic stimulation of central muscarinic receptors influences the release of adrenocorticotrophic hormone (ACTH) and corticosterone. We previously reported that oxotremorine administration to male and female M(2) receptor knockout and wild-type mice increased ACTH to a significantly greater degree in knockout males compared to all other groups, and that M(2) knockout mice of both sexes were significantly more responsive to the mild stress of saline injection than were wild-type mice. These results accord with the primary function of M(2) receptors as presynaptic autoreceptors. In the present study, we explored the role of the M(1) receptor in pituitary-adrenal responses to oxotremorine and saline in male and female M(1) knockout and wild-type mice. Because these mice responded differently to the mild stress of saline injection than did the M(2) knockout and wild-type mice, we also determined hormone responses to restraint stress in both M(1) and M(2) knockout and wild-type mice. Male and female M(1) knockout and wild-type mice were equally unresponsive to the stress of saline injection. Oxotremorine increased both ACTH and corticosterone in M(1) wild-type mice to a significantly greater degree than in knockout mice. In both M(1) knockout and wild-type animals, ACTH responses were greater in males compared to females, and corticosterone responses were greater in females compared to males. Hormone responses to restraint stress were increased in M(2) knockout mice and decreased in M(1) knockout mice compared to their wild-type counterparts. These findings suggest that M(1) and M(2) muscarinic receptor subtypes differentially influence male and female pituitary-adrenal responses to cholinergic stimulation and stress. The decreased pituitary-adrenal sensitivity to oxotremorine and restraint stress noted in M(1) knockout mice is consistent with M(1) being primarily a postsynaptic receptor. Conversely, the increased pituitary-adrenal sensitivity to these challenges noted in M(2) knockout mice is consistent with M(2) being primarily a presynaptic autoreceptor.     

Sex‐related differences in behavioral and amygdalar responses to compound facial threat cues

During face perception, we integrate facial expression and eye gaze to take advantage of their shared signals. For example, fear with averted gaze provides a congruent avoidance cue, signaling both threat presence and its location, whereas fear with direct gaze sends an incongruent cue, leaving threat location ambiguous. It has been proposed that the processing of different combinations of threat cues is mediated by dual processing routes: reflexive processing via magnocellular (M) pathway and reflective processing via parvocellular (P) pathway. Because growing evidence has identified a variety of sex differences in emotional perception, here we also investigated how M and P processing of fear and eye gaze might be modulated by observer's sex, focusing on the amygdala, a structure important to threat perception and affective appraisal. We adjusted luminance and color of face stimuli to selectively engage M or P processing and asked observers to identify emotion of the face. Female observers showed more accurate behavioral responses to faces with averted gaze and greater left amygdala reactivity both to fearful and neutral faces. Conversely, males showed greater right amygdala activation only for M‐biased averted‐gaze fear faces. In addition to functional reactivity differences, females had proportionately greater bilateral amygdala volumes, which positively correlated with behavioral accuracy for M‐biased fear. Conversely, in males only the right amygdala volume was positively correlated with accuracy for M‐biased fear faces. Our findings suggest that M and P processing of facial threat cues is modulated by functional and structural differences in the amygdalae associated with observer's sex.     

Differential effect of amphetamine on c-fos expression in female aromatase knockout (ArKO) mice compared to wildtype controls

Estrogen may be involved in psychosis by an interaction with central dopaminergic activity. Aromatase knockout mice are unable to produce estrogen and have been shown to display altered behavioural responses and effects of the dopamine releaser, amphetamine. This study investigates the effect of gonadal status on amphetamine-induced c-fos expression in the brains of female aromatase knockout and wildtype mice. Six groups of mice were treated intraperitoneally with saline or 5mg/kg amphetamine. Fos immunoreactivity was assessed in the cingulate cortex, caudate putamen and nucleus accumbens. Aromatase knockout mice showed markedly reduced amphetamine-induced Fos immunoreactivity compared to wildtype mice. However, the amphetamine response was restored in aromatase-knockout mice after ovariectomy, which reduced this effect in wildtype controls. Estrogen supplementation reversed the effect of ovariectomy in wildtype mice but had no additional significant effect in aromatase-knockout mice. These results indicate that mechanisms involved in amphetamine-induced c-fos expression are altered in aromatase knockout mice and that the primary hormone involved in this effect is not estrogen, but may be another factor released from the ovaries, such as an androgen. These results provide new insight into the effect of gonadal hormones on amphetamine induced c-fos expression in this mouse model of estrogen deficiency. These results could be important for our understanding of the role of sex steroid hormones in psychosis.     

Effects of morphine on pentobarbital-induced responses in mu-opioid receptor knockout mice

Effects of morphine on the potentiation of pentobarbital-induced responses were investigated using mu-opioid receptor knockout mice. The duration of loss of righting reflex, hypothermia, and loss of motor coordination induced by pentobarbital were measured after pretreatment with either morphine or saline. Morphine pretreatment failed to show potentiation of both pentobarbital-induced loss of righting reflex and hypothermia in mu-opioid receptor knockout mice, while it significantly potentiated these responses in the wild-type controls. For motor incoordination test, morphine potentiated pentobarbital-induced motor incoordination in the wild-type mice. However, morphine may have opposite effects in the mu-opioid receptor knockout mice. These results demonstrate that synergism between morphine and pentobarbital is not detected in mu-opioid receptor knockout mice and that potentiation of pentobarbital-induced loss of righting reflex and hypothermia by morphine is mediated through mu-opioid receptor. It was interesting to note that pentobarbital-induced decrease in body temperature was less severe in mu-opioid receptor knockout mice than in wild-type mice.     

The development of the nociceptive responses in neurokinin-1 receptor knockout mice

An important yet unanswered question is how neonates respond to painful stimuli, given the immaturity of their neural pathways. We examined the development of the neurokinin system using a novel approach, examining changes of this system by observing the pain responses of mice lacking the NK1 receptor at different stages of development We show that the NK1 receptor is not involved in nociception to heat, mechanical or chemical stimuli, at 3 days. In contrast, the NK1 receptor is involved in nociceptive responses to high intensity heat and mechanical stimuli, and mediates the second phase of the formalin response in 21-day-old mice. This indicates that nociception in neonates does not require the NK1 receptor and that the functional maturation of the NK1 receptor allows diversity in both the type of stimuli that activate the pain system and the types of responses elicited by nociceptive stimuli.     

Mating-induced Fos and aromatase are not co-localized in the preoptic area.

Male sexual behavior is determined by the interaction of endocrine and environmental stimuli originating from the female, yet it is unknown how and where these stimuli are integrated within the brain. Activation of copulatory behavior by testosterone is limited by its central aromatization into an estrogen in the preoptic area. We investigated whether mating-induced neuronal activation as identified by the expression of the immediate early gene Fos occurs in aromatase-immunoreactive (ARO-ir) cells of the male quail preoptic area. Fos-immunoreactive (ir) cells were observed within and lateral to these ARO-ir cells groups but few ARO-ir cells contained Fos-ir indicating that mating-related stimuli do not directly affect estrogen-synthesizing cells.     

Posture and balance responses to a sensory challenge are related to anxiety in mice

Anxiety disorders and balance disorders share common clinical features related to perception such as spatial disorientation or dizziness. The search for the mechanism underlying this core of symptoms led us to investigate impairments in multisensory integration. In mice, the 'rotating beam test' allows analysis of changes in balance control and posture in response to a multisensory challenge. We used the BALB/c and C57BL/6 inbred strains of mice, known for their contrasted anxiety-related behavior. The level of anxiety was also manipulated using anxiolytic and anxiogenic pharmacological compounds. Despite equal sensori-motor abilities, anxious mice were more prone to fall off the rotating beam and showed more imbalance than non-anxious mice. Striking inter-strain differences in posture were also observed. Diazepam and beta-CCM reversed these strain-specific responses in opposite directions. We demonstrated that balance and postural strategies developed in response to a multisensory challenge vary as a function of the level of anxiety in mice.     

Rapid approach responses to alcohol cues in heavy drinkers

Incentive-motivational models of addiction predict that alcohol cues should elicit approach behaviours in heavy drinkers. In this study we compared heavy and light social drinkers' response latencies when required to make approach or avoidance responses to alcohol pictures. Participants completed a stimulus-response compatibility (SRC) task, which requires participants to move a manikin towards or away from alcohol-related and matched control pictures, together with self-report measures of alcohol consumption and craving. Results demonstrated that heavy drinkers, but not light drinkers, were faster to approach than avoid alcohol pictures on the SRC task. The bias to approach alcohol pictures was also associated with alcohol craving.     

Behavioral responses to social stress in noradrenaline transporter knockout mice: effects on social behavior and depression

Noradrenaline has been implicated in the pathogenesis of depression and the noradrenaline transporter (NET) is a target for some antidepressants. Therefore, mice with disrupted NET gene expression (NET-KO) appear especially suitable for studying this behavioral disorder. We have examined the interaction between social stress (an etiological factor of depression) and the resulting depressive behaviors in NET-KO mice. Social stress was induced by daily defeats from larger resident mice while depression was assessed by the behavioral despair model. Animals subjected to repeated social stress showed reduced weight gain and a gradual shift from offensive to defensive behaviors. The latter may be considered a situation-specific depressive-like behavior. NET gene disruption did not prevent these changes that developed in a homotypic situation (i.e., during the repeated application of the same stressor). In contrast, stressed NET-KO mice showed more struggling in the behavioral despair model than stressed wild type (WT) animals. Thus, NET gene disruption inhibited depression-like behavior in chronically stressed animals tested in a situation heterotypic to the original cause of chronic stress. We suggest that the behavioral effects of NET gene disruption were overruled by experience and learning in the homotypic situation but manifested fully in the heterotypic situation. Tentatively, our data suggest that enhanced noradrenergic function does not prevent situation-specific social learning but impedes the generalization of depression to heterotypic circumstances.     

Enteric neurons from postnatal Fgf2 knockout mice differ in neurite outgrowth responses

The enteric nervous system (ENS) consists of several neuronal subclasses with distinct functional properties. The formation and maintenance of these distinct populations during development and aging is dependent on the support of appropriate neurotrophic factors. During early postnatal development, the ENS has to adept continuously to changing alimentation situations, which might also affect neuronal maturation and differentiation. There is evidence that basic-fibroblast-growth-factor (Fgf2) exerts neurotrophic effects in the ENS. In this study primary myenteric plexus cultures from both wild type and Fgf2-knockout mice were investigated under the influence of Fgf2 and glial-cell-line-derived-factor (GDNF). It could be demonstrated, that the influence of neurotrophic support is decreased in the Fgf2-knockouts, while the neuronal cultures of wild type revealed a more pronounced receptiveness for trophic support. These data show that Fgf2 plays a role in the development of the ENS.     

Failure of neural responses to safety cues in schizophrenia

CONTEXT Abnormalities in associative memory processes, such as Pavlovian fear conditioning and extinction, have been observed in schizophrenia. The retrieval of fear extinction memories (safety signals) may be particularly affected; although schizophrenic patients can extinguish conditioned fear, they show a deficit in retrieving fear extinction memories after a delay. The neurobiological basis of this abnormality is unknown, but clues have emerged from studies in rodents and humans demonstrating that the ventromedial prefrontal cortex (vmPFC) is a key mediator of extinction memory retrieval. OBJECTIVE To measure autonomic and neural responses during the acquisition and extinction of conditioned fear and the delayed recall of fear and extinction memories in patients with schizophrenia and healthy control participants. DESIGN Cross-sectional case control, functional magnetic resonance imaging study. SETTING Academic medical center. PARTICIPANTS Twenty schizophrenic patients and 17 healthy control participants demographically matched to the patient group. MAIN OUTCOME MEASURES Skin conductance and blood oxygen level-dependent responses. RESULTS During fear conditioning, schizophrenic patients showed blunted autonomic responses and abnormal blood oxygen level-dependent responses, relative to control participants, within the posterior cingulate gyrus, hippocampus, and other regions. Several of these abnormalities were linked to negative symptoms. During extinction learning, patients with schizophrenia and control participants showed comparable autonomic and neural responses. Twenty-four hours after the learning phases, the control subjects exhibited decreased fear and increased vmPFC responses in the extinction (safe) context as expected, indicating successful retention of the extinction memory. In contrast, the schizophrenic patients showed inappropriately elevated fear and poor vmPFC responses in the safe context. CONCLUSION Failure of extinction memory retrieval in schizophrenia is associated with vmPFC dysfunction. In future studies, abnormalities in fear learning and extinction recall may serve as quantitative phenotypes that can be linked to genetic, symptom, or outcome profiles in schizophrenia and those at risk for the disorder.     

Differences in human evoked potentials related to olfactory or trigeminal chemosensory activation.

The aim of the present study was to determine, whether there are differences in the topographical distribution of chemosensory evoked potentials (CSEPs) due to stimulation with different odorous substances. The odorants used in the study which mainly excited the olfactory nerve were vanillin and acetaldehyde; those which additionally excited the trigeminal nerve were sulphur dioxide and ammonia. Twelve subjects participated in the study. The subjects separately estimated the intensity of the odorous and of the painful/pricking sensation caused by the stimuli, and described the odorous qualities in their own words. CSEPs were recorded from 7 positions. After stimulation with "olfactory" substances maximum CSEP amplitudes were recorded at parieto-central sites, and after stimulation with "trigeminal" substances maximum amplitudes were obtained at the vertex. Following stimulation with ammonia and sulphur dioxide amplitudes were largest contralateral to the stimulated nostril. In contrast, little difference in CSEP amplitudes was observed between hemispheres after stimulation with vanillin or acetaldehyde. Thus, the topographical distribution of CSEP amplitudes may provide information with regard to the sensory system (olfactory or trigeminal) activated by the presentation of an odorous stimulus.     

Neurocognitive processes of linguistic cues related to death

Consciousness of the finiteness of one's personal existence influences human thoughts and behaviors tremendously. However, the neural substrates underlying the processing of death-related information remain unclear. The current study addressed this issue by scanning 20 female adults, using functional magnetic resonance imaging, in a modified Stroop task that required naming colors of death-related, negative-valence, and neutral-valence words. We found that, while both death-related and negative-valence words increased activity in the precuneus/posterior cingulate and lateral frontal cortex relative to neutral-valence words, the neural correlate of the processing of death-related words was characterized by decreased activity in bilateral insula relative to both negative-valence and neutral-valence words. Moreover, the decreased activity in the left insula correlated with subjective ratings of death relevance of death-related words and the decreased activity in the right insula correlated with subjective ratings of arousal induced by death-related words. Our fMRI findings suggest that, while both death-related and negative-valence words are associated with enhanced arousal and emotion regulation, the processing of linguistic cues related to death is associated with modulations of the activity in the insula that mediates neural representation of the sentient self.