Oxytocin receptors in the Magel2 mouse model of autism: Specific region, age, sex and oxytocin treatment effects.

The neurohormone oxytocin (OXT) has been implicated in the regulation of social behavior and is intensively investigated as a potential therapeutic treatment in neurodevelopmental disorders characterized by social deficits. In the Magel2-knockout (KO) mouse, a model of Schaaf-Yang Syndrome, an early postnatal administration of OXT rescued autistic-like behavior and cognition at adulthood, making this model relevant for understanding the actions of OXT in (re)programming postnatal brain development. The oxytocin receptor (OXTR), the main brain target of OXT, was dysregulated in the hippocampus of Magel2-KO adult males, and normalized upon OXT treatment at birth. Here we have analyzed male and female Magel2-KO brains at postnatal day 8 (P8) and at postnatal day 90 (P90), investigating age, genotype and OXT treatment effects on OXTR levels in several regions of the brain. We found that, at P8, male and female Magel2-KOs displayed a widespread, substantial, down-regulation of OXTR levels compared to wild type (WT) animals. Most intriguingly, the postnatal OXT treatment did not affect Magel2-KO OXTR levels at P8 and, consistently, did not rescue the ultrasonic vocalization deficits observed at this age. On the contrary, the postnatal OXT treatment reduced OXTR levels at P90 in male Magel2-KO in a region-specific way, restoring normal OXTR levels in regions where the Magel2-KO OXTR was upregulated (central amygdala, hippocampus and piriform cortex). Interestingly, Magel2-KO females, previously shown to lack the social deficits observed in Magel2-KO males, were characterized by a different trend in receptor expression compared to males; as a result, the dimorphic expression of OXTR observed in WT animals, with higher OXTR expression observed in females, was abolished in Magel2-KO mice. In conclusion, our data indicate that in Magel2-KO mice, OXTRs undergo region-specific modifications related to age, sex and postnatal OXT treatment. These results are instrumental to design precisely-timed OXT-based therapeutic strategies that, by acting at specific brain regions, could modify the outcome of social deficits in Schaaf-Yang Syndrome patients.

Region-Specific KCC2 Rescue by rhIGF-1 and Oxytocin in a Mouse Model of Rett Syndrome.

Rett syndrome (RTT) is characterized by dysfunction in neuronal excitation/inhibition (E/I) balance, potentially impacting seizure susceptibility via deficits in K+/Cl- cotransporter 2 (KCC2) function. Mice lacking the Methyl-CpG binding protein 2 (MeCP2) recapitulate many symptoms of RTT, and recombinant human insulin-like growth factor-1 (rhIGF-1) restores KCC2 expression and E/I balance in MeCP2 KO mice. However, clinical trial outcomes of rhIGF-1 in RTT have been variable, and increasing its therapeutic efficacy is highly desirable. To this end, the neuropeptide oxytocin (OXT) is promising, as it also critically modulates KCC2 function during early postnatal development. We measured basal KCC2 expression levels in MeCP2 KO mice and identified 3 key frontal brain regions showing KCC2 alterations in young adult mice, but not in postnatal P10 animals. We hypothesized that deficits in an IGF-1/OXT signaling crosstalk modulating KCC2 may occur in RTT during postnatal development. Consistently, we detected alterations of IGF-1 receptor and OXT receptor levels in those brain areas. rhIGF-1 and OXT treatments in KO mice rescued KCC2 expression in a region-specific and complementary manner. These results suggest that region-selective combinatorial pharmacotherapeutic strategies could be most effective at normalizing E/I balance in key brain regions subtending the RTT pathophysiology.

Oxytocin Signaling in the Central Amygdala Modulates Emotion Discrimination in Mice.

Recognition of other's emotions influences the way social animals interact and adapt to the environment. The neuropeptide oxytocin (OXT) has been implicated in different aspects of emotion processing. However, the role of endogenous OXT brain pathways in the social response to different emotional states in conspecifics remains elusive. Here, using a combination of anatomical, genetic, and chemogenetic approaches, we investigated the contribution of endogenous OXT signaling in the ability of mice to discriminate unfamiliar conspecifics based on their emotional states. We found that OXTergic projections from the paraventricular nucleus of the hypothalamus (PVN) to the central amygdala (CeA) are crucial for the discrimination of both positively and negatively valenced emotional states. In contrast, blocking PVN OXT release into the nucleus accumbens, prefrontal cortex, and hippocampal CA2 did not alter this emotion discrimination. Furthermore, silencing each of these PVN OXT pathways did not influence basic social interaction. These findings were further supported by the demonstration that virally mediated enhancement of OXT signaling within the CeA was sufficient to rescue emotion discrimination deficits in a genetic mouse model of cognitive liability. Our results indicate that CeA OXT signaling plays a key role in emotion discrimination both in physiological and pathological conditions.

Impaired approach to novelty and striatal alterations in the oxytocin receptor deficient mouse model of autism.

Long-standing studies established a role for the oxytocin system in social behavior, social reward, pair bonding and affiliation. Oxytocin receptors, implicated in pathological conditions affecting the social sphere such as autism spectrum disorders, can also modulate cognitive processes, an aspect generally overlooked. Here we examined the effect of acute (pharmacological) or genetic (Oxtr-/-) inactivation of oxytocin receptor-mediated signaling, in male mice, in several cognitive tests. In the novel object recognition test, both oxytocin receptor antagonist treated wild type animals and Oxtr-/- mice lacked the typical preference for novelty. Oxtr-/- mice even preferred the familiar object; moreover, their performance in the Morris water maze did not differ from wild types, suggesting that oxytocin receptor inactivation did not disrupt learning. Because the preference for novel objects could be rescued in Oxtr-/- mice with longer habituation periods, we propose that the loss of novelty preferences following Oxtr inactivation is due to altered processing of novel contextual information. Finally, we observed an increased expression of excitatory synaptic markers in the striatum of Oxtr-/- mice and a greater arborization and higher number of spines/neuron in the dorsolateral area of this structure, which drives habit formation. Our data also indicate a specific reshaping of dorsolateral striatal spines in Oxtr-/- mice after exposure to a novel environment, which might subtend their altered approach to novelty, and support previous work pointing at this structure as an important substrate for autistic behaviors.

Regional specific modulation of neuronal activation associated with nitric oxide synthase inhibitors in an animal model of antidepressant activity.

OBJECTIVE: The regional specific modulation of neuronal activation following drug administration is of interest to determine brain areas involved in the behavioural effects of experimental test compounds. In the current investigation the effects of the L-arginine related NOS inhibitor Nω-l-nitroarginine (L-NA) and the structurally unrelated selective neuronal NOS inhibitor 1-(2-Trifluoro-methyl-phenyl) imidazole (TRIM) were assessed in the rat for changes in regional c-FOS immunoreactivity, a marker of neuronal activation, upon exposure to the forced swimming test (FST). Behaviour and regional FOS and FosB/ΔFosB expression was assessed in naive animals and in animals exposed to stress with central serotonin-depletion which exhibit a stress related phenotype in the FST. METHODS: Male Sprague-Dawley rats (n=5- 6 per group) were treated with the irreversible tryptophan hydroxylase inhibitor, DL-4-p-chlorophenylalanine (pCPA, 150mg/kg, i.p.), to achieve central serotonin-depletion followed by repeated exposures to restraint stress and were then subjected to the FST. 24, 5 and 1h prior to the test, animals were treated with either L-NA (10mg/kg, i.p.), TRIM (50mg/kg, i.p.) or saline vehicle (1mg/kg i.p). RESULTS: pCPA treatment coupled with restraint stress increased immobility in the FST compared to naïve controls. Both NOS inhibitors decreased immobility time in 5-HT depleted and stressed animals only in keeping with their antidepressant-like properties. Brain regions analyzed for c-FOS immunoreactivity included the pre-limbic cortex, lateral septum (LS), nucleus accumbens, paraventricular hypothalamic nucleus (PVN), central amygdala, hippocampus (dorsal dentate gyrus and ventral CA1), and the dorsal raphe nucleus (DRN). Exposure to the FST increased c-FOS immunoreactivity in the LS, PVN, dentate gyrus, vCA1 and the DRN when compared to non-FST exposed controls. FST-induced c-FOS immunoreactivity was further increased in the LS following treatment with L-NA or TRIM when compared to vehicle-treated FST controls. By contrast, FST-induced c-FOS immunoreactivity was reduced in dorsal dentate gyrus, vCA1 and the DRN following treatment with L-NA or TRIM when compared to vehicle-treated FST controls. There was no difference observed in FST-induced expression of c-FOS between naïve animals and animals exposed to pCPA and restraint stress. This combination however provoked an increase in FosB/ΔFosB immunoreactivity in the infra-limbic cortex and nucleus accumbens with a concomitant reduction in the lateral septum, suggesting alterations to long-term, adaptive neuronal activation. CONCLUSION: This study identified a pattern of enhanced and reduced FST-related c-FOS immunoreactivity indicative of a NO-regulated network where inhibition of NO leads to activation of the septum with concomitant inhibition of the hippocampus, and the DRN. No link between FST-induced regional expression of c-FOS and increased immobility in the FST was observed in animals exposed to pCPA and stress. However, the 5-HT depletion regime combined with restraint stress provoked regional changes in the expression of ΔFosB which may relate to increased immobility in the FST.

Soluble beta amyloid evokes alteration in brain norepinephrine levels: role of nitric oxide and interleukin-1.

Strong evidence showed neurotoxic properties of beta amyloid (Aß) and its pivotal role in the Alzheimer's disease (AD) pathogenesis. Beside, experimental data suggest that Aß may have physiological roles considering that such soluble peptide is produced and secreted during normal cellular activity. There is now suggestive evidence that neurodegenerative conditions, like AD, involve nitric oxide (NO) in their pathogenesis. Nitric oxide also possess potent neuromodulatory actions in brain regions, such as prefrontal cortex (PFC), hippocampus (HIPP), and nucleus accumbens (NAC). In the present study, we evaluated the effect of acute Aß injection on norepinephrine (NE) content before and after pharmacological manipulations of nitrergic system in above mentioned areas. Moreover, effects of the peptide on NOS activity were evaluated. Our data showed that 2 h after i.c.v. soluble Aß administration, NE concentrations were significantly increased in the considered areas along with increased iNOS activity. Pre-treatment with NOS inhibitors, 7-Nitroindazole (7-NI), and N6-(1-iminoethyl)-L-lysine-dihydrochloride (L-NIL), reversed Aß-induced changes. Ultimately, pharmacological block of interleukin1 (IL-1) receptors prevented NE increase in all brain regions. Taken together our findings suggest that NO and IL-1 are critically involved in regional noradrenergic alterations induced by soluble Aß injection.

Region specific up-regulation of oxytocin receptors in the opioid oprm1 (-/-) mouse model of autism.

Autism spectrum disorders (ASDs) are characterized by impaired communication, social impairments, and restricted and repetitive behaviors and interests. Recently, altered motivation and reward processes have been suggested to participate in the physiopathology of ASDs, and µ-opioid receptors (MORs) have been investigated in relation to social reward due to their involvement in the neural circuitry of reward. Mice lacking a functional MOR gene (Oprm1 (-/-) mice) display abnormal social behavior and major autistic-like core symptoms, making them an animal model of autism. The oxytocin (OXT) system is a key regulator of social behavior and co-operates with the opioidergic system in the modulation of social behavior. To better understand the opioid-OXT interplay in the central nervous system, we first determined the expression of the oxytocin receptor (OXTR) in the brain of WT C57BL6/J mice by quantitative autoradiography; we then evaluated OXTR regional alterations in Oprm1 (-/-) mice. Moreover, we tested these mice in a paradigm of social behavior, the male-female social interaction test, and analyzed the effects of acute intranasal OXT treatment on their performance. In autoradiography, Oprm1 (-/-) mice selectively displayed increased OXTR expression in the Medial Anterior Olfactory Nucleus, the Central and Medial Amygdaloid nuclei, and the Nucleus Accumbens. Our behavioral results confirmed that Oprm1 (-/-) male mice displayed social impairments, as indicated by reduced ultrasonic calls, and that these were rescued by a single intranasal administration of OXT. Taken together, our results provide evidence of an interaction between OXT and opioids in socially relevant brain areas and in the modulation of social behavior. Moreover, they suggest that the oxytocinergic system may act as a compensative mechanism to bypass and/or restore alterations in circuits linked to impaired social behavior.

Characterisation of the antidepressant properties of nitric oxide synthase inhibitors in the olfactory bulbectomised rat model of depression.

Nitric oxide synthase (NOS) inhibitors possess antidepressant-like properties in preclinical tests and in the current investigation the brain penetrant NOS inhibitor N(ω)-nitro-L-arginine (l-NA) and the preferential inhibitor of neuronal NOS (nNOS) 1-(2-trifluoromethylphenyl) imidazole (TRIM) were assessed in the olfactory bulbectomised (OB) rat, a well-established animal model of depression. Magnetic resonance imaging (MRI) was employed to assess regional brain volumes, blood perfusion and T1 and T2 relaxometry times both with and without drug treatment. l-NA (10 mg/kg, once daily p.o. for 10 days) attenuated OB-related hyperactivity in the "open field" test in a comparable fashion to the tricyclic antidepressant imipramine (20 mg/kg, once daily p.o. for 14 days) indicative of an antidepressant-like response in the model. Treatment with TRIM (50 mg/kg, once daily s.c.) attenuated OB-related hyperactivity following 7 days of treatment when compared to vehicle treated controls. OB is associated with enlarged ventricular volume, increased periventicular perfusion and a decrease in T2 relaxation times in cortical and hippocampal regions, with enhanced perfusion and reduced T2 times attenuated by L-NA treatment. L-NA treatment was also associated with an increase in T1 relaxation times in limbic and cortical regions and found to reduce resting state hippocampal blood perfusion in OB animals. Behavioural observations are consistent with an antidepressant action of NOS inhibitors where associated changes in perfusion and T2 relaxation times may be related to the antidepressant action of L-NA in the model.

Ketamine elicits sustained antidepressant-like activity via a serotonin-dependent mechanism.

RATIONALE: Behavioural antidepressant-like effects of ketamine have been reported in the forced swimming test (FST). The mechanisms mediating such effects are unknown. OBJECTIVES: As serotonin (5-HT) is an important transmitter mediating antidepressant responsiveness in the FST, the influence of 5-HT depletion on the antidepressant-like effect of ketamine was assessed. METHODS: The effect of ketamine (25 mg/kg, i.p., 1 or 24 h prior to test) was assessed in the FST in naive rats or animals subjected to 5-HT depletion, repeated stress or following a combination of 5-HT depletion and stress. Endogenous 5-HT was depleted using the tryptophan hydroxylase inhibitor para-chlorophenylalanine (3 × 150 mg/kg, i.p.). Stress was induced by physical restraint (2 h/day for 10 days). RESULTS: In naive rats, ketamine administered 24 or 1 h prior to test produced a characteristic antidepressant-like reduction in immobility time in the FST. Depletion of 5-HT blocked this reduction in immobility when ketamine was administered 24 h prior FST, indicative of 5-HT dependency. The increase in immobility provoked by repeated restraint stress (2 h/day for 10 days) was blocked by ketamine when administered 24 h prior to FST, but this effect dissipated when animals were subjected to 5-HT depletion. CONCLUSIONS: These observations are consistent with a role for 5-HT in mediating sustained antidepressant activity of ketamine in the FST. Molecular and cellular changes induced by ketamine may produce a rapid adaptation of 5-HT transmission which underlies the antidepressant response.

A role for serotonin in the antidepressant activity of NG-Nitro-L-arginine, in the rat forced swimming test.

The present study determined regional serotonin (5-HT) synthesis and metabolism changes associated with the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine (L-NA) and the influence of 5-HT receptor blockade in the antidepressant-like actions of L-NA in the forced swimming test (FST). Regional effects of L-NA (5,10 and 20mg/kg i.p.) on tryptophan hydroxylase (TPH) activity, the rate limiting enzyme for 5-HT synthesis, were determined by measuring accumulation of the transient intermediate 5-hydoxytryptophan (5-HTP) following in vivo administration of the amino acid decarboxylase inhibitor, NSD 1015 (100mg/kg). L-NA (5-20mg/kg) dose dependently increased 5-HTP accumulation, particularly in the amygdaloid cortex, following exposure to the FST. L-NA also provoked an increase in regional brain 5-HIAA concentrations and in the 5-HIAA:5-HT metabolism ratio. Co-treatment with NSD-1015 failed to consistently modify the antidepressant-like effects of L-NA in the FST. Sub-active doses of L-NA (1mg/kg) and the 5-HT re-uptake inhibitor fluoxetine (2.5mg/kg) acted synergistically to increase swimming in the test. Co-treatment with the non-selective 5-HT receptor antagonist metergoline (1, 2 and 4mg/kg), attenuated the L-NA (20mg/kg)-induced reduction in immobility and increase in swimming behaviours. Metergoline alone however provoked an increase in immobility and reduction in swimming behaviours in the test. A similar response was obtained following co-treatment with the preferential 5-HT(2A) receptor antagonist ketanserin (5mg/kg) and the 5-HT(2C) receptor antagonist RO-430440 (5mg/kg). Co-treatment with the 5-HT(1A) receptor antagonist WAY 100635 (0.3mg/kg) or the 5-HT(1B) receptor antagonist GR 127935 (4mg/kg) failed to influence the antidepressant-like activity of L-NA. Taken together these data provide further support for a role for 5-HT in the antidepressant-like properties of NOS inhibitors.