Yoshiyuki Kasahara

Oxytocin receptor-deficient mice developed late-onset obesity.

The oxytocin receptor has been suggested to be involved in energy metabolism, such as food intake and energy consumption. Here, we demonstrate that oxytocin receptor-deficient (Oxtr−/−) male mice exhibited late-onset obesity with increases in abdominal fat pads and fasting plasma triglycerides. Daily food intake and spontaneous motor activity of Oxtr−/− mice were not significantly different as compared with wild-type mice. In contrast, brown adipose tissue in Oxtr−/− mice contained large lipid droplets and cold-induced thermogenesis was impaired. This study demonstrates that oxytocin receptor plays essential roles in the regulation of energy homeostasis.

New aspects of oxytocin receptor function revealed by knockout mice: sociosexual behaviour and control of energy balance

To further define the function of the oxytocin receptor (OXTR) in vivo, we generated mice deficient in the Oxtr gene (Oxtr-/-). Oxtr-/- mice had no obvious deficits in fertility or sexual behaviour, but displayed several aberrations in social behaviours, including male aggression, and mother-offspring interaction. In addition, they showed novel physiological defects including obesity, and dysfunction in body temperature control when exposed to cold. We review here our new findings with Oxtr-/- mice, and introduce newly generated Oxtr-Venus knockin mice as a potential tool for examining molecular physiology of Oxtr-neurons.

Impaired Thermoregulatory Ability of Oxytocin-Deficient Mice during Cold-Exposure

We analyzed temperature homeostasis in oxytocin-deficient (Oxt−/−) mice and found that Oxt−/− mice exhibited lower body temperatures than wild-type animals when they were exposed to cold. Oxt−/− mice also showed slightly more weight gain, but there were no obvious differences in the morphology of white and brown adipose tissues as between wild-type and Oxt−/− mice. In cold-exposed conditions, oxytocin neurons containing c-Fos immunoreactivity existed in the paraventricular nucleus of the hypothalamus. These results suggest that the central oxytocin neurons constitute part of the thermoregulatory system involved in maintaining body temperature in cold environments.

Chapter 3 Monoamine Transporter as a Target Molecule for Psychostimulants

Methamphetamine (MAP), a drug of abuse known worldwide for its addictive effects and neurotoxicity, causes somatic and psychiatric disorders. MAP enters terminals/neurons via monoamine transporters, displaces both vesicular and intracellular monoamines, and facilitates the release of monoamines into the extraneuronal space through synaptic transport via the monoamine transporters. Chronic psychostimulant abusers exhibit psychotic features, including delusions and auditory hallucinations. The dopamine transporter (DAT) and the vesicular monoamine transporter 2 (VMAT2) play pivotal roles in the action of MAP, including locomotor effects. The deletion of DAT attenuates the locomotor effects of MAP and may play larger role in behavioral responses to MAP compared to the deletion of VMAT2. MAP produces hyperthermia and/or neuronal toxicity in most species. The effects of MAP in DAT or serotonin transporter (SERT) single knockout (KO) mice and DAT/SERT double KO mice suggested that DAT and SERT are key molecules for hyperthermia and neuronal toxicity of MAP.

Impaired cliff avoidance reaction in dopamine transporter knockout mice

RationaleImpulsivity is a key feature of disorders that include attention-deficit/hyperactivity disorder (ADHD). The cliff avoidance reaction (CAR) assesses maladaptive impulsive rodent behavior. Dopamine transporter knockout (DAT-KO) mice display features of ADHD and are candidates in which to test other impulsive phenotypes.ObjectivesImpulsivity of DAT-KO mice was assessed in the CAR paradigm. For comparison, attentional deficits were also assessed in prepulse inhibition (PPI) in which DAT-KO mice have been shown to exhibit impaired sensorimotor gating.ResultsDAT-KO mice exhibited a profound CAR impairment compared to wild-type (WT) mice. As expected, DAT-KO mice showed PPI deficits compared to WT mice. Furthermore, the DAT-KO mice with the most impaired CAR exhibited the most severe PPI deficits. Treatment with methylphenidate or nisoxetine ameliorated CAR impairments in DAT-KO mice.ConclusionThese results suggest that DAT-KO mice exhibit impulsive CAR behavior that correlates with their PPI deficits. Blockade of monoamine transporters, especially the norepinephrine transporter (NET) in the prefrontal cortex (PFC), may contribute to pharmacological improvement of impulsivity in these mice.

Oxytocin Receptor in the Hypothalamus Is Sufficient to Rescue Normal Thermoregulatory Function in Male Oxytocin Receptor Knockout Mice

Oxytocin (OXT) and OXT receptor (OXTR) have been implicated in the regulation of energy homeostasis, but the detailed mechanism is still unclear. We recently showed late-onset obesity and impaired cold-induced thermogenesis in male OXTR knockout (Oxtr(-/-)) mice. Here we demonstrate that the OXTR in the hypothalamus has important functions in thermoregulation. Male Oxtr(-/-) mice failed to maintain their body temperatures during exposure to a cold environment. Oxtr(-/-) mice also showed decreased neuronal activation in the thermoregulatory hypothalamic region during cold exposure. Normal cold-induced thermogenesis was recovered in Oxtr(-/-) mice by restoring OXTR to the hypothalamus with an adeno-associated virus-Oxtr vector. In addition, brown adipose tissue (BAT) in Oxtr(-/-) mice contained larger lipid droplets in both 10- and 20-week-old compared with BAT from age-matched Oxtr(+/+) control mice. In BAT, the expression level of β3-adrenergic receptor at normal temperature was lower in Oxtr(-/-) mice than that in control mice. In contrast, α2A-adrenergic receptor expression level was higher in BAT from Oxtr(-/-) mice in both normal and cold temperatures. Because β3- and α2A-adrenergic receptors are known to have opposite effects on the thermoregulation, the imbalance of adrenergic receptors is suspected to affect this dysfunction in the thermoregulation. Our study is the first to demonstrate that the central OXT/OXTR system plays important roles in the regulation of body temperature homeostasis.

Cortico-Subcortical Neuromodulation Involved in the Amelioration of Prepulse Inhibition Deficits in Dopamine Transporter Knockout Mice

Prepulse inhibition (PPI) deficits are among the most reproducible phenotypic markers found in schizophrenic patients. We recently reported that nisoxetine, a selective norepinephrine transporter (NET) inhibitor, reversed the PPI deficits that have been identified in dopamine transporter (DAT) knockout (KO) mice. However, the mechanisms underlying nisoxetine-induced PPI recovery in DAT KO mice were unclear in previous experiments. To clarify these mechanisms, PPI was tested after microinjections of nisoxetine into the medial prefrontal cortex (mPFc) or nucleus accumbens (NAc) in wildtype (WT) and DAT KO mice. c-Fos immunohistochemistry provided an indicator of neural activation. Multiple-fluorescent-labeling procedures and the retrograde tracer fluorogold were employed to identify nisoxetine-activated neurons and circuits. Systemic nisoxetine activated the mPFc, the NAc shell, the basolateral amygdala, and the subiculum. Infusions of nisoxetine into the mPFc reversed PPI deficits in DAT KO mice, but produced no changes in WT mice, while infusion of nisoxetine into the NAc had no effect on PPI in both WT and DAT KO mice. Experiments using multiple-fluorescent labeling/fluorogold revealed that nisoxetine activates presumed glutamatergic pyramidal cells that project from the mPFc to the NAc. Activated glutamatergic projections from the mPFc to the NAc appear to have substantial roles in the ability of a NET inhibitor to normalize PPI deficits in DAT KO. Thus, this data suggest that selective NET inhibitors such as nisoxetine might improve information processing deficits in schizophrenia via regulation of cortico-subcortical neuromodulation.

The Regulation of Oxytocin Receptor Gene Expression during Adipogenesis

Although it has been reported that oxytocin stimulates lipolysis in adipocytes, changes in the expression of oxytocin receptor (OTR) mRNA in adipogenesis are still unknown. The present study aimed to investigate the expression of OTR mRNA during adipocyte differentiation and fat accumulation in adipocytes. OTR mRNA was highly expressed in adipocytes prepared from mouse adipose tissues compared to stromal‐vascular cells. OTR mRNA expression was increased during the adipocyte differentiation of 3T3‐L1 cells. OTR expression levels were higher in subcutaneous and epididymal adipose tissues of 14‐week‐old male mice compared to 7‐week‐old male mice. Levels of OTR mRNA expression were higher in adipose tissues at four different sites of mice fed a high‐fat diet than in those of mice fed a normal diet. The OTR expression level was also increased by refeeding for 4 h after fasting for 16 h. Oxytocin significantly induced lipolysis in 3T3‐L1 adipocytes. In conclusion, a new regulatory mechanism is demonstrated for oxytocin to control the differentiation and fat accumulation in adipocytes via activation of OTR as a part of the hypothalamic‐pituitary‐adipose axis.

Microglial production of TNF-alpha is a key element of sustained fear memory.

The proinflammatory cytokine productions in the brain are altered in a process of fear memory formation, indicating a possibility that altered microglial function may contribute to fear memory formation. We aimed to investigate whether and how microglial function contributes to fear memory formation. Expression levels of M1- and M2-type microglial marker molecules in microglia isolated from each conditioned mice group were assessed by real-time PCR and immunohistochemistry. Levels of tumor necrosis factor (TNF)-α, but not of other proinflammatory cytokines produced by M1-type microglia, increased in microglia from mice representing retention of fear memory, and returned to basal levels in microglia from mice representing extinction of fear memory. Administration of inhibitors of TNF-α production facilitated extinction of fear memory. On the other hand, expression levels of M2-type microglia-specific cell adhesion molecules, CD206 and CD209, were decreased in microglia from mice representing retention of fear memory, and returned to basal levels in microglia from mice representing extinction of fear memory. Our findings indicate that microglial TNF-α is a key element of sustained fear memory and suggest that TNF-α inhibitors can be candidate molecules for mitigating posttraumatic reactions caused by persistent fear memory.

Role of the Oxytocin Receptor Expressed in the Rostral Medullary Raphe in Thermoregulation During Cold Conditions

Recent papers have reported that oxytocin (Oxt) and the oxytocin receptor (Oxtr) may be involved in the regulation of food intake in mammals. We therefore suspected the Oxt/Oxtr system to be involved in energy homeostasis. In previous studies, we found a tendency toward obesity in Oxtr-deficient (Oxtr−/−) mice, as well as impaired thermoregulation when these mice were exposed to cold conditions. In the present study, we observed the expression of Oxtr in the rostral medullary raphe (RMR), the brain region known to control thermogenesis in brown adipose tissue (BAT). Through immunohistochemistry, we detected neurons expressing Oxtr and c-Fos in the RMR of mice exposed to cold conditions. Up to 40% of Oxtr-positive neurons in RMR were classified as glutamatergic neurons, as shown by immunostaining using anti-VGLUT3 antibody. In addition, mice with exclusive expression of Oxtr in the RMR were generated by injecting an AAV-Oxtr vector into the RMR region of Oxtr−/− mice. We confirmed the recovery of thermoregulatory ability in the manipulated mice during exposure to cold conditions. Moreover, mice with RMR-specific expression of Oxtr lost the typical morphological change in BAT observed in Oxtr−/− mice. Additionally, increased expression of the β3-adrenergic receptor gene, Adrb3, was observed in BAT. These results are the first to show the critical role of RMR Oxtr expression in thermoregulation during cold conditions.