Sex differences in pain-induced modulation of corticotropin-releasing hormone neurons in the dorsolateral part of the stria terminalis in mice.

We earlier reported female-biased, sex-specific involvement of the dorsolateral bed nucleus of the stria terminalis (dl BST) in the formalin-induced pain response in rats. The present study investigated pain effects on mice behaviors. Because the dl BST is densely populated with corticotropin-releasing hormone (CRH) neurons, we examined sex differences in these parameters for the dl BST CRH neurons in male and female mice of a mouse line for which the CRH gene promoter (corticotropin-releasing factor [CRF]-Venus ΔNeo) controls the expression of the modified yellow fluorescent protein (Venus). Approximately 92% of Venus-positive cells in the dl BST were also CRH mRNA-positive, irrespective of sex. Therefore, the cells identified using Venus fluorescence were regarded as CRH neurons. A female-biased sex difference was observed in pain-induced behaviors during the interphase (5-15 min after formalin injection) but not during the later phase (phase 2, 15-60 min) in wild-type mice. In CRF-Venus ΔNeo mice, a female-biased difference was observed in either the earlier phase (phase 1, 0-5 min) or the interphase, but not in phase 2. Patch-clamp recordings taken using an acute BST slice obtained from a CRF-Venus ΔNeo mouse after formalin injection showed miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs). Remarkably, the mEPSCs frequency was higher in the Venus-expressing cells of formalin-injected female mice than in vehicle-treated female mice. Male mice showed no increase in mEPSC frequency by formalin injection. Formalin injection had no effect on mEPSC or mIPSC amplitudes in either sex. Pain-induced changes in mEPSC frequency in putative CRH neurons were phase-dependent. Results show that excitatory synaptic inputs to BST CRH neurons are temporally enhanced along with behavioral sex differences in pain response, suggesting that pain signals alter the BST CRH neurons excitability in a sex-dependent manner.

Ketogenic diet feeding improves aerobic metabolism property in extensor digitorum longus muscle of sedentary male rats.

Recent studies of the ketogenic diet, an extremely high-fat diet with extremely low carbohydrates, suggest that it changes the energy metabolism properties of skeletal muscle. However, ketogenic diet effects on muscle metabolic characteristics are diverse and sometimes countervailing. Furthermore, ketogenic diet effects on skeletal muscle performance are unknown. After male Wistar rats (8 weeks of age) were assigned randomly to a control group (CON) and a ketogenic diet group (KD), they were fed for 4 weeks respectively with a control diet (10% fat, 10% protein, 80% carbohydrate) and a ketogenic diet (90% fat, 10% protein, 0% carbohydrate). After the 4-week feeding period, the extensor digitorum longus (EDL) muscle was evaluated ex vivo for twitch force, tetanic force, and fatigue. We also analyzed the myosin heavy chain composition, protein expression of metabolic enzymes and regulatory factors, and citrate synthase activity. No significant difference was found between CON and KD in twitch or tetanic forces or muscle fatigue. However, the KD citrate synthase activity and the protein expression of Sema3A, citrate synthase, succinate dehydrogenase, cytochrome c oxidase subunit 4, and 3-hydroxyacyl-CoA dehydrogenase were significantly higher than those of CON. Moreover, a myosin heavy chain shift occurred from type IIb to IIx in KD. These results demonstrated that the 4-week ketogenic diet improves skeletal muscle aerobic capacity without obstructing muscle contractile function in sedentary male rats and suggest involvement of Sema3A in the myosin heavy chain shift of EDL muscle.

Short-term fasting decreases excitatory synaptic inputs to ventromedial tuberoinfundibular dopaminergic neurons and attenuates their activity in male mice.

Tuberoinfundibular dopaminergic (TIDA) neurons in the arcuate nucleus (ARC) of the hypothalamus play a role in inhibiting prolactin (PRL) secretion from the anterior pituitary. PRL is involved in a variety of behaviors, including feeding. Consequently, we hypothesized that fasting might reduce the activity of TIDA neurons, which might alter PRL secretion. However, direct examinations of TIDA neuron activity are difficult. Recently, transgenic mice were generated that expressed green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase gene. We first determined that GFP in the dorsomedial ARC was a reliable marker of TIDA neurons. Then, we performed electrophysiology and immunocytochemistry in GFP-labeled TIDA neurons to examine whether different feeding conditions could change their activity. Eight-week-old male mice were fed or fasted for 24 h. After sacrifice, we prepared acutely isolated brain slices for conducting whole-cell voltage-clamp recordings. TIDA neurons were identified with fluorescence microscopy. The mean amplitude of miniature excitatory postsynaptic currents (mEPSCs) was significantly reduced in fasting mice compared to fed mice, but different feeding conditions did not affect the mean mEPSC intervals. This result suggested that fasting reduced the number of excitatory synaptic inputs to TIDA neurons. To determine whether a reduction in excitatory synaptic inputs would cause a reduction in TIDA neuron activity, we examined the effect of 24-h fasting on c-Fos expression in the ARC. We found that fasting significantly reduced the number of Fos-positive TIDA neurons. In addition, serum PRL levels were significantly increased. Taken together, the present findings suggested that short-term fasting attenuated TIDA neuron activity.

Prostaglandin E2 modulates presynaptic regulation of GnRH neurons via EP4 receptors in accordance with estrogen milieu.

Prostaglandin E2 (PGE2) promotes gonadotropin secretion by regulating the activity of neurons that release gonadotropin-releasing hormone (GnRH) in the hypothalamus. However, the mechanisms of action of PGE2 at these neurons have yet to be fully explored. We examined the effects of PGE2 on the generation of miniature excitatory postsynaptic currents (mEPSCs) at GnRH neurons as measured by whole-cell, patch-clamp recordings. GnRH neurons were identified in slices prepared from the preoptic areas of female GnRH-EGFP rats. Exposure to PGE2 significantly increased the frequency, but not the amplitude, of the mEPSCs generated on the day of proestrus, but neither frequency nor amplitude was altered on day 1 of diestrus. These data suggest that the action of PGE2 on mEPSC frequency varies depending on the stage of estrous. An estrogen-dependence of PGE2's action was further supported by the increased frequency, but not amplitude, of mEPSCs generated at GnRH neurons prepared from estrogen-primed ovariectomized rats. Conversely, PGE2 had no effect on mEPSC frequency or amplitude at GnRH neurons in cholesterol-treated rats. Subsequent experiments to identify candidate receptors for PG2E's action revealed that exposure to a PGE2 receptor 4 (EP4) agonist, but not EP1 or EP2 agonists, mimicked the effects achieved by PGE2 exposure. These effects of mEPSCs could be reversed using an EP4 antagonist, illustrating the specificity of the effect. Collectively, these data demonstrate that PGE2 can alter excitatory synaptic neurotransmission at GnRH neurons via EP4 signaling at presynaptic site(s) in an estrogen-dependent fashion during proestrus.

Early Growth Response 3 (Egr3) Contributes a Maintenance of C2C12 Myoblast Proliferation.

Satellite cell proliferation is a crucially important process for adult myogenesis. However, its regulatory mechanisms remain unknown. Early growth response 3 (Egr3) is a zinc-finger transcription factor that regulates different cellular functions. Reportedly, Egr3 interacts with multiple signaling molecules that are also known to regulate satellite cell proliferation. Therefore, it is possible that Egr3 is involved in satellite cell proliferation. Results of this study have demonstrated that Egr3 transcript levels are upregulated in regenerating mouse skeletal muscle after cardiotoxin injury. Using C2C12 myoblast culture (a model of activated satellite cells), results show that inhibition of Egr3 by shRNA impairs the myoblast proliferation rate. Results also show reduction of NF-кB transcriptional activity in Egr3-inhibited cells. Inhibition of Egr3 is associated with an increase in annexin V+ cell fraction and apoptotic protein activity including caspase-3 and caspase-7, and Poly-ADP ribose polymerase. By contrast, the reduction of cellular proliferation by inhibition of Egr3 was partially recovered by treatment of pan-caspase inhibitor Z-VAD-FMK. Collectively, these results suggest that Egr3 is involved in myoblast proliferation by interaction with survival signaling. J. Cell. Physiol. 232: 1114-1122, 2017. © 2016 Wiley Periodicals, Inc.

Involvement of Transient Receptor Potential Cation Channel Vanilloid 1 (TRPV1) in Myoblast Fusion.

The mechanisms that underlie the complex process of muscle regeneration after injury remain unknown. Transient receptor potential cation channel vanilloid 1 (TRPV1) is expressed in several cell types, including skeletal muscle, and is activated by high temperature and by certain molecules secreted during tissue inflammation. Severe inflammation and local temperature perturbations are induced during muscle regeneration, which suggests that TRPV1 might be activated and involved in the process. The aim of this study, was to clarify the role of TRPV1 in the myogenic potential of satellite cells responsible for muscle regeneration. We found that mRNA and protein levels of TRPV1 increased during regeneration after cardiotoxin (CTX)-induced muscle injury in mice. Using isolated mouse satellite cells (i.e., myoblasts), we observed that activation of TRPV1 by its agonist capsaicin (CAP) augmented myogenin protein levels. Whereas CAP did not alter myoblast proliferation, it facilitated myoblast fusion (evaluated using myonucleii number per myotube and fusion index). In contrast, suppression of TRPV1 by siRNA impaired myoblast fusion. Using mice, we also demonstrated that intramuscular injection of CAP facilitated muscle repair after CTX-induced muscle injury. Moreover, we showed that these roles of TRPV1 might be mediated by interleukin-4 and calcium signaling during myoblast fusion. Collectively, these results suggest that TRPV1 underlies normal myogenesis through promotion of myoblast fusion. J. Cell. Physiol. 231: 2275-2285, 2016. © 2016 Wiley Periodicals, Inc.

Ketogenic diet does not impair spatial ability controlled by the hippocampus in male rats.

A ketogenic diet was recently shown to reduce glutamate accumulation in synaptic vesicles, decreasing glutamate transmission. We questioned whether a ketogenic diet affects hippocampal function, as glutamate transmission is critically involved in visuospatial ability. In the present study, male Wistar rats were maintained on a ketogenic diet containing 10% protein and 90% fat with complements for 3 weeks to change their energy expenditure from glucose-dependent to fat-dependent. Control rats were fed a diet containing 10% protein, 10% fat, and 80% carbohydrates. The fat-dependent energy expenditure induced by the ketogenic diet led to decreased body weight and increased blood ketone production, though the rats in the two groups consumed the same number of calories. The ketogenic diet did not alter food preferences for the control or high-fat diet containing 10% protein, 45% fat, and 45% carbohydrates. Anxiety in the open field was not altered by ingestion the ketogenic diet. However, rats fed the ketogenic diet performed better in the Y-maze test than rats fed the control diet. No difference was observed between the two groups in the Morris water maze test. Finally, Western blot revealed that the hippocampal expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor subunit 1 (GluR1) was significantly increased in mice fed a ketogenic diet. These results suggest that hippocampal function is not impaired by a ketogenic diet and we speculate that the fat-dependent energy expenditure does not impair visuospatial ability.

Sex differences in feeding behavior in rats: the relationship with neuronal activation in the hypothalamus.

There is general agreement that the central nervous system in rodents differs between sexes due to the presence of gonadal steroid hormone during differentiation. Sex differences in feeding seem to occur among species, and responses to fasting (i.e., starvation), gonadal steroids (i.e., testosterone and estradiol), and diet (i.e., western-style diet) vary significantly between sexes. The hypothalamus is the center for controlling feeding behavior. We examined the activation of feeding-related peptides in neurons in the hypothalamus. Phosphorylation of cyclic AMP response element-binding protein (CREB) is a good marker for neural activation, as is the Fos antigen. Therefore, we predicted that sex differences in the activity of melanin-concentrating hormone (MCH) neurons would be associated with feeding behavior. We determined the response of MCH neurons to glucose in the lateral hypothalamic area (LHA) and our results suggested MCH neurons play an important role in sex differences in feeding behavior. In addition, fasting increased the number of orexin neurons harboring phosphorylated CREB in female rats (regardless of the estrous day), but not male rats. Glucose injection decreased the number of these neurons with phosphorylated CREB in fasted female rats. Finally, under normal spontaneous food intake, MCH neurons, but not orexin neurons, expressed phosphorylated CREB. These sex differences in response to fasting and glucose, as well as under normal conditions, suggest a vulnerability to metabolic challenges in females.

Exposure to social defeat stress in adolescence improves the working memory and anxiety-like behavior of adult female rats with intrauterine growth restriction, independently of hippocampal neurogenesis.

Intrauterine growth restriction (IUGR) is a risk factor for memory impairment and emotional disturbance during growth and adulthood. However, this risk might be modulated by environmental factors during development. Here we examined whether exposing adolescent male and female rats with thromboxane A2-induced IUGR to social defeat stress (SDS) affected their working memory and anxiety-like behavior in adulthood. We also used BrdU staining to investigate hippocampal cellular proliferation and BrdU and NeuN double staining to investigate neural differentiation in female IUGR rats. In the absence of adolescent stress, IUGR female rats, but not male rats, scored significantly lower in the T-maze test of working memory and exhibited higher anxiety-like behavior in the elevated-plus maze test compared with controls. Adolescent exposure to SDS abolished these behavioral impairments in IUGR females. In the absence of adolescent stress, hippocampal cellular proliferation was significantly higher in IUGR females than in non-IUGR female controls and was not influenced by adolescent exposure to SDS. Hippocampal neural differentiation was equivalent in non-stressed control and IUGR females. Neural differentiation was significantly increased by adolescent exposure to SDS in controls but not in IUGR females. There was no significant difference in the serum corticosterone concentrations between non-stressed control and IUGR females; however, adolescent exposure to SDS significantly increased serum corticosterone concentration in control females but not in IUGR females. These results demonstrate that adolescent exposure to SDS improves behavioral impairment independent of hippocampal neurogenesis in adult rats with IUGR.

Expression of phosphorylated cyclic AMP response element-binding protein in melanin-concentrating hormone neurons and orexin neurons in male and female rats during ad-libitum feeding.

Using phosphorylated cyclic AMP response element-binding protein (pCREB) as a marker of neural activity, we previously suggested that orexin neurons and melanin-concentrating hormone (MCH) neurons play distinct roles in feeding behavior. In the present study, we examined the expression of pCREB during ad-libitum feeding; previously, only fasted animals were examined. MCH neurons, but not orexin neurons, expressed pCREB during spontaneous food intake. The induction of pCREB expression did not differ by sex, but attenuation seemed to occur faster in females than in males. On the basis of the results of the present study, we speculate that MCH neurons respond to nutrition-related feeding, but the feeding-related activity of orexin was not evident unless hunger was accompanied by stress, such as the stress caused by the absence of food in the case of fasting. Therefore, the desire to eat under normal conditions does not drive orexin neurons, but it does drive MCH neurons. We tested this hypothesis by examining the effects of consuming glucose or saccharin, a nonmetabolized sweetener, in fasted male and female rats. Glucose and saccharin were equally effective in reducing pCREB expression in the orexin neurons of female rats. In MCH neurons, glucose attenuated the expression of pCREB, but saccharin had no effect, irrespective of sex. Taken together, the results indicate that MCH and orexin peptides play physiologically distinct roles in feeding behavior.