The voltage-gated calcium channel UNC-2 is involved in stress-mediated regulation of tryptophan hydroxylase

Migraine is an episodic pain disorder whose pathophysiology is related to deficiency of serotonin signaling and abnormal function of the P/Q-type calcium channel, CACNA1A. Because the relationship of the CACNA1A channel to serotonin signaling is unknown and potentially of therapeutic interest we have used genetic analysis of the Caenorhabditis elegans ortholog of this calcium channel, UNC-2, to help identify candidate downstream effectors of the human channel. By genetic dissection of the lethargic mutant phenotype of unc-2, we have established an epistasis pathway showing that UNC-2 function antagonizes a transforming growth factor (TGF)-beta pathway influencing movement rate. This same UNC-2/TGF-beta pathway is required for accumulation of normal serotonin levels and stress-induced modulation of tryptophan hydroxylase (tph) expression in the serotonergic chemosensory ADF neurons, but not the NSM neurons. We also show that transgenic expression of the migraine-associated Ca2+ channel, CACNA1A, in unc-2 animals can functionally substitute for UNC-2 in stress-activated regulation of tph expression. The demonstration that these evolutionarily related channels share a conserved ability to modulate tph expression through their effects on TGF-beta signaling provides the first specific example of how CACNA1A function may influence levels of the critical migraine neurotransmitter serotonin.     

Effects of treadmill exercise on brain insulin signaling and β-amyloid in intracerebroventricular streptozotocin induced-memory impairment in rats

[Purpose]

The purpose of the study is to explore effect of 6 weeks treadmill exercise on brain insulin signaling and β-amyloid(Aβ).

[Methods]

The rat model of Alzheimer’s disease(AD) used in the present study was induced by the intracerebroventricular(ICV) streptozotocin(STZ). To produce the model of animal with AD, STZ(1.5mg/kg) was injected to a cerebral ventricle of both cerebrums of Sprague-Dawley rat(20 weeks). The experimental animals were divided into ICV-Sham(n=7), ICV-STZ CON(n=7), ICV-STZ EXE(n=7). Treadmill exercise was done for 30 min a day, 5 days a week for 6 weeks. Passive avoidance task was carried out before and after treadmill exercise.

[Results]

The results of this study show that treadmill exercise activated Protein kinase B(AKT)/ Glycogen synthase kinase 3α (GSK3α), possibly via activation of insulin receptor(IR) and insulin receptor substrate(IRS) and reduced Aβ in the brain of ICV-STZ rats. More interestingly, treadmill exercise improved cognitive function of ICV-STZ rats. Finally, physical exercise or physical activity gave positive influences on brain insulin signaling pathway.

[Conclusion]

Therefore, treadmill exercise can be applied to improve AD as preventive and therapeutic method.     

The Effects of Mechanical Loading on Tendons - An In Vivo and In Vitro Model Study

Mechanical loading constantly acts on tendons, and a better understanding of its effects on the tendons is essential to gain more insights into tendon patho-physiology. This study aims to investigate tendon mechanobiological responses through the use of mouse treadmill running as an in vivo model and mechanical stretching of tendon cells as an in vitro model. In the in vivo study, mice underwent moderate treadmill running (MTR) and intensive treadmill running (ITR) regimens. Treadmill running elevated the expression of mechanical growth factors (MGF) and enhanced the proliferative potential of tendon stem cells (TSCs) in both patellar and Achilles tendons. In both tendons, MTR upregulated tenocyte-related genes: collagen type I (Coll. I ∼10 fold) and tenomodulin (∼3–4 fold), but did not affect non-tenocyte-related genes: LPL (adipocyte), Sox9 (chondrocyte), Runx2 and Osterix (both osteocyte). However, ITR upregulated both tenocyte (Coll. I ∼7–11 fold; tenomodulin ∼4–5 fold) and non-tenocyte-related genes (∼3–8 fold). In the in vitro study, TSCs and tenocytes were stretched to 4% and 8% using a custom made mechanical loading system. Low mechanical stretching (4%) of TSCs from both patellar and Achilles tendons increased the expression of only the tenocyte-related genes (Coll. I ∼5–6 fold; tenomodulin ∼6–13 fold), but high mechanical stretching (8%) increased the expression of both tenocyte (Coll. I ∼28–50 fold; tenomodulin ∼14–48 fold) and non-tenocyte-related genes (2–5-fold). However, in tenocytes, non-tenocyte related gene expression was not altered by the application of either low or high mechanical stretching. These findings indicate that appropriate mechanical loading could be beneficial to tendons because of their potential to induce anabolic changes in tendon cells. However, while excessive mechanical loading caused anabolic changes in tendons, it also induced differentiation of TSCs into non-tenocytes, which may lead to the development of degenerative tendinopathy frequently seen in clinical settings.     

The C1473G polymorphism in gene tph2 is the main factor mediating the genetically defined variability of tryptophan hydroxylase-2 activity in the mouse brain

Brain neurotransmitter serotonin is involved in the regulation of many physiological functions and types of behavior. The key enzyme of serotonin synthesis in the brain is tryptophan hydroxylase-2 (TPH-2). An association of the C1473G polymorphism in gene tph2 causing the replacement of Pro447 by Arg447 in TPH-2 molecule with enzyme activity in the mouse brain of 10 inbred strains was found. Association of the polymorphism with the TPH-2 activity in the brain of F2 hybrids between strains C57BL/6 and CC57BR was shown. The results indicate that the C1473G polymorphism in gene tph2 is the main factor determining the genetic defined variability of enzyme activity in the mouse brain.     

Differential effects of jump versus running exercise on trabecular architecture during remobilization after suspension-induced osteopenia in growing rats

High-impact exercise is considered to be very beneficial for bones. We investigated the ability of jump exercise to restore bone mass and structure after the deterioration induced by tail suspension in growing rats and made comparisons with treadmill running exercise. Five-week-old male Wistar rats (n = 28) were randomly assigned to four body weight-matched groups: a spontaneous recovery group after tail suspension (n = 7), a jump exercise group after tail suspension (n = 7), a treadmill running group after tail suspension (n = 7), and age-matched controls without tail suspension or exercise (n = 7). Treadmill running was performed at 25 m/min, 1 h/day, 5 days/wk. The jump exercise protocol consisted of 10 jumps/day, 5 days/wk, with a jump height of 40 cm. Bone mineral density (BMD) of the total right femur was measured by dual-energy X-ray absorptiometry. Three-dimensional trabecular bone architecture at the distal femoral metaphysis was evaluated using microcomputed tomography. After 5 wk of free remobilization, right femoral BMD, right hindlimb muscle weight, and body weight returned to age-matched control levels, but trabeculae remained thinner and less connected. Although both jump and running exercises during the remobilization period increased trabecular bone mass, jump exercise increased trabecular thickness, whereas running exercise increased trabecular number. These results indicate that restoration of trabecular bone architecture induced by jump exercise during remobilization is predominantly attributable to increased trabecular thickness, whereas running adds trabecular bone mass through increasing trabecular number, and suggest that jumping and running exercises have different mechanisms of action on structural characteristics of trabecular bone.     

The <Emphasis Type="Italic">C1473G</Emphasis> polymorphism in gene <Emphasis Type="Italic">tph2</Emphasis> is the main factor mediating the genetically defined variability of tryptophan hydroxylase-2 activity in the mouse brain

Brain neurotransmitter serotonin is involved in the regulation of many physiological functions and types of behavior. The key enzyme of serotonin synthesis in the brain is tryptophan hydroxylase-2 (TPH-2). Linkage between the C1473G polymorphism in gene tph2 causing the replacement of Pro⁴⁴⁷ by Arg⁴⁴⁷ in TPH-2 molecule and enzyme activity in the mouse brain of 10 inbred strains was found. Association of the polymorphism with the TPH-2 activity in the brain of F₂ hybrids between strains C57BL/6 and CC57BR was shown. The results indicate that the C1473G polymorphism in gene tph2 is the main factor determining the genetically defined variability of enzyme activity in the mouse brain.     

Effect of Treadmill Exercise on Serotonin Immunoreactivity in Medullary Raphe Nuclei and Spinal Cord Following Sciatic Nerve Transection in Rats

The serotoninergic system modulates nociceptive and locomotor spinal cord circuits. Exercise improves motor function and changes dopaminergic, noradrenergic, and serotonergic central systems. However, the direct relationship between serotonin, peripheral nerve lesion and aerobic treadmill exercise has not been studied. Using immunohistochemistry and optic densitometry, this study showed that the sciatic nerve transection increased the serotoninergic immunoreactivity in neuronal cytoplasm of the magnus raphe nuclei of trained and sedentary rats. In the dorsal raphe nucleus the increase only occurred in sedentary-sham-operated rats. In the spinal cord of trained, transected rats, the ventral horn showed significant changes, while the change in dorsal horn was insignificant. Von Frey’s test indicated analgesia in all exercise-trained rats. The sciatic nerve functional index indicated recovery in the trained group. Thus, both the aerobic treadmill exercise training and the nervous lesion appear to contribute to changes in serotonin immunoreactivity.     

Neuroprotective effects of treadmill exercise on BDNF and PI3-K/Akt signaling pathway in the cortex of transgenic mice model of Alzheimer’s disease

(AD). Although physical exercise and AD have received attention in the scientific literature, the mechanism through which treadmill exercise may impact the brain insulin signaling of AD has not been elucidated. This study aimed to evaluate the neuroprotective effects of treadmill exercise on apoptotic factors (Bcl-2/Bax ratio, caspase-3), HSP70, COX-2, BDNF and PI3-K/Akt signaling pathway in the cortex of NSE/hPS2m transgenic mice model of AD. Treadmill exercise ameliorated cognitive function in water maze test and significantly increased the level of Bcl-2/Bax ratio and HSP-70 in Tg-exe group compared to Tg-con group; on the other hand, it significantly decreased the expression of caspase-3 and COX-2 in Tg-exe group compared to Tg-con group. In addition, treadmill exercise significantly increased the expression of BDNF and PI3K/Akt in Tg-exe group compared to Tg-con group. Consequently, treadmill exercise improves cognitive function possibly via activating neurotrophic factor, BDNF and PI3k/Akt signaling pathway, and Aβ-induced neuronal cell death in the cortex of Tg mice was markedly suppressed following treadmill exercise. These results suggest that treadmill exercise may be beneficial in preventing or treating Alzheimer’s disease.     

Two Strategies for Response to 14°C Cold-Water Immersion: Is there a Difference in the Response of Motor,Cognitive, Immune and Stress Markers?

Here, we address the question of why some people have a greater chance of surviving and/or better resistance to cold-related-injuries in prolonged exposure to acute cold environments than do others, despite similar physical characteristics. The main aim of this study was to compare physiological and psychological reactions between people who exhibited fast cooling (FC; n = 20) or slow cooling (SC; n = 20) responses to cold water immersion. Individuals in whom the T_re decreased to a set point of 35.5°C before the end of the 170-min cooling time were indicated as the FC group; individuals in whom the T_re did not decrease to the set point of 35.5°C before the end of the 170-min cooling time were classified as the SC group. Cold stress was induced using intermittent immersion in bath water at 14°C. Motor (spinal and supraspinal reflexes, voluntary and electrically induced skeletal muscle contraction force) and cognitive (executive function, short term memory, short term spatial recognition) performance, immune variables (neutrophils, leucocytes, lymphocytes, monocytes, IL-6, TNF-α), markers of hypothalamic–pituitary–adrenal axis activity (cortisol, corticosterone) and autonomic nervous system activity (epinephrine, norepinephrine) were monitored. The data obtained in this study suggest that the response of the FC group to cooling vs the SC group response was more likely an insulative–hypothermic response and that the SC vs the FC group displayed a metabolic–insulative response. The observations that an exposure time to 14°C cold water—which was nearly twice as short (96-min vs 170-min) with a greater rectal temperature decrease (35.5°C vs 36.2°C) in the FC group compared with the SC group—induces similar responses of motor, cognitive, and blood stress markers were novel. The most important finding is that subjects with a lower cold-strain-index (SC group) showed stimulation of some markers of innate immunity and suppression of markers of specific immunity.     

Characterization of wild-type and mutant forms of human tryptophan hydroxylase 2

Tryptophan hydroxylase (TPH) catalyses the rate-limiting step in the biosynthesis of serotonin. In vertebrates, the homologous genes tph1 and tph2 encode two different enzymes with distinct patterns of expression, enzyme kinetics and regulation. Variants of TPH2 have recently reported to be associated with reduced serotonin production and behavioural alterations in man and mice. We have produced the human forms of these enzymes in Esherichia coli and in human embryonic kidney cell lines (HEK293) and examined the effects of mutations on their heterologous expression levels, solubility, thermal stability, secondary structure, and catalytic properties. Pure human TPH2 P449R (corresponds to mouse P447R) had comparable catalytic activity (V(max)) and solubility relative to the wild type, but had decreased thermal stability; whereas human TPH2 R441H had decreased activity, solubility and stability. Thus, we consider the variations in kinetic values between wild-type and TPH2 mutants to be of secondary importance to their effects on protein stability and solubility. These findings provide potential molecular explanations for disorders related to the central serotonergic system, such as depression or suicidal behaviour.     

Alpha2 adrenergic and high affinity serotonergic receptor changes in the brain stem of streptozotocin-induced diabetic rats.

The brain stems (BS) of streptozotocin (STZ)-diabetic rats were studied to see the changes in neurotransmitter content and their receptor regulation. The norepinephrine (NE) content determined in the diabetic brain stems did not show an increase, while epinephrine (EPI) content increased significantly compared with control. The NE to EPI turnover showed a significant increase. The alpha2 adrenergic receptor kinetics revealed that the receptor affinity was significantly reduced during diabetes. In insulin treated rats the NE content decreased while EPI content remained increased as in the diabetic state. Insulin treatment increased the Bmax for alpha2 adrenergic receptors significantly while the increase in Kd reversed to normal. Unlabelled clonidine inhibited [3H]NE binding in BS of control diabetic and insulin treated diabetic rats showed that alpha2 adrenergic receptors consisted of two populations of binding sites with Hill slopes significantly away from unity. In diabetic animals the ligand bound weaker to the low affinity site than in controls. Insulin treatment reversed this alteration to control levels. The displacement analysis using (-)-epinephrine against [3H]yohimbine in control and diabetic animals revealed two populations of receptor affinity states. In control animals, when GTP analogue added with epinephrine, the curve fitted for a single affinity model; but in the diabetic BS this effect was not observed. In both the diabetic and control BS the effects of monovalent cations on affinity alterations were intact. Our data thus show that alpha2 adrenergic receptors have a reduced affinity due to an altered post receptor affinity regulation The serotonin (5-HT) content in the brain stem increased. Its precursor (5-hydroxy) tryptophan (5-HTP) showed an increase and its breakdown metabolite (5-hydroxy) indoleacetic acid (5-HIAA) showed a significant decrease. This showed that in serotonergic nerves there is a disturbance in both synthetic and breakdown pathways which lead to an increased 5-HT. The high affinity serotonin receptor numbers remained unaltered with a decrease in the receptor affinity. The insulin treatment reversed these altered serotonergic receptor kinetic parameters to control level. Thus our study shows a decreased serotonergic receptor function. These changes in adrenergic and serotonergic receptor function were suggested to be important in insulin function during STZ diabetes.     

The effects of glycogen synthase kinase-3beta in serotonin neurons

Glycogen synthase kinase-3 (GSK3) is a constitutively active protein kinase in brain. Increasing evidence has shown that GSK3 acts as a modulator in the serotonin neurotransmission system, including direct interaction with serotonin 1B (5-HT1B) receptors in a highly selective manner and prominent modulating effect on 5-HT1B receptor activity. In this study, we utilized the serotonin neuron-selective GSK3β knockout (snGSK3β-KO) mice to test if GSK3β in serotonin neurons selectively modulates 5-HT1B autoreceptor activity and function. The snGSK3β-KO mice were generated by crossbreeding GSK3β-floxed mice and ePet1-Cre mice. These mice had normal growth and physiological characteristics, similar numbers of tryptophan hydroxylase-2 (TpH2)-expressing serotonin neurons, and the same brain serotonin content as in littermate wild type mice. However, the expression of GSK3β in snGSK3β-KO mice was diminished in TpH2-expressing serotonin neurons. Compared to littermate wild type mice, snGSK3β-KO mice had a reduced response to the 5-HT1B receptor agonist anpirtoline in the regulation of serotonergic neuron firing, cAMP production, and serotonin release, whereas these animals displayed a normal response to the 5-HT1A receptor agonist 8-OH-DPAT. The effect of anpirtoline on the horizontal, center, and vertical activities in the open field test was differentially affected by GSK3β depletion in serotonin neurons, wherein vertical activity, but not horizontal activity, was significantly altered in snGSK3β-KO mice. In addition, there was an enhanced anti-immobility response to anpirtoline in the tail suspension test in snGSK3β-KO mice. Therefore, results of this study demonstrated a serotonin neuron-targeting function of GSK3β by regulating 5-HT1B autoreceptors, which impacts serotonergic neuron firing, serotonin release, and serotonin-regulated behaviors.     

Expression of Components of the Serotonergic System in Folliculogenesis and Preimplantation Development in Mice

The functions of serotonin include the growth and development regulation of female germ cells as well as early embryo development. RT-PCR analysis of mRNA expression of the genes of the enzymes for synthesis and degradation and transporters and receptors of serotonin during folliculogenesis and preimplantation development of mice was performed to discover the particular mechanisms of these functions. The mRNA of tryptophan hydroxylase tph1 and monoaminoxidase maoa; membrane transporter sert and vesicular transporter vmat2; and serotonin receptors htr1b, htr1d, htr2a, htr5b, and htr7 were revealed in granulosa cells. The expression of mRNA of the aromatic amino acid decarboxylase ddc and the htr2b receptor additionally appears in the yellow body. The expression of mRNA of the genes of the tph2, ddc, and maoa enzymes; the sert, vmat1, and vmat2 transporters; and quite a number of receptors is observed during the preimplantation development, and it is transitory in most of them. The expression of all components and its dynamics suggest that the serotonergic signaling system is functionally active in mouse folliculogenesis and preimplantation development.     

Influence of a single loading episode on gene expression in healing rat Achilles tendons

Mechanical loading stimulates tendon healing via mechanisms that are largely unknown. Genes will be differently regulated in loaded healing tendons, compared with unloaded, just because of the fact that healing processes have been changed. To avoid such secondary effects and study the effect of loading per se, we therefore studied the gene expression response shortly after a single loading episode in otherwise unloaded healing tendons. The Achilles tendon was transected in 30 tail-suspended rats. The animals were let down from the suspension to load their tendons on a treadmill for 30 min once, 5 days after tendon transection. Gene expression was studied by Affymetrix microarray before and 3, 12, 24, and 48 h after loading. The strongest response in gene expression was seen 3 h after loading, when 150 genes were up- or downregulated (fold change ≥2, P ≤ 0.05). Twelve hours after loading, only three genes were upregulated, whereas 38 were downregulated. Fewer than seven genes were regulated after 24 and 48 h. Genes involved in the inflammatory response were strongly regulated at 3 and 12 h after loading; this included upregulation of iNOS, PGE synthase, and IL-1β. Also genes involved in wound healing/coagulation, angiogenesis, and production of reactive oxygen species were strongly regulated by loading. Microarray results were confirmed for 16 selected genes in a repeat experiment (N = 30 rats) using real-time PCR. It was also confirmed that a single loading episode on day 5 increased the strength of the healing tendon on day 12. In conclusion, the fact that there were hardly any regulated genes 24 h after loading suggests that optimal stimulation of healing requires a mechanical loading stimulus every day.     

Acute effects of heroin and morphine on newly synthesized serotonin in rat brain.

Heroin and morphine, in acute intraperitoneal doses of 2 and 10 mg/kg respectively, produced significant increments in the formation of newly formed brain serotonin from tritiated (³H)-L-tryptophan to ³H-serotonin. Opiate analgesia, Straub tail sign and catatonia, were observed during the increase in the synthesis of serotonin. The transport of radio-labelled tryptophan into the rat brain was not increased by the acute injection of the opiates, but brain levels of ³H-serotonin and of its main metabolite, 5-hydroxyindoleacetic acid, were significantly elevated. These opiates do not interfere with the accumulation of serotonin or with the transport of its metabolite in serotonergic neurons after inhibition of monoamine oxidases with Pargyline. An increase in the activity of tryptophan hydroxylases was more pronounced in the forebrain than in the brain stem. Stimulation of newly synthesized serotonin is probably mediated by an increase in tryptophan hydroxylase activity and not by an increase in the transport of tryptophan into the brain.     

Serotonin and Prefrontal Cortex Function: Neurons,Networks, and Circuits

Higher-order executive tasks such as learning, working memory, and behavioral flexibility depend on the prefrontal cortex (PFC), the brain region most elaborated in primates. The prominent innervation by serotonin neurons and the dense expression of serotonergic receptors in the PFC suggest that serotonin is a major modulator of its function. The most abundant serotonin receptors in the PFC, 5-HT1A, 5-HT2A and 5-HT3A receptors, are selectively expressed in distinct populations of pyramidal neurons and inhibitory interneurons, and play a critical role in modulating cortical activity and neural oscillations (brain waves). Serotonergic signaling is altered in many psychiatric disorders such as schizophrenia and depression, where parallel changes in receptor expression and brain waves have been observed. Furthermore, many psychiatric drug treatments target serotonergic receptors in the PFC. Thus, understanding the role of serotonergic neurotransmission in PFC function is of major clinical importance. Here, we review recent findings concerning the powerful influences of serotonin on single neurons, neural networks, and cortical circuits in the PFC of the rat, where the effects of serotonin have been most thoroughly studied.