Coenzyme Q10 supplementation improves the motor function of middle-aged mice by restoring the neuronal activity of the motor cortex.

Physiological aging causes motor function decline and anatomical and biochemical changes in the motor cortex. We confirmed that middle-aged mice at 15-18 months old show motor function decline, which can be restored to the young adult level by supplementing with mitochondrial electron transporter coenzyme Q10 (CoQ10) as a water-soluble nanoformula by drinking water for 1 week. CoQ10 supplementation concurrently improved brain mitochondrial respiration but not muscle strength. Notably, we identified an age-related decline in field excitatory postsynaptic potential (fEPSP) amplitude in the pathway from layers II/III to V of the primary motor area of middle-aged mice, which was restored to the young adult level by supplementing with CoQ10 for 1 week but not by administering CoQ10 acutely to brain slices. Interestingly, CoQ10 with high-frequency stimulation induced NMDA receptor-dependent long-term potentiation (LTP) in layer V of the primary motor cortex of middle-aged mice. Importantly, the fEPSP amplitude showed a larger input‒output relationship after CoQ10-dependent LTP expression. These data suggest that CoQ10 restores the motor function of middle-aged mice by improving brain mitochondrial function and the basal fEPSP level of the motor cortex, potentially by enhancing synaptic plasticity efficacy. Thus, CoQ10 supplementation may ameliorate the age-related decline in motor function in humans.

Volatile anesthetic sevoflurane pretreatment alleviates hypoxia-induced potentiation of excitatory inputs to striatal medium spiny neurons of mice.

Sevoflurane, a commonly used anesthetic in surgery, has drawn attention because of its preconditioning effects in hypoxic conditions. To investigate the preconditioning effects in the striatum, a common site for ischemic stroke, we collected whole-cell current-clamp recordings from striatal medium spiny neurons. In our in vitro brain slice experiments, deprivation of oxygen and glucose depolarized the striatal neurons to subthreshold potentials, and the pre-administration of sevoflurane (4%, 15 min) prolonged the time to depolarization. Furthermore, transient hypoxia induced the potentiation of excitatory postsynaptic potentials, which play a part in post-ischemic excitotoxicity. Glibenclamide, a KATP channel inhibitor, reversed the prolonged time to depolarization and the prevention of the pathological potentiation of excitatory responses, indicating that the short exposure to sevoflurane likely participates in neuroprotection against hypoxia via activation of KATP channels. A monocarboxylate transporter blocker, 4-CIN, also depolarized striatal neurons. Interestingly, the blockade of monocarboxylate transporters that supply lactate to neurons caused the pathological potentiation, even in the presence of enough oxygen and glucose. In this case, sevoflurane could not prevent the pathological potentiation, suggesting the involvement of monocarboxylate transporters in the sevoflurane-mediated effects. These results indicate that sevoflurane protects striatal neurons from hypoxic damage and alleviates the pathological potentiation. Under these conditions, sevoflurane may become an effective intervention for patients undergoing surgery.

Modulation of hyperpolarization-activated cation current Ih by volatile anesthetic sevoflurane in the mouse striatum during postnatal development.

Volatile anesthetics have been reported to inhibit hyperpolarization-activated cyclic-nucleotide gated channels underlying the hyperpolarization-activated cation current (Ih) that contributes to generation of synchronized oscillatory neural rhythms. Meanwhile, the developmental change of Ih has been speculated to play a pivotal role during maturation. In this study, we examined the effect of the volatile anesthetic sevoflurane, which is widely used in pediatric surgery, on Ih and on functional Ih activation kinetics of cholinergic interneurons in developing striatum. Our analyses showed that the changes in Ih of cholinergic interneurons occurred in conjunction with maturation. Sevoflurane application (1-4%) caused significant inhibition of Ih in a dose-dependent manner, and apparently slowed Ih activation. In current-clamp recordings, sevoflurane significantly decreased spike firing during the rebound activation, which is essential for responses to the sensory inputs from the cortex and thalamus. The sevoflurane-induced inhibition of Ih in striatal cholinergic interneurons may lead to alterations of the acetylcholine-dopamine balance in the neural circuits during the early postnatal period.

Phosphoproteomics of the Dopamine Pathway Enables Discovery of Rap1 Activation as a Reward Signal In Vivo.

Dopamine (DA) type 1 receptor (D1R) signaling in the striatum presumably regulates neuronal excitability and reward-related behaviors through PKA. However, whether and how D1Rs and PKA regulate neuronal excitability and behavior remain largely unknown. Here, we developed a phosphoproteomic analysis method to identify known and novel PKA substrates downstream of the D1R and obtained more than 100 candidate substrates, including Rap1 GEF (Rasgrp2). We found that PKA phosphorylation of Rasgrp2 activated its guanine nucleotide-exchange activity on Rap1. Cocaine exposure activated Rap1 in the nucleus accumbens in mice. The expression of constitutively active PKA or Rap1 in accumbal D1R-expressing medium spiny neurons (D1R-MSNs) enhanced neuronal firing rates and behavioral responses to cocaine exposure through MAPK. Knockout of Rap1 in the accumbal D1R-MSNs was sufficient to decrease these phenotypes. These findings demonstrate a novel DA-PKA-Rap1-MAPK intracellular signaling mechanism in D1R-MSNs that increases neuronal excitability to enhance reward-related behaviors.

Effects of the volatile anesthetic sevoflurane on tonic GABA currents in the mouse striatum during postnatal development.

The volatile anesthetic sevoflurane, which is widely used in pediatric surgery, has proposed effects on GABAA receptor-mediated extrasynaptic tonic inhibition. In the developing striatum, medium-sized spiny projection neurons have tonic GABA currents, which function in the excitatory/inhibitory balance and maturation of striatal neural circuits. In this study, we examined the effects of sevoflurane on the tonic GABA currents of medium spiny neurons in developing striatal slices. Sevoflurane strongly increased GABAA receptor-mediated tonic conductance at postnatal days 3-35. The antagonist of the GABA transporter-1, 1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride further increased tonic GABA conductance during the application of sevoflurane, thereby increasing the total magnitude of tonic currents. Both GABA (5 µM) and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride, the δ-subunit-containing GABAA receptor agonist, induced tonic GABA currents in medium spiny neurons but not in cholinergic neurons. However, sevoflurane additively potentiated the tonic GABA currents in both cells. Interestingly, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride-sensitive neurons made a large current response to sevoflurane, indicating the contribution of the δ-subunit on sevoflurane-enhanced tonic GABA currents. Our findings suggest that sevoflurane can affect the tone of tonic GABA inhibition in a developing striatal neural network.

Postnatal development of tyrosine hydroxylase mRNA-expressing neurons in mouse neostriatum.

The striatum harbors a small number of tyrosine hydroxylase (TH) mRNA-containing GABAergic neurons that express TH immunoreactivity after dopamine depletion, some of which reportedly resembled striatal medium spiny projection neurons (MSNs). To clarify whether the TH mRNA-expressing neurons were a subset of MSNs, we characterized their postnatal development of electrophysiological and morphological properties using a transgenic mouse strain expressing enhanced green fluorescent protein (EGFP) under the control of the rat TH gene promoter. At postnatal day (P)1, EGFP-TH+ neurons were present as clusters in the striatum and, thereafter, gradually scattered ventromedially by P18 without regard to the striatal compartments. They were immunonegative for calbindin, but immunopositive for enkephalin (54.5%) and dynorphin (80.0%). Whole-cell patch-clamp recordings revealed at least two distinct neuronal types, termed EGFP-TH+ Type A and B. Whereas Type B neurons were aspiny and negative for the MSN marker dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa (DARPP-32), Type A neurons constituted 75% of the EGFP+ cells, had dendritic spines (24.6%), contained DARPP-32 (73.6%) and a proportion acquired TH immunoreactivity after injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 3-nitropropionic acid. The membrane properties and N-methyl-d-aspartate : non-N-methyl-d-aspartate excitatory postsynaptic current ratio of Type A neurons were very similar to MSNs at P18. However, their resting membrane potentials and spike widths were statistically different from those of MSNs. In addition, the calbindin-like, DARPP-32-like and dynorphin B-like immunoreactivity of Type A neurons developed differently from that of MSNs in the matrix. Thus, Type A neurons closely resemble MSNs, but constitute a cell type distinct from classical MSNs.

Activin plays a key role in the maintenance of long-term memory and late-LTP.

A recent study has revealed that fear memory may be vulnerable following retrieval, and is then reconsolidated in a protein synthesis-dependent manner. However, little is known about the molecular mechanisms of these processes. Activin betaA, a member of the TGF-beta superfamily, is increased in activated neuronal circuits and regulates dendritic spine morphology. To clarify the role of activin in the synaptic plasticity of the adult brain, we examined the effect of inhibiting or enhancing activin function on hippocampal long-term potentiation (LTP). We found that follistatin, a specific inhibitor of activin, blocked the maintenance of late LTP (L-LTP) in the hippocampus. In contrast, administration of activin facilitated the maintenance of early LTP (E-LTP). We generated forebrain-specific activin- or follistatin-transgenic mice in which transgene expression is under the control of the Tet-OFF system. Maintenance of hippocampal L-LTP was blocked in the follistatin-transgenic mice. In the contextual fear-conditioning test, we found that follistatin blocked the formation of long-term memory (LTM) without affecting short-term memory (STM). Furthermore, consolidated memory was selectively weakened by the expression of follistatin during retrieval, but not during the maintenance phase. On the other hand, the maintenance of memory was also influenced by activin overexpression during the retrieval phase. Thus, the level of activin in the brain during the retrieval phase plays a key role in the maintenance of long-term memory.

Synthetic fibril peptide promotes clearance of scrapie prion protein by lysosomal degradation.

Transmissible spongiform encephalopathies are infectious and neurodegenerative disorders that cause neural deposition of aggregates of the disease-associated form of PrP(Sc). PrP(Sc) reproduces by recruiting and converting the cellular PrP(C), and ScN2a cells support PrP(Sc) propagation. We found that incubation of ScN2a cells with a fibril peptide named P9, which comprises an intrinsic sequence of residues 167-184 of mouse PrP(C), significantly reduced the amount of PrP(Sc) in 24 hr. P9 did not affect the rates of synthesis and degradation of PrP(C). Interestingly, immunofluorescence analysis showed that the incubation of ScN2a cells with P9 induced colocalization of the accumulation of PrP with cathepsin D-positive compartments, whereas the accumulation of PrP in the cells without P9 colocalized mainly with lysosomal associated membrane proteins (LAMP)-1-positive compartments but rarely with cathepsin D-positive compartments in perinuclear regions. Lysosomal enzyme inhibitors attenuated the anti-PrP(Sc) activity; however, a proteasome inhibitor did not impair P9 activity. In addition, P9 neither promoted the ubiquitination of cellular proteins nor caused the accumulation of LC3-II, a biochemical marker of autophagy. These results indicate that P9 promotes PrP(Sc) redistribution from late endosomes to lysosomes, thereby attaining PrP(Sc) degradation.

Roles of micro-opioid receptors in GABAergic synaptic transmission in the striosome and matrix compartments of the striatum.

The striatum is divided into two compartments, the striosomes and extrastriosomal matrix, which differ in several cytochemical markers, input-output connections, and time of neurogenesis. Since it is thought that limbic, reward-related information and executive aspects of behavioral information may be differentially processed in the striosomes and matrix, respectively, intercompartmental communication should be of critical importance to proper functioning of the basal ganglia-thalamocortical circuits. Cholinergic interneurons are in a suitable position for this communication since they are preferentially located in the striosome-matrix boundaries and are known to elicit a conditioned pause response during sensorimotor learning. Recently, micro-opioid receptor (MOR) activation was found to presynaptically suppress the amplitude of GABAergic inhibitory postsynaptic currents in striosomal cells but not in matrix cells. Disinhibition of cells in the striosomes is further enhanced by inactivation of the protein kinase C cascade. We discuss in this review the possibility that MOR activation in the striosomes affects the activity of cholinergic interneurons and thus leads to changes in synaptic efficacy in the striatum.

Recombinant human fibrinogen expressed in the yeast Pichia pastoris was assembled and biologically active.

Fibrinogen is a large plasma glycoprotein with a molecular mass of 340kDa that plays a critical role in the final stage of blood coagulation. Human plasma fibrinogen is a dimeric molecule comprising two sets of three different polypeptides (Aalpha, 66kDa; Bbeta, 55kDa; gamma, 48kDa). To express recombinant human fibrinogen in the methylotrophic yeast Pichia pastoris, we constructed an expression vector containing three individual fibrinogen chain cDNAs under the control of the mutated AOX2 (mAOX2) promoter. First, P. pastoris GTS115 was transformed with the vector, but the expressed recombinant fibrinogen suffered severe degradation by yeast-derived proteases under conventional nutrient culture conditions. Fibrinogen degradation was prevented by using the protease A-deficient strain SMD1168 as a host strain and regulating the pH of the culture to between 5.5 and 7.0. Western blot analysis revealed that the Aalpha, Bbeta and gamma chains of recombinant fibrinogen were assembled and secreted as a complete molecule. The Bbeta chain of the recombinant fibrinogen was N-glycosylated but the Aalpha chain, as in plasma fibrinogen, was not. The gamma chains however were heterologous, one being N-glycosylated and the other not. The recombinant fibrinogen was capable of forming a thrombin-induced clot in the presence of factor XIIIa and both the glycosylated and the non-glycosylated gamma chains were involved in the formation of cross-linking fibrin. The present study indicates that the recombinant fibrinogen expressed in P. pastoris, although different from plasma fibrinogen in post-translational modification, is correctly assembled and biologically active.

Compartment-specific modulation of GABAergic synaptic transmission by mu-opioid receptor in the mouse striatum with green fluorescent protein-expressing dopamine islands.

The striatum is a heterogeneous mosaic of two neurochemically, developmentally, and functionally distinct compartments: the mu-opioid receptor (MOR)-enriched striosomes and the matrix. Preferential activation of the striosomes and persistent suppression of the matrix have recently been suggested to represent neural correlates of motor stereotypy. However, little is known concerning the physiological properties of the striosomes. We made patch-clamp recordings from medium spiny neurons in identified MOR-immunoreactive "dopamine islands" as striosomes in a slice preparation taken from transgenic mice expressing green fluorescent protein in tyrosine hydroxylase mRNA-containing neurons. Striosomal neurons differed electrophysiologically from cells in the matrix in having significantly less hyperpolarized resting membrane potentials and larger input resistances, suggesting developmental differences between the two types of cells. Moreover, corticostriatal EPSCs were inhibited by MOR activation to similar extents in the two compartments, although inhibition of IPSCs was observed only in the striosomes. This MOR-induced inhibition of IPSCs was presynaptically mediated, because MOR agonist invariably decreased IPSC amplitudes when postsynaptic G-protein was inactivated, significantly increased the paired-pulse ratio of the IPSCs, and decreased the frequency but not the amplitude of miniature IPSCs. These effects of MOR were mediated principally by 4-aminopyridine-sensitive K+ conductance via a cAMP-dependent pathway, which was further augmented by previous blockade of the protein kinase C cascade. These findings suggest that MOR activation by endogenous and/or exogenous MOR-selective opioid substances differentially regulates the activities of the striosome and matrix compartments and thus plays an important role in motivated behavior and learning.

Chronic nicotine treatment increases GABAergic input to striatal neurons.

Recent studies suggest the involvement of the dorsal striatum in the advanced stages of drug addiction as well as motor functions. We investigated the effect of chronic nicotine treatment on GABAergic synaptic transmission in the striatum of mice. Intrastriatal stimulation evoked GABAA receptor-mediated polysynaptic inhibitory postsynaptic currents more frequently in medium-sized spiny projection neurons of mice treated chronically with nicotine (1 mg/kg, twice-daily subcutaneous injections for 10-15 days) than in those of PBS-treated mice. The multiphasic inhibitory postsynaptic currents consisted of monosynaptic early and polysynaptic, nicotinic acetylcholine receptor-mediated late components. Dihydro-beta-erythroidine, an antagonist of the non-alpha7nicotinic acetylcholine receptor, suppressed only the late inhibitory postsynaptic current. These results suggest that chronic nicotine treatment increases GABAergic input to projection neurons in the dorsal striatum.

A potent adjuvant monophosphoryl lipid A triggers various immune responses, but not secretion of IL-1beta or activation of caspase-1.

Lipid A, the membrane anchor portion of LPS, is responsible for the endotoxin activity of LPS and induces many inflammatory responses in macrophages. Monophosphoryl lipid A (MPL), a lipid A derivative lacking a phosphate residue, induces potent immune responses with low toxicity. To elucidate the mechanism underlying the low toxicity of MPL, we examined the effects of MPL on the secretion of proinflammatory cytokines by mouse peritoneal macrophages, a murine macrophage-like cell line (RAW 264.7), and a human macrophage-like cell line (THP-1). MPL enhanced the secretion of TNF-alpha, but not that of IL-1beta, whereas Escherichia coli-type lipid A (natural source-derived and chemically synthesized lipid A) enhanced the secretion of both cytokines. Although MPL enhanced the levels of IL-1beta mRNA and IL-1beta precursor protein to levels similar to those induced by lipid A, IL-1beta precursor processing in MPL-treated cells was much lower than that in E. coli-type lipid A-treated ones. Moreover, MPL, unlike E. coli-type lipid A, failed to induce activation of caspase-1, which catalyzes IL-1beta precursor processing. These results suggest that an immune response without activation of caspase-1 or secretion of IL-1beta results in the low toxicity of this adjuvant.

A case of small cell carcinoma of the common bile duct.

Small cell carcinoma occasionally occurs in the gastrointestinal tract, but rarely in the biliary tract. We report a case of small cell carcinoma which occurred in the common bile duct. A 66-year-old female complained of epigastralgia and weight loss. Computed tomography and ultrasonography showed a mass near the pancreas head and dilatation of the intrahepatic bile ducts. Endoscopic nasobiliary drainage was undertaken, and it revealed obstruction of the common bile duct. The patient was diagnosed preoperatively as having extrahepatic bile duct cancer. Upon laparotomy, a tumor was found to be located in the middle common bile duct. Pylorus-preserving pancreaticoduodenectomy was performed. The main trunk of the portal vein and the right hepatic artery were resected concomitantly because of tumor involvement. Postoperative pathological examination revealed well-differentiated papillary adenocarcinoma on the surface of the bile duct lumen, but a large part of the extraductal component was small cell carcinoma. Upon immunohistochemical examination, synaptophysin and chromogranin A were found to be focally positive in small cell carcinoma, but negative for L-26 and CEA. The patient then underwent two postoperative courses of systemic chemotherapy. Nevertheless, she died of cancer recurrence eight months after the operation, which showed that the tumor had a highly lethal nature, with rapid and widespread dissemination. Further therapeutic trials are needed to improve survival in such cases.

Dopamine D4 receptor-induced postsynaptic inhibition of GABAergic currents in mouse globus pallidus neurons.

Dopamine D4 receptors (D4R) are localized in the globus pallidus (GP), but their function remains unknown. In contrast, dopamine D2 receptor activation hyperpolarizes medium spiny neurons projecting from the striatum to the GP and inhibits GABA release. However, using slice preparations from D2R-deficient [D2 knock-out (D2KO)] mice, we found that dopamine inhibited GABA(A)-receptor-mediated currents in GP neurons. The paired-pulse ratio was statistically unchanged after dopamine application but was significantly elevated in D2KO wild-type littermates (WT). Furthermore, in D2KO mice, outward currents elicited by iontophoretically applied GABA were suppressed by dopamine. Dopamine (30 microm) decreased the amplitude of miniature IPSCs in both WT and D2KO mice, but the decrease in the frequency was observed only in the former but not significantly in the latter. Dopamine-induced suppression of IPSCs was blocked by selective D4R antagonists (clozapine or 3-[4-(4-iodophenyl)piperazin-1-yl]methyl-1H-pyrrolo[2,3-b]pyridine trihydrochloride), and a D4R-selective agonist N-[[4-(2-cyanophenyl)-1-piperazinyl]methyl]-3-methyl-benzamide reversibly and dose-dependently suppressed IPSCs, whereas agonists [SKF38,393 ((+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride) or (+)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol] or antagonists [SCH23,390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) or sulpiride] of other receptor subtypes had little effect. In GP neurons from D4R-deficient mice, dopamine-induced inhibition of GABAergic outward currents was undetectable. D4R activation suppressed the activity of protein kinase A in GP neurons, resulting in a decrease in the amplitude of GABAergic IPSCs. These findings showed that postsynaptic activation of D4R on the GP neurons reduces GABAergic currents through the suppression of PKA activity.

Hepatic resection under in situ hypothermic hepatic perfusion.

BACKGROUND/AIMS: Temporary inflow occlusion of the portal triad has been used frequently in hepatectomy to minimize bleeding. On the other hand, Pringle's maneuver produces ischemic-reperfusion injury especially in patients with underlying liver disease. METHODOLOGY: Thirty-seven cases of hepatic resections were performed with intermittent Pringle's maneuver (IP group; n = 17) and in situ hypothermic perfusion (CP group; n = 20). In the CP group, hepatic inflow was continuously occluded, and 4-degree Centigrade Ringer's lactate was administered by drip during resection. Hepatic outflow occlusion was not performed. RESULTS: All patients tolerated the procedures well. Cold perfusion technique significantly decreased both the times required and the blood loss in hepatectomy (p < 0.05). Serum hyaluronic acid levels gradually increased after the induction of hepatectomy and peaked 10 minutes after reperfusion in the both groups. Thereafter, it decreased and showed a significantly lower level in the CP group until 60 minutes after reperfusion (p < 0.05). Hepaplastin levels remained significantly higher in the CP group one week after operation (p < 0.05). CONCLUSIONS: Using the technique of in situ hypothermic perfusion, we can prolong the ischemic time safely with minimal systemic influence even in cases with underlying liver diseases. This may compare favorably with intermittent Pringle's maneuver in terms of reducing hepatic sinusoidal endothelial cell damage during hepatectomy and reperfusion.