Wfs1‐deficient animals have brain‐region‐specific changes of Na+, K+‐ATPase activity and mRNA expression of α1 and β1 subunits

Mutations in the WFS1 gene, which encodes the endoplasmic reticulum (ER) glycoprotein, cause Wolfram syndrome, a disease characterized by juvenile‐onset diabetes mellitus, optic atrophy, deafness, and different psychiatric abnormalities. Loss of neuronal cells and pancreatic β‐cells in Wolfram syndrome patients is probably related to the dysfunction of ER stress regulation, which leads to cell apoptosis. The present study shows that Wfs1‐deficient mice have brain‐region‐specific changes in Na⁺,K⁺‐ATPase activity and in the expression of the α₁ and β₁ subunits. We found a significant (1.6‐fold) increase of Na‐pump activity and β₁ subunit mRNA expression in mice lacking the Wfs1 gene in the temporal lobe compared with their wild‐type littermates. By contrast, exposure of mice to the elevated plus maze (EPM) model of anxiety decreased Na‐pump activity 1.3‐fold in the midbrain and dorsal striatum and 2.0‐fold in the ventral striatum of homozygous animals compared with the nonexposed group. Na‐pump α₁‐subunit mRNA was significantly decreased in the dorsal striatum and midbrain of Wfs1‐deficient homozygous animals compared with wild‐type littermates. In the temporal lobe, an increase in the activity of the Na‐pump is probably related to increased anxiety established in Wfs1‐deficient mice, whereas the blunted dopamine function in the forebrain of Wfs1‐deficient mice may be associated with a decrease of Na‐pump activity in the dorsal and ventral striatum and in the midbrain after exposure to the EPM. © 2014 Wiley Periodicals, Inc.     

Evaluation of (‐)‐[18F]Flubatine‐specific binding: Implications for reference region approaches

We aimed to characterize changes in binding of (‐)‐[¹⁸F]Flubatine to α₄β₂*‐nicotinic acetylcholine receptors (α₄β₂*‐nAChRs) during a tobacco cigarette smoking challenge. Displacement of (‐)‐[¹⁸F]Flubatine throughout the brain was quantified as change in (‐)‐[¹⁸F]Flubatine distribution volume (V_T), with particular emphasis on regions with low V_T. Three tobacco smokers were imaged with positron emission tomography (PET) during a 210 min bolus‐plus‐constant infusion of (‐)‐[¹⁸F]Flubatine. A tobacco cigarette was smoked in the PET scanner ～125 min after the start of (‐)‐[¹⁸F]Flubatine injection. Equilibrium analysis was used to estimate V_T at baseline (90‐120 min) and after cigarette challenge (180‐210 min), at the time of greatest receptor occupancy by nicotine. Smoking reduced V_T by 21 ± 9% (average ±SD) in corpus callosum, 17 ± 9% in frontal cortex, 36 ± 11% in cerebellum, and 22 ± 10% in putamen. The finding of displaceable (‐)‐[¹⁸F]Flubatine binding throughout the brain is an important consideration for reference region‐based quantification approaches with this tracer. We observed displacement of (‐)‐[¹⁸F]Flubatine binding to α₄β₂*‐nicotinic acetylcholine receptors in corpus callosum by a tobacco cigarette challenge. We conclude that reference region approaches utilizing corpus callosum should first perform careful characterization of displaceable (‐)‐[¹⁸F]Flubatine binding and nondisplaceable kinetics in this putative reference region.     

Differential contributions of Ca2+‐activated K+ channels and Na+/K+‐ATPases to the generation of the slow afterhyperpolarization in CA1 pyramidal cells

In many types of CNS neurons, repetitive spiking produces a slow afterhyperpolarization (sAHP), providing sustained, intrinsically generated negative feedback to neuronal excitation. Changes in the sAHP have been implicated in learning behaviors, in cognitive decline in aging, and in epileptogenesis. Despite its importance in brain function, the mechanisms generating the sAHP are still controversial. Here we have addressed the roles of M‐type K⁺ current (I_M), Ca²⁺‐gated K⁺ currents (I_Ca(K)'s) and Na⁺/K⁺‐ATPases (NKAs) current to sAHP generation in adult rat CA1 pyramidal cells maintained at near‐physiological temperature (35 °C). No evidence for I_M contribution to the sAHP was found in these neurons. Both I_Ca(K)'s and NKA current contributed to sAHP generation, the latter being the predominant generator of the sAHP, particularly when evoked with short trains of spikes. Of the different NKA isoenzymes, α₁‐NKA played the key role, endowing the sAHP a steep voltage‐dependence. Thus normal and pathological changes in α₁‐NKA expression or function may affect cognitive processes by modulating the inhibitory efficacy of the sAHP.     

Endogenous purines modulate K+‐evoked ACh secretion at the mouse neuromuscular junction

At the mouse neuromuscular junction, adenosine triphosphate (ATP) is co‐released with the neurotransmitter acetylcholine (ACh), and once in the synaptic cleft, it is hydrolyzed to adenosine. Both ATP/adenosine diphosphate (ADP) and adenosine modulate ACh secretion by activating presynaptic P2Y₁₃ and A₁, A_2A, and A₃ receptors, respectively. To elucidate the action of endogenous purines on K⁺‐dependent ACh release, we studied the effect of purinergic receptor antagonists on miniature end‐plate potential (MEPP) frequency in phrenic diaphragm preparations. At 10 mM K⁺, the P2Y₁₃ antagonist N‐[2‐(methylthio)ethyl]‐2‐[3,3,3‐trifluoropropyl]thio‐5′‐adenylic acid, monoanhydride with (dichloromethylene)bis[phosphonic acid], tetrasodium salt (AR‐C69931MX) increased asynchronous ACh secretion while the A₁, A₃, and A_2A antagonists 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), (3‐Ethyl‐5‐benzyl‐2‐methyl‐4‐phenylethynyl‐6‐phenyl‐1, 4‐(±)‐dihydropyridine‐3,5‐, dicarboxylate (MRS‐1191), and 2‐(2‐Furanyl)‐7‐(2‐phenylethyl)‐7H‐pyrazolo[4,3‐e][1,2,4]triazolo[1,5‐c]pyrimidin‐5‐amine (SCH‐58261) did not modify neurosecretion. The inhibition of equilibrative adenosine transporters by S‐(p‐nitrobenzyl)‐6‐thioinosine provoked a reduction of 10 mM K⁺‐evoked ACh release, suggesting that the adenosine generated from ATP is being removed from the synaptic space by the transporters. At 15 and 20 mM K⁺, endogenous ATP/ADP and adenosine bind to inhibitory P2Y₁₃ and A₁ and A₃ receptors since AR‐C69931MX, DPCPX, and MRS‐1191 increased MEPP frequency. Similar results were obtained when the generation of adenosine was prevented by using the ecto‐5′‐nucleotidase inhibitor α,β‐methyleneadenosine 5′‐diphosphate sodium salt. SCH‐58261 only reduced neurosecretion at 20 mM K⁺, suggesting that more adenosine is needed to activate excitatory A_2A receptors. At high K⁺ concentration, the equilibrative transporters appear to be saturated allowing the accumulation of adenosine in the synaptic cleft. In conclusion, when motor nerve terminals are depolarized by increasing K⁺ concentrations, the ATP/ADP and adenosine endogenously generated are able to modulate ACh secretion by sequential activation of different purinergic receptors.     

Lambert–Eaton syndrome antibodies target multiple subunits of voltage‐gated Ca2+ channels

Introduction: Lambert–Eaton myasthenic syndrome (LEMS) is an autoimmune presynaptic neuromuscular disorder. Autoantibodies against subunits of voltage‐gated calcium channels (VGCCs) associated with acetylcholine release are thought to cause LEMS. Methods: HEK293 cells expressing specific individual recombinant subunits of α_1A, α_1B, α_1C, and α_1E; β₃; and α₂δ of human neuronal VGCCs were exposed to antibodies from 3 LEMS patients, 1 patient with small‐cell lung carcinoma, and 1 with myasthenia gravis. Results: All LEMS patient antibodies bound to cells containing any of the α₁ or β₃ subunits alone or combined with α₂δ subunits, but not α₂δ alone. Autoantibodies from the patient with small‐cell lung carcinoma but not the myasthenia gravis patient targeted the same VGCC subunits. Conclusions: Autoantibodies from LEMS patients bind directly to multiple VGCC α₁ subunits as well as the β₃ subunit. Thus, multiple components of the presynaptic VGCC complex are prospective targets for antibodies in LEMS. Muscle Nerve 51 : 176–184, 2015     

Estimation of skeletal muscle energy metabolism in Machado‐Joseph disease using 31P‐MR spectroscopy

The aim of this study was to determine if muscle energy metabolism, as measured by ³¹P‐magnetic resonance spectroscopy (MRS), is a metabolic marker for the efficacy of treatment of Machado‐Joseph disease (MJD). We obtained ³¹P‐MRS in the calf muscle of 8 male patients with MJD and 11 healthy men before, during, and after a 4 minute plantar flexion exercise in a supine position. The data showed that there was a significant difference between the groups in terms of the PCr/(Pi + PCr) ratio at rest (P = 0.03) and the maximum rate of mitochondrial ATP production (V_max) (P < 0.01). In addition, V_max was inversely correlated with the scale for the assessment and rating of ataxia score (r = ?0.34, P = 0.04). The MJD group also showed a reduction in V_max over the course of 2 years (P < 0.05). These data suggest that this noninvasive measurement of muscle energy metabolism may represent a surrogate marker for MJD. © 2010 Movement Disorder Society     

Evaluation in monkey of two candidate PET radioligands, [11C]RX‐1 and [18F]RX‐2, for imaging brain 5‐HT4 receptors

The serotonin subtype‐4 (5‐HT₄) receptor, which is known to be involved physiologically in learning and memory, and pathologically in Alzheimer's disease, anxiety, and other neuropsychiatric disorders—has few radioligands readily available for imaging in vivo. We have previously reported two novel 5‐HT₄ receptor radioligands, namely [methoxy‐¹¹C](1‐butylpiperidin‐4‐yl)methyl 4‐amino‐3‐methoxybenzoate; [¹¹C]RX‐1), and the [¹⁸F]3‐fluoromethoxy analog ([¹⁸F]RX‐2), and in this study we evaluated them by PET in rhesus monkey. Brain scans were performed at baseline, receptor preblock or displacement conditions using SB 207710, a 5‐HT₄ receptor antagonist, on the same day for [¹¹C]RX‐1 and on different days for [¹⁸F]RX‐2. Specific‐to‐nondisplaceable ratio (BP_ND) was measured with the simplified reference tissue model from all baseline scans. To determine specific binding, total distribution volume (V_T) was also measured in some monkeys by radiometabolite‐corrected arterial input function after ex vivo inhibition of esterases from baseline and blocked scans. Both radioligands showed moderate to high peak brain uptake of radioactivity (2–6 SUV). Regional BP_ND values were in the rank order of known 5‐HT₄ receptor distribution with a trend for higher BP_ND values from [¹⁸F]RX‐2. One‐tissue compartmental model provided good fits with well identified V_T values for both radioligands. In the highest 5‐HT₄ receptor density region, striatum, 50–60% of total binding was specific. The V_T in receptor‐poor cerebellum reached stable values by about 60 min for both radioligands indicating little influence of radiometabolites on brain signal. In conclusion, both [¹¹C]RX‐1 and [¹⁸F]RX‐2 showed positive attributes for PET imaging of brain 5‐HT₄ receptors, validating the radioligand design strategy. Synapse 68:613–623, 2014 . © 2014 Wiley Periodicals, Inc.     

α2‐Adrenergic drugs modulate the binding of [18F]fallypride to dopamine D2/3 receptors in striatum of living mouse

Aim. To test for α₂ adrenergic modulation of dopamine D_2/3 receptor availability in striatum of living mice using the high‐affinity ligand [¹⁸F]fallypride and microPET. Methods. Groups of anesthetized mice were pretreated with saline, the α₂‐agonist clonidine (1 mg/kg), and the α₂‐antagonists RX821002 (1 mg/kg) and yohimbine (1 mg/kg). Dynamic microPET recordings lasting 120 min were then initiated upon i.v. tracer injection of [¹⁸F]fallypride. Parametric maps of [¹⁸F]fallypride binding potential (BP_ND) were calculated using the Logan method, with cerebellum serving as the reference region. Results. Mean striatal [¹⁸F]fallypride BP_ND was 10.6 ± 1.7 in the saline control animals, 8.9 ± 1.7 (?16%; P < 0.05) in the RX821002 group, 8.3 ± 2.6 (?22%; P < 0.05) in the yohimbine group and 10.3 ± 2.2 (n.s.) in the clonidine group. Conclusions. These findings are consistent with a tonic inhibition of dopamine release by α₂ adrenergic receptors, such that α₂ blockade increased the competition from endogenous dopamine at D_2/3 receptors, thus reducing the [¹⁸F]fallypride BP_ND by about 20%. Absent effects of clonidine suggest a ceiling effect in the tonic inhibition of dopamine release. This in vivo PET evidence for α₂/dopaminergic interaction may be relevant to putative actions of atypical antipsychotic medications via adrenergic receptors. Synapse, 2010. © 2010 Wiley‐Liss, Inc.     

Regulatory volume increase in astrocytes exposed to hypertonic medium requires β1‐adrenergic Na+/K+‐ATPase stimulation and glycogenolysis

The cotransporter of Na⁺, K⁺, 2Cl^–, and water, NKKC1, is activated under two conditions in the brain, exposure to highly elevated extracellular K⁺ concentrations, causing astrocytic swelling, and regulatory volume increase in cells shrunk in response to exposure to hypertonic medium. NKCC1‐mediated transport occurs as secondary active transport driven by Na⁺/K⁺‐ATPase activity, which establishes a favorable ratio for NKCC1 operation between extracellular and intracellular products of the concentrations of Na⁺, K⁺, and Cl^– × Cl^–. In the adult brain, astrocytes are the main target for NKCC1 stimulation, and their Na⁺/K⁺‐ATPase activity is stimulated by elevated K⁺ or the β‐adrenergic agonist isoproterenol. Extracellular K⁺ concentration is normal during regulatory volume increase, so this study investigated whether the volume increase occurred faster in the presence of isoproterenol. Measurement of cell volume via live cell microscopic imaging fluorescence to record fluorescence intensity of calcein showed that this was the case at isoproterenol concentrations of ≥1 µM in well‐differentiated mouse astrocyte cultures incubated in isotonic medium with 100 mM sucrose added. This stimulation was abolished by the β₁‐adrenergic antagonist betaxolol, but not by ICI118551, a β₂‐adrenergic antagonist. A large part of the β₁‐adrenergic signaling pathway in astrocytes is known. Inhibitors of this pathway as well as the glycogenolysis inhibitor 1,4‐dideoxy‐1,4‐imino‐D‐arabinitol hydrochloride and the NKCC1 inhibitors bumetanide and furosemide abolished stimulation by isoproterenol, and it was weakened by the Na⁺/K⁺‐ATPase inhibitor ouabain. These observations are of physiological relevance because extracellular hypertonicity occurs during intense neuronal activity. This might trigger a regulatory volume increase, associated with the post‐excitatory undershoot. © 2014 Wiley Periodicals, Inc.     

Comparing α7 nicotinic acetylcholine receptor binding,amyloid‐β deposition,and mitochondria complex‐I function in living brain: A PET study in aged monkeys

This study was aimed to assess the correlations among α7 nicotinic acetylcholine receptor (α7‐nAChR) binding, amyloid‐β (Aβ) deposition, and mitochondrial complex I (MC‐I) activity in the brain of aged monkeys (Macaca mulatta). Positron emission tomography (PET) measurements with [¹¹C](R)‐MeQAA, [¹¹C]PIB, and [¹⁸F]BCPP‐EF were conducted in monkeys in a conscious condition. [¹¹C](R)‐MeQAA binding was analyzed by a simplified reference tissue model to calculate nondisplaceable binding potential (BP_ND), [¹¹C]PIB uptake was calculated by standard uptake value ratio (SUVR), and [¹⁸F]BCPP‐EF binding was determined by Logan graphical analysis to calculate total distribution volume (V_T) with arterial blood sampling. Higher brain uptake was determined in the thalamus, hippocampus, striatum, and cortical regions for [¹¹C](R)‐MeQAA, while being lower in the cerebellum. Significant age‐related reduction of [¹¹C](R)‐MeQAA binding to α7‐nAChR was determined only in the occipital cortex. The plot of Vt of [¹⁸F]BCPP‐EF against BP_ND of [¹¹C](R)‐MeQAA indicated a significant negative correlation in the hippocampus and cortical regions in aged animals. Plotting of SUVR of [¹¹C]PIB against BP_ND of [¹¹C](R)‐MeQAA showed a positive correlation. The in vivo binding of [¹¹C](R)‐MeQAA could reflect the upregulation of α7‐nAChR induced by neurodegenerative damage determined by Aβ deposition as well as impaired MC‐I activity in living brain. Synapse 69:475–483, 2015. © 2015 Wiley Periodicals, Inc.     

Cytochrome P450 2D6 enzyme neuroprotects against 1‐methyl‐4‐phenylpyridinium toxicity in SH‐SY5Y neuronal cells

Cytochrome P450 (CYP) 2D6 is an enzyme that is expressed in liver and brain. It can inactivate neurotoxins such as 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine, 1,2,3,4‐tetrahydroisoquinoline and β‐carbolines. Genetically slow CYP2D6 metabolizers are at higher risk for developing Parkinson’s disease, a risk that increases with exposure to pesticides. The goal of this study was to investigate the neuroprotective role of CYP2D6 in an in‐vitro neurotoxicity model. SH‐SY5Y human neuroblastoma cells express CYP2D6 as determined by western blotting, immunocytochemistry and enzymatic activity. CYP2D6 metabolized 3‐[2‐(N,N‐diethyl‐N‐methylammonium)ethyl]‐7‐methoxy‐4‐methylcoumarin and the CYP2D6‐specific inhibitor quinidine (1 μm ) blocked 96 ± 1% of this metabolism, indicating that CYP2D6 is functional in this cell line. Treatment of cells with CYP2D6 inhibitors (quinidine, propanolol, metoprolol or timolol) at varying concentrations significantly increased the neurotoxicity caused by 1‐methyl‐4‐phenylpyridinium (MPP⁺) at 10 and 25 μm by between 9 ± 1 and 22 ± 5% (P < 0.01). We found that CYP3A is also expressed in SH‐SY5Y cells and inhibiting CYP3A with ketoconazole significantly increased the cell death caused by 10 and 25 μm of MPP⁺ by between 8 ± 1 and 30 ± 3% (P < 0.001). Inhibiting both CYP2D6 and CYP3A showed an additive effect on MPP⁺ neurotoxicity. These data further support a possible role for CYP2D6 in neuroprotection from Parkinson’s disease‐causing neurotoxins, especially in the human brain where expression of CYP2D6 is high in some regions (e.g. substantia nigra).     

A novel Na+‐Independent alanine‐serine‐cysteine transporter 1 inhibitor inhibits both influx and efflux of D‐Serine

NMDA receptor dysfunctions are hypothesized to underlie the pathophysiology of schizophrenia, and treatment with D‐serine (D‐Ser), an NMDA receptor coagonist, may improve the clinical symptoms of schizophrenia. Thus, upregulating the synaptic D‐Ser level is a novel strategy for schizophrenia treatment. Na⁺‐independent alanine‐serine‐cysteine transporter 1 (asc‐1) is a transporter responsible for regulating the extracellular D‐Ser levels in the brain. In this study, we discovered a novel asc‐1 inhibitor, (+)‐amino(1‐(3,5‐dichlorophenyl)‐3,5‐dimethyl‐1H‐pyrazol‐4‐yl)acetic acid (ACPP), and assessed its pharmacological profile. ACPP inhibited the D‐[³H]Ser uptake in human asc‐1‐expressing CHO cells and rat primary neurons with IC₅₀ values of 0.72 ± 0.13 and 0.89 ± 0.30 μM, respectively. In accordance with the lower asc‐1 expression levels in astrocytes, ACPP did not inhibit D‐Ser uptake in rat primary astrocytes. In a microdialysis study, ACPP dose dependently decreased the extracellular D‐Ser levels in the rat hippocampus under the same conditions in which the asc‐1 inhibitor S‐methyl‐L‐cysteine (SMLC) increased it. To obtain insights into this difference, we conducted a D‐[³H]Ser efflux assay using asc‐1‐expressing CHO cells. ACPP inhibited D‐[³H]Ser efflux, whereas SMLC increased it. These results suggest that ACPP is a novel inhibitor of asc‐1. © 2016 Wiley Periodicals, Inc.     

Astrocytic poly(ADP‐ribose) polymerase‐1 activation leads to bioenergetic depletion and inhibition of glutamate uptake capacity

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a ubiquitous nuclear enzyme involved in genomic stability. Excessive oxidative DNA strand breaks lead to PARP‐1‐induced depletion of cellular NAD⁺, glycolytic rate, ATP levels, and eventual cell death. Glutamate neurotransmission is tightly controlled by ATP‐dependent astrocytic glutamate transporters, and thus we hypothesized that astrocytic PARP‐1 activation by DNA damage leads to bioenergetic depletion and compromised glutamate uptake. PARP‐1 activation by the DNA alkylating agent, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), caused a significant reduction of cultured cortical astrocyte survival (EC₅₀ = 78.2 ± 2.7 μM). HPLC revealed MNNG‐induced time‐dependent reductions in NAD⁺ (98%, 4 h), ATP (71%, 4 h), ADP (63%, 4 h), and AMP (66%, 4 h). The maximal [³H]glutamate uptake rate (V_max) also declined in a manner that corresponded temporally with ATP depletion, falling from 19.3 ± 2.8 in control cells to 2.1 ± 0.8 nmol/min/mg protein 4 h post‐MNNG. Both bioenergetic depletion and loss of glutamate uptake capacity were attenuated by genetic deletion of PARP‐1, directly indicating PARP‐1 involvement, and by adding exogenous NAD⁺ (10 mM). In mixed neurons/astrocyte cultures, MNNG neurotoxicity was partially mediated by extracellular glutamate and was reduced by co‐culture with PARP‐1^−/− astrocytes, suggesting that impairment of astrocytic glutamate uptake by PARP‐1 can raise glutamate levels sufficiently to have receptor‐mediated effects at neighboring neurons. Taken together, these experiments showed that PARP‐1 activation leads to depletion of the total adenine nucleotide pool in astrocytes and severe reduction in neuroprotective glutamate uptake capacity. © 2009 Wiley‐Liss, Inc.     

Differential influence of propofol and isoflurane anesthesia in a non‐human primate on the brain kinetics and binding of [18F]DPA‐714, a positron emission tomography imaging marker of glial activation

Translocator protein 18 kDa (TSPO) expression at the mitochondrial membrane of glial cells is related to glial activation. TSPO radioligands such as [¹⁸F]DPA‐714 are useful for the non‐invasive study of neuroimmune processes using positron emission tomography (PET). Anesthetic agents were shown to impact mitochondrial function and may influence [¹⁸F]DPA‐714 binding parameters and PET kinetics. [¹⁸F]DPA‐714 PET imaging was performed in Papio anubis baboons anesthetized using either intravenous propofol (n = 3) or inhaled isoflurane (n = 3). Brain kinetics and metabolite‐corrected input function were measured to estimate [¹⁸F]DPA‐714 brain distribution (V_T). Displacement experiments were performed using PK11195 (1.5 mg/kg). In vitro [¹⁸F]DPA‐714 binding experiments were performed using baboon brain tissue in the absence and presence of tested anesthetics. Brain radioactivity peaked higher in isoflurane‐anesthetized animals compared with propofol (SUV_max = 2.7 ± 0.5 vs. 1.3 ± 0.2, respectively) but was not different after 30 min. Brain V_T was not different under propofol and isoflurane. Displacement resulted in a 35.8 ± 8.4% decrease of brain radioactivity under propofol but not under isoflurane (0.1 ± 7.0%). In vitro, the presence of propofol increased TSPO density and dramatically reduced its affinity for [¹⁸F]DPA‐714 compared with control. This in vitro effect was not significant with isoflurane. Exposure to propofol and isoflurane differentially influences TSPO interaction with its specific radioligand [¹⁸F]DPA‐714 with subsequent impact on its tissue kinetics and specific binding estimated in vivo using PET. Therefore, the choice of anesthetics and their potential influence on PET data should be considered for the design of imaging studies using TSPO radioligands, especially in a translational research context.     

Comparative pharmacokinetic analysis of USL255, a new once‐daily extended‐release formulation of topiramate

Purpose: To compare the pharmacokinetics of USL255, a once‐daily extended‐release (ER) formulation of topiramate (TPM), with Topamax (immediate‐release TPM) in healthy subjects after oral dosing and evaluate the effect of food on USL255 bioavailability and pharmacokinetics. Methods: This randomized, single‐center, open‐label, cross‐over design study had three dosing periods separated by 21 days of washout between treatments. Thirty‐six volunteers received single doses of USL255 (200 mg) in fasted and fed conditions and two doses of Topamax (100 mg) administered 12 h apart. TPM plasma samples were analyzed by liquid chromatography–mass spectroscopy. Pharmacokinetic parameters were calculated by noncompartmental methods. Key Findings: USL255 fasted pharmacokinetic parameters [point estimate (90% confidence interval, CI) compared to Topamax] were: relative bioavailability (F´) 91.2% (84–99%), peak plasma concentration (C_max) USL255/Topamax‐ratio 59% (53–65%), time to reach C_max (t_max) 19.5 ± 7.2 h, accumulation ratio (R_ac) 3.9 ± 1.2, effective half‐life (t_1/2,eff) 55.7 ± 19.9 h, terminal half‐life (t_1/2,z) 80.2 ± 14.2 h, and peak‐occupancy‐time (POT) 12.1 ± 4.0 h. Although the F´ and C_max were unaffected by food, R_ac and t_1/2,eff increased to 4.9 ± 0.9, and 72.5 ± 15.4 h, respectively. In contrast to t_1/2,z, t_1/2,eff reflects absorption rate; therefore, USL255’s t_1/2,eff was significantly longer than Topamax’s t_1/2,eff (37.1 ± 6.5 h). Significance: Although bioequivalent to Topamax in extent of absorption, USL255 had a slower absorption rate as reflected in its lower C_max and longer t_max, larger POT and longer t_1/2,eff, and similar R_ac values to that of Topamax (q12 h). This relative flat plasma profile allows for once‐daily dosing with diminished fluctuations in TPM plasma levels. In addition, neither USL255’s peak nor extent of plasma exposure of TPM was affected by food.     

α4βδ‐GABAA receptors in dorsal hippocampal CA1 of adolescent female rats traffic to the plasma membrane of dendritic spines following voluntary exercise and contribute to protection of animals from activity‐based anorexia through localization at excitatory synapses

In hippocampal CA1 of adolescent female rodents, α4βδ‐GABA_A receptors (α4βδ‐GABA_ARs) suppress excitability of pyramidal neurons through shunting inhibition at excitatory synapses. This contributes to anxiolysis of stressed animals. Socially isolated adolescent female rats with 8 days of wheel access, the last 4 days of which entail restricted food access, have been shown to exhibit excessive exercise, choosing to run instead of eat (activity‐based anorexia [ABA]). Upregulation of α4βδ‐GABA_ARs in the dorsal hippocampal CA1 (DH), seen among some ABA animals, correlates with suppression of excessive exercise. We used electron microscopic immunocytochemistry to show that exercise alone (EX), but not food restriction alone (FR), also augments α4βδ‐GABA_AR expression at axospinous excitatory synapses of the DH (67%, P = 0.027), relative to socially isolated controls without exercise or food restriction (CON). Relative to CON, ABA animals' synaptic α4βδ‐GABA_AR elevation was modestly elevated (37%), but this level correlated strongly and negatively with individual differences in ABA vulnerability—i.e., food restriction–evoked hyperactivity (Pearson R = ?0.902, P = 0.002) and weight changes (R = 0.822, P = 0.012). These correlations were absent from FR and EX brains or ventral hippocampus of ABA brains. Comparison to CON of α4βδ‐GABA_AR location in the DH indicated that ABA induces trafficking of α4βδ‐GABA_AR from reserve pools in spine cytoplasm to excitatory synapses. Pair‐housing CON animals reduced cytoplasmic α4βδ‐GABA_AR without reducing synaptic α4βδ‐GABA_AR. Thus, exercise induces trafficking of α4βδ‐GABA_ARs to excitatory synapses, while individual differences in ABA vulnerability are linked most strongly to trafficking of α4βδ‐GABA_ARs in the reverse direction—from excitatory synapses to the reserve pool during co‐occurring food restriction. © 2017 Wiley Periodicals, Inc.     

Involvement of R‐type Ca2+ channels in neurotransmitter release from spinal dorsolateral funiculus terminals synapsing motoneurons

Molecular studies have revealed the presence of R‐type voltage‐gated Ca²⁺ channels at pre‐ and postsynaptic regions; however, no evidence for the participation of these channels in transmitter release has been presented for the spinal cord. Here we characterize the effects of SNX‐482, a selective R channel blocker, on the monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in motoneurons by stimulation of dorsolateral funiculus (DLF) terminals in a slice preparation from the adult turtle spinal cord. SNX‐482 inhibited neurotransmission in a dose‐dependent manner, with an IC₅₀ of ～9 ± 1 nM. The EPSP time course and membrane time constant of the motoneurons were not altered, suggesting a presynaptic mechanism. The toxin inhibited the residual component of the EPSPs recorded in the presence of N‐ and P/Q‐type Ca²⁺ channel blockers, strongly suggesting a role for the R channels in neurotransmission at the spinal cord DLF terminals. Consistently with this, RT‐PCR analysis of turtle spinal cord segments revealed the expression of the Ca_V2.3 pore‐forming (α_1E) subunit of R channels, whereas the use of anti‐α_1E‐specific antibodies resulted in its localization in the DLF fibers as demonstrated by immunohistochemistry coupled with laser confocal microscopy. J. Comp. Neurol. 513:188–196, 2009. © 2009 Wiley‐Liss, Inc.     

Validation of [11C]ORM‐13070 as a PET tracer for alpha2c‐adrenoceptors in the human brain

This study explored the use of the α_2C‐adrenoceptor PET tracer [¹¹C]ORM‐13070 to monitor α_2C‐AR occupancy in the human brain. The subtype‐nonselective α₂‐AR antagonist atipamezole was administered to eight healthy volunteer subjects to determine its efficacy and potency (E_max and EC₅₀) at inhibiting tracer uptake. We also explored whether the tracer could reveal changes in the synaptic concentrations of endogenous noradrenaline in the brain, in response to several pharmacological and sensory challenge conditions. We assessed occupancy from the bound‐to‐free ratio measured during 5–30 min post injection. Based on extrapolation of one‐site binding, the maximal extent of inhibition of striatal [¹¹C]ORM‐13070 uptake (E_max) achievable by atipamezole was 78% (95% CI 69–87%) in the caudate nucleus and 65% (53–77%) in the putamen. The EC₅₀ estimates of atipamezole (1.6 and 2.5 ng/ml, respectively) were in agreement with the drug's affinity to α_2C‐ARs. These findings represent clear support for the use of [¹¹C]ORM‐13070 for monitoring drug occupancy of α_2C‐ARs in the living human brain. Three of the employed noradrenaline challenges were associated with small, approximately 10–16% average reductions in tracer uptake in the dorsal striatum (atomoxetine, ketamine, and the cold pressor test; P < 0.05 for all), but insulin‐induced hypoglycemia did not affect tracer uptake. The tracer is suitable for studying central nervous system receptor occupancy by α_2C‐AR ligands in human subjects. [¹¹C]ORM‐13070 also holds potential as a tool for in vivo monitoring of synaptic concentrations of noradrenaline, but this remains to be further evaluated in future studies. Synapse 69:172–181, 2015. © 2014 Wiley Periodicals, Inc.