Trine Meldgaard Lund

Astrocyte glycogen metabolism is required for neural activity during aglycemia or intense stimulation in mouse white matter

We tested the hypothesis that inhibiting glycogen degradation accelerates compound action potential (CAP) failure in mouse optic nerve (MON) during aglycemia or high‐intensity stimulation. Axon function was assessed as the evoked CAP, and glycogen content was measured biochemically. Isofagomine, a novel inhibitor of central nervous system (CNS) glycogen phosphorylase, significantly increased glycogen content under normoglycemic conditions. When MONs were bathed in artificial cerebrospinal fluid (aCSF) containing 10 mM glucose, the CAP failed 16 min after exposure to glucose‐free aCSF. MONs bathed in aCSF plus isofagomine displayed accelerated CAP failure on glucose removal. Similar results were obtained in MONs bathed in 30 mM glucose, which increased baseline glycogen concentration. The ability of isofagomine to increase glycogen content thus was not translated into delayed CAP failure. This is likely due to the inability of the tissue to metabolize glycogen in the presence of isofagomine, highlighting the importance of glycogen in sustaining neural function during aglycemia. The hypothesis that glycogen breakdown supports intense neural activity was tested by blocking glycogen breakdown during periods of high‐frequency stimulation. The CAP area declined more rapidly when glycogen metabolism was inhibited by isofagomine, explicitly showing an important physiological role for glycogen metabolism during neural activity.

Changes in brain levels of N-acylethanolamines and 2-arachidonoylglycerol in focal cerebral ischemia in mice

The N‐acylethanolamines (NAEs) and 2‐arachidonoylglycerol (2‐AG) are bioactive lipids that can modulate inflammatory responses and protect neurons against glutamatergic excitotoxicity. We have used a model of focal cerebral ischemia in young adult mice to investigate the relationship between focal cerebral ischemia and endogenous NAEs. Over the first 24 h after induction of permanent middle cerebral artery occlusion, we observed a time‐dependent increase in all the investigated NAEs, except for anandamide. Moreover, we found an accumulation of 2‐AG at 4 h that returned to basal level 12 h after induction of ischemia. Accumulation of NAEs did not depend on regulation of N‐acylphosphatidylethanolamine‐hydrolyzing phospholipase D or fatty acid amide hydrolase. Treatment with the fatty acid amide hydrolase inhibitor URB597 (cyclohexyl carbamic acid 3′‐carbamoyl‐biphenyl‐3‐yl ester; 1 mg/kg; i.p.) 1.5 h before arterial occlusion decreased the infarct volume in our model system. Our results suggest that NAEs and 2‐AG may be involved in regulation of neuroprotection during focal cerebral ischemia in mice.

Design, synthesis and pharmacology of model compounds for indirect elucidation of the topography of AMPA receptor sites

Abstract Based on structure-activity studies on excitatory amino acids with specific agonist effect at ( RS )-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors we have earlier proposed a simple model of the AMPA receptor pharmacophore. In order to judge the capacity of this empirical model we have now synthesized and tested 3 model compounds derived from the AMPA receptor agonists, AMPA and ( RS )-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4- c ]pyridine-7-carboxylic acid (7-HPCA). These model compounds, ( RS )-2-amino-3-(5-ethyl-3-hydroxy-4-isoxazolyl)propionic acid (Et-AMPA), ( RS )-2-amino-4-(3-hydroxy-5-methyl-4-isoxazolyl)butyric acid (Homo-AMPA) and ( RS )-3-hydroxy-5,6,7,8-tetrahydro-4 H -isoxazolo[5,4- c ]azepine-8-carboxylic acid (Homo-7-HPCA) were tested electrophysiologically and in receptor binding assays. Et-AMPA was slightly more potent than AMPA as an AMPA agonist (EC 50 = 2.3 μM compared to 3.5 μM for AMPA) and as a specific inhibitor of [ 3 H]AMPA binding (IC 50 = 0.030 μM compared with 0.040 μM for AMPA), whereas Homo-AMPA was essentially inactive. Homo-7-HPCA was much weaker than 7-HPCA. These data support the view that the AMPA recognition site(s) comprise a confined region, which tightly binds the charged structure-elements of agonists molecules, and a cavity capable of accommodating bulky lipophilic groups in such compounds.

A review of morphine and morphine-6-glucuronide's pharmacokinetic-pharmacodynamic relationships in experimental and clinical pain.

Morphine is a widely used opioid for treatment of moderate to severe pain, but large interindividual variability in patient response and no clear guidance on how to optimise morphine dosage regimen complicates treatment strategy for clinicians. Population pharmacokinetic-pharmacodynamic models can be used to quantify dose-response relationships for the population as well as interindividual and interoccasion variability. Additionally, relevant covariates for population subgroups that deviate from the typical population can be identified and help clinicians in dose optimisation. This review provides a detailed overview of the published human population pharmacokinetic-pharmacodynamic studies for morphine analgesia in addition to basic drug disposition and pharmacological properties of morphine and its analgesic active metabolite, morphine-6-glucuronide, that may help identify future covariates. Furthermore, based on simulations from key pharmacokinetic-pharmacodynamic models, the contribution of morphine-6-glucuronide to the analgesic response in patients with renal insufficiency was investigated. Simulations were also used to examine the impact of effect-site equilibration half-life on time course of response. Lastly, the impact of study design on the likelihood of determining accurate pharmacodynamic parameters for morphine response was evaluated.

Availability of neurotransmitter glutamate is diminished when β‐hydroxybutyrate replaces glucose in cultured neurons

Ketone bodies serve as alternative energy substrates for the brain in cases of low glucose availability such as during starvation or in patients treated with a ketogenic diet. The ketone bodies are metabolized via a distinct pathway confined to the mitochondria. We have compared metabolism of [2,4‐13C]β‐hydroxybutyrate to that of [1,6‐13C]glucose in cultured glutamatergic neurons and investigated the effect of neuronal activity focusing on the aspartate–glutamate homeostasis, an essential component of the excitatory activity in the brain. The amount of 13C incorporation and cellular content was lower for glutamate and higher for aspartate in the presence of [2,4‐13C]β‐hydroxybutyrate as opposed to [1,6‐13C]glucose. Our results suggest that the change in aspartate–glutamate homeostasis is due to a decreased availability of NADH for cytosolic malate dehydrogenase and thus reduced malate–aspartate shuttle activity in neurons using β‐hydroxybutyrate. In the presence of glucose, the glutamate content decreased significantly upon activation of neurotransmitter release, whereas in the presence of only β‐hydroxybutyrate, no decrease in the glutamate content was observed. Thus, the fraction of the glutamate pool available for transmitter release was diminished when metabolizing β‐hydroxybutyrate, which is in line with the hypothesis of formation of transmitter glutamate via an obligatory involvement of the malate–aspartate shuttle.

Localization and pharmacological characterization of voltage dependent calcium channels in cultured neocortical neurons

The physiological significance and subcellular distribution of voltage dependent calcium channels was defined using calcium channel blockers to inhibit potassium induced rises in cytosolic calcium concentration in cultured mouse neocortical neurons. The cytosolic calcium concentration was measured using the fluorescent calcium chelator fura‐2. The types of calcium channels present at the synaptic terminal were determined by the inhibitory action of calcium channel blockers on potassium‐induced [3H]GABA release in the same cell preparation. L‐, N‐, P‐, Q‐ and R‐/T‐type voltage dependent calcium channels were differentially distributed in somata, neurites and nerve terminals. ω‐conotoxin MVIIC (ω‐CgTx MVIIC) inhibited approximately 40% of the Ca2+‐rise in both somata and neurites and 60% of the potassium induced [3H]GABA release, indicating that the Q‐type channel is the quantitatively most important voltage dependent calcium channel in all parts of the neuron. After treatment with thapsigargin the increase in cytosolic calcium was halved, indicating that calcium release from thapsigargin sensitive intracellular calcium stores is an important component of the potassium induced rise in cytosolic calcium concentration. The results of this investigation demonstrate that pharmacologically distinct types of voltage dependent calcium channels are differentially localized in cell bodies, neurites and nerve terminals of mouse cortical neurons but that the Q‐type calcium channel appears to predominate in all compartments.

Hydroxylated analogues of 5-aminovaleric acid as 4-aminobutyric acidB receptor antagonists: stereostructure-activity relationships.

Abstract: The (R) and (S) forms of 5‐amino‐2‐hydroxyvaleric acid (2‐OH‐DAVA) and 5‐amino‐4‐hydroxyvaleric acid (4‐OH‐DAVA) were designed as structural hybrids of the 4‐aminobutyric acidB (GABAB) agonist (R)‐(–)‐4‐amino‐3‐hydroxybutyric acid [(R)‐(–)‐3‐OH‐GABA] and the GABAB antagonist 5‐aminovaleric acid (DAVA). (S)‐(–)‐2‐OH‐DAVA and (R)‐(−)‐4‐OH‐DAVA showed a moderately potent affinity for GABAB receptor sites in rat brain and showed GABAB antagonist effects in a guinea pig ileurn preparation. The respective enantiomers, (R)‐(+)‐2‐OH‐DAVA and (S)‐ (+)‐4‐OH‐DAVA, were markedly weaker in both test systems. AH four compounds were weak inhibitors of GABAA receptor binding in rat brain, and none of them significantly affected synaptosomal GABA uptake. Based on molecular modeling studies it has been demonstrated that low‐energy conformations of (R)‐(–)‐3‐OH‐GABA, (S)(–)‐2‐OH‐DAVA, and (R)‐(–)4‐OH‐DAVA can be superimposed. These conformations may reflect the shapes adopted by these conforma‐tionally flexible compounds during their interaction with GABAB receptors. The present studies emphasize the similar, but distinct, constraints imposed on agonists and antagonists for GABAB receptors.

Repeated Time-to-event Analysis of Consecutive Analgesic Events in Postoperative Pain

Background:Reduction in consumption of opioid rescue medication is often used as an endpoint when investigating analgesic efficacy of drugs by adjunct treatment, but appropriate methods are needed to analyze analgesic consumption in time. Repeated time-to-event (RTTE) modeling is proposed as a way to describe analgesic consumption by analyzing the timing of consecutive analgesic events. Methods:Retrospective data were obtained from 63 patients receiving standard analgesic treatment including morphine on request after surgery following hip fracture. Times of analgesic events up to 96 h after surgery were extracted from hospital medical records. Parametric RTTE analysis was performed with exponential, Weibull, or Gompertz distribution of analgesic events using NONMEM®, version 7.2 (ICON Development Solutions, USA). The potential influences of night versus day, sex, and age were investigated on the probability. Results:A Gompertz distribution RTTE model described the data well. The probability of having one or more analgesic events within 24 h was 80% for the first event, 55% for the second event, 31% for the third event, and 18% for fourth or more events for a typical woman of age 80 yr. The probability of analgesic events decreased in time, was reduced to 50% after 3.3 days after surgery, and was significantly lower (32%) during night compared with day. Conclusions:RTTE modeling described analgesic consumption data well and could account for time-dependent changes in probability of analgesic events. Thus, RTTE modeling of analgesic events is proposed as a valuable tool when investigating new approaches to pain management such as opioid-sparing analgesia.

Role of GluR2 expression in AMPA-induced toxicity in cultured murine cerebral cortical neurons

α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPA‐R)‐mediated neurotoxicity was studied in relation to subunit expression and the presence of Ca2+‐permeable receptor channels. AMPA‐mediated toxicity had two components: 1) a direct AMPA‐R‐mediated component, which was not due to Ca2+ influx through voltage‐gated Ca2+ channels, reversal of the Na+/Ca2+ exchanger or release of calcium from dantrolene‐sensitive intracellular Ca2+ stores, and 2) a minor, indirect component involving activation of NMDA receptor channels, because of glutamate release and removal of the Mg2+ block of the NMDA receptor on AMPA‐R stimulation. The involvement of Ca2+ influx through AMPA‐R was also examined. The number of neurons possessing Ca2+‐permeable AMPA‐R increased during culture development, concurrently with an increasing susceptibility for AMPA‐induced toxicity during development. GluR2(R) levels also increased during development, and channel blockers of Ca2+‐permeable AMPA‐R lacking the GluR2(R) subunit (spermine and philanthotoxin) failed to prevent neurotoxicity or increases in [Ca2+]i. Thus, the direct AMPA‐R‐mediated toxicity may be explained by initiation of cell death by Ca2+ fluxing through AMPA‐R containing GluR2(R). The components of direct AMPA‐R‐mediated toxicity are proposed to be 1) toxicity mediated by GluR2(R)‐lacking AMPA‐R and 2) toxicity mediated by low‐Ca2+‐permeability AMPA‐R containing GluR2(R). J. Neurosci. Res. 65:267–277, 2001.

Co-administration of morphine and gabapentin leads to dose dependent synergistic effects in a rat model of postoperative pain.

Despite much evidence that combination of morphine and gabapentin can be beneficial for managing postoperative pain, the nature of the pharmacological interaction of the two drugs remains unclear. The aim of this study was to assess the interaction of morphine and gabapentin in range of different dose combinations and investigate whether co-administration leads to synergistic effects in a preclinical model of postoperative pain. The pharmacodynamic effects of morphine (1, 3 and 7mg/kg), gabapentin (10, 30 and 100mg/kg) or their combination (9 combinations in total) were evaluated in the rat plantar incision model using an electronic von Frey device. The percentage of maximum possible effect (%MPE) and the area under the response curve (AUC) were used for evaluation of the antihyperalgesic effects of the drugs. Identification of synergistic interactions was based on Loewe additivity response surface analyses. The combination of morphine and gabapentin resulted in synergistic antihyperalgesic effects in a preclinical model of postoperative pain. The synergistic interactions were found to be dose dependent and the increase in observed response compared to the theoretical additive response ranged between 26 and 58% for the synergistic doses. The finding of dose-dependent synergistic effects highlights that choosing the right dose-dose combination is of importance in postoperative pain therapy. Our results indicate benefit of high doses of gabapentin as adjuvant to morphine. If these findings translate to humans, they might have important implications for the treatment of pain in postoperative patients.