Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Computational Modeling of Retinal Neurons for Visual Prosthesis Research - Fundamental Approaches

Computational modeling has become an increasingly important method in neural engineering due to its capacity to predict behaviors of in vivo and in vitro ...

Western Sydney University

The University of Sydney

Ketamine treatments and social isolation of rats reflect certain features of schizophrenia, among them altered pain sensitivity. To study the underlying mechanisms of these phenomena, rats were either housed individually or grouped for 33 days after weaning, and treated with either ketamine or saline for 14 days. After one month re-socialization, the urinary bladder capacity by ultrasound examination in the anesthetized animals, and changes of μ-opioid receptors by saturation binding experiments using a specific μ-opioid agonist [(3)H]DAMGO were determined. G-protein signaling was investigated in DAMGO-stimulated [(35)S]GTPγS functional assays. Ketamine treatment significantly decreased the bladder volume and isolation decreased the receptor density in cortical membranes. Among all groups, the only change in binding affinity was an increase induced by social isolation in the cortex. G-protein signaling was significantly decreased by either ketamine or social isolation in this tissue. Ketamine treatment, but not housing, significantly increased μ-opioid receptor densities in hippocampal membranes. Both ketamine and isolation increased the efficacy, while the potency of signaling was decreased by any treatment. Ketamine increased the receptor density and G-protein activation; while isolation decreased the efficacy of G-protein signaling in hippocampal membranes. The changes in the co-treated group were similar to those of the isolated animals in most tests. The distinct changes of opioid receptor functioning in different areas of the CNS may, at least partially, explain the augmented nociceptive threshold and morphine potency observed in these animals. Changes in the relative urinary bladder suggest a detrusor hyperreflexia, another sign of schizophrenia.

Long-lasting, distinct changes in central opioid receptor and urinary bladder functions in models of schizophrenia in rats.

We report for the first time on neuronal signaling for the evaluation of interactions between native plasmamembrane and polyamidoamine (PAMAM) dendrimers. Generation 5 polycationic (G5-NH(2)), novel β-D-glucopyranose-conjugated G5-NH(2) and generation 4.5 polyanionic (G4.5-COONa) polyamidoamine (PAMAM) dendrimers (1-0.0001 mg/ml) were applied in acute brain slices. Functional toxicity assessments-validated by fluorescence imaging of dead cells-were performed by employing electrophysiological indicators of plasma membrane breakdown and synaptic transmission relapse. Irreversible membrane depolarization and decrease of membrane resistance predicted substantial functional neurotoxicity of unmodified G5-NH(2), but not of the G4.5-COONa PAMAM dendrimers. Model calculations suggested that freely moving protonated NH(2) groups of terminal monomeric units of PAMAM dendrimers may be able directly destroy the membrane or inhibit important K(+) channel function via contacting the positively charged NH(2). In accordance, conjugation of surface amino groups by β-D-glucopyranose units reduced functional neurotoxicity that may hold great potential for biomedical applications.

Assessing toxicity of polyamidoamine dendrimers by neuronal signaling functions.

Glutamate and γ-aminobutyric acid (GABA) transporters play important roles in balancing excitatory and inhibitory signals in the brain. Increasing evidence suggest that they may act concertedly to regulate extracellular levels of the neurotransmitters.

Astrocytes convert network excitation to tonic inhibition of neurons.

Here we have studied regulatory changes of µ-opioid receptors accompanying in vivo 14-methoxymetopon treatments of rats. Previously, this ligand has been shown to be an extremely potent, centrally acting µ-opioid specific analgesic with low physical dependence, tolerance, respiratory depression, constipation and other side effects. Our work shows that it is a highly potent full agonist of µ-opioid receptor coupled G-protein signaling in vitro, alike the well-known opioid agonist, etorphine. However, unlike etorphine, which desensitized and down-regulated the endogenous µ-opioid receptors, 14-methoxymetopon, given to rats intraperitoneally (i.p.) either acutely or chronically, did not change the binding or G-protein signaling of µ-opioid receptors in rat brain subcellular membranes. Thereby, these data provide further evidence that there is no direct relationship between the efficacy of the ligand in signaling and its ability to internalize or desensitize the receptor. Viewed collectively with published work, it is discussed that µ-opioid receptors display functional selectivity, also called 'biased agonism'. This concept implies that each ligand may induce unique, ligand-specific receptor conformation that can result in distinct agonist- directed trafficking and/or signal transduction pathways associated with the receptor. Ligand-specific signaling may open up new directions for designing potent analgesics that do not interact with unwanted signaling pathways, which mediate undesired side-effects, such as tolerance and dependence.

Lack of regulatory changes of µ-opioid receptors by 14-methoxymetopon treatment in rat brain. Further evidence for functional selectivity.

Since the discovery of the endomorphins (EM), the postulated endogenous peptide agonists of the mu-opioid receptors, several analogues have been synthesized to improve their binding and pharmacological profiles. We have shown previously that a new analogue, cis-1S,2R-aminocyclohexanecarboxylic acid(2)-endomorphin-2 (ACHC-EM2), had elevated mu-receptor affinity, selectivity, and proteolytic stability over the parent compound. In the present work, we have studied its antinociceptive effects and receptor regulatory processes. ACHC-EM2 displayed a somewhat higher (60%) acute antinociceptive response than the parent peptide, EM2 (45%), which peaked at 10 min after intracerebroventricular (icv) administration in the rat tail-flick test. Analgesic tolerance developed to the antinociceptive effect of ACHC-EM2 upon its repeated icv injection that was complete by a 10-day treatment. This was accompanied by attenuated coupling of mu-sites to G-proteins in subcellular fractions of rat brain. Also, the density of mu-receptors was upregulated by about 40% in the light membrane fraction, with no detectable changes in surface binding. Distinct receptor regulatory processes were noted in subcellular fractions of rat brains made tolerant by the prototypic full mu-agonist peptide, DAMGO, and its chloromethyl ketone derivative, DAMCK. These results are discussed in light of the recently discovered phenomenon, that is, the "so-called biased agonism" or "functional selectivity".

Ligand-specific regulation of the endogenous mu-opioid receptor by chronic treatment with mu-opioid peptide agonists.

Several studies have reported that statins occasionally cause impairment of liver functions characterized by elevated serum bilirubin levels, which might be due to altered function of the multidrug resistance-associated proteins (Mrp2/3). We aimed to study the modulation of the hepatobiliary transport of bilirubin by four statin derivatives, atorvastatin, fluvastatin, pravastatin, and rosuvastatin in sandwich-cultured rat hepatocytes. All statins except pravastatin significantly inhibited the uptake of bilirubin. The biliary efflux of bilirubin conjugates was increased by pravastatin and rosuvastatin concentration dependently. Rosuvastatin stimulated not only the Mrp2 mediated biliary, but the Mrp3 mediated sinusoidal elimination, resulting in decreased intracellular bilirubin accumulation. The significantly induced Mrp2/3 protein levels (ranging from 1.5 to 1.8-fold) accounted for the elevated efflux. Cell polarization, the formation of biliary network was also significantly increased by fluvastatin, pravastatin and rosuvastatin (151%, 216% and 275% of the control, respectively). The simultaneous inhibition of the uptake and the stimulation of the sinusoidal and canalicular elimination may explain, at least in part, the clinical observation of elevated serum bilirubin levels. In conclusion, our results suggest that in spite of the elevated serum bilirubin levels, the altered Mrp2 and Mrp3 functions by statins is probably not associated with hepatotoxic effects.

Statins alter the hepatobiliary transport of unconjugated and conjugated bilirubin in sandwich-cultured rat hepatocytes.

Increasing evidence suggest that astrocytes significantly modulate neuronal function at the level of the tripartite synapse both in physiological and pathophysiological conditions. The global control of the astrocytic syncytium over neuronal networks, however, is still less recognized. Here we examined astrocytic signaling during epileptiform activity which is generally attributed to large-scale neuronal synchronization. We show that seizure-like events in the low-[Mg(2+)] in vitro epilepsy model initiate massive, long-range astrocytic synchronization which is spatiotemporally coupled to the synchronized neuronal activity reaching its maximum at the electrographic tonic/clonic transition. Cross-correlation analysis of neuronal and astrocytic Ca(2+) signaling demonstrates that high degree of synchronization arises not only among astrocytes, but also between neuronal and astrocyte populations, manifesting in astrocytic seizure-like events. We further show that astrocytic gap junction proteins contribute to astrocytic synchronization since their inhibition by carbenoxolone (CBX) or Cx43 antibody increased the interictal interval and in 41% of slices completely prevented recurrent seizure-like activity. In addition, CBX also induced unsynchronized Ca(2+) transients associated with decreasing incidence of epileptiform discharges afterwards. We propose therefore that local, unsynchronized astrocytic Ca(2+) transients inhibit, while long-range, synchronized Ca(2+) signaling contributes to the propagation of recurrent seizure-like events.

Recurrent seizure-like events are associated with coupled astroglial synchronization.

Supply of major metabolites such as γ-aminobutyric acid (GABA), β-alanine and taurine is an essential instrument that shapes signalling, proper cell functioning and survival in the brain and peripheral organs. This background motivates the synthesis of novel classes of compounds regulating their selective transport through various fluid-organ barriers via the low-affinity γ-aminobutyric acid (GABA) transporter subtype 2 (GAT2). Natural and synthetic spirocyclic compounds or therapeutics with a range of structures and biological activity are increasingly recognised in this regard. Based on pre-validated GABA transport activity, straightforward and efficient synthesis method was developed to provide an azaspiro[4.5]decane scaffold, holding a variety of charge, substituent and 3D constrain of spirocyclic amine. Investigation of the azaspiro[4.5]decane scaffold in cell lines expressing the four GABA transporter subtypes led to the discovery of a subclass of a GAT2-selective compounds with acyl-substituted azaspiro[4.5]decane core.

Straightforward and effective synthesis of γ-aminobutyric acid transporter subtype 2-selective acyl-substituted azaspiro[4.5]decanes.

Calcium-imaging is a sensitive method for monitoring calcium dynamics during neuronal activity. As intracellular calcium concentration is correlated to physiological and pathophysiological activity of neurons, calcium imaging with fluorescent indicators is one of the most commonly used techniques in neuroscience today. Current methodologies for loading calcium dyes into the tissue require prolonged incubation time (45-150 min), in addition to dissection and recovery time after the slicing procedure. This prolonged incubation curtails experimental time, as tissue is typically maintained for 6-8 hours after slicing. Using a recently introduced recovery chamber that extends the viability of acute brain slices to more than 24 hours, we tested the effectiveness of calcium AM staining following long incubation periods post cell loading and its impact on the functional properties of calcium signals in acute brain slices and wholemount retinae. We show that calcium dyes remain within cells and are fully functional >24 hours after loading. Moreover, the calcium dynamics recorded >24 hrs were similar to the calcium signals recorded in fresh tissue that was incubated for <4 hrs. These results indicate that long exposure of calcium AM dyes to the intracellular cytoplasm did not alter the intracellular calcium concentration, the functional range of the dye or viability of the neurons. This data extends our previous work showing that a custom recovery chamber can extend the viability of neuronal tissue, and reliable data for both electrophysiology and imaging can be obtained >24hrs after dissection. These methods will not only extend experimental time for those using acute neuronal tissue, but also may reduce the number of animals required to complete experimental goals.

Orsolya Kekesi

The MARCS Institute,
School of Medicine

Western Sydney University

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Computational Modeling of Retinal Neurons for Visual Prosthesis Research - Fundamental Approaches

Orsolya Kekesi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}The MARCS Institute,School of Medicine

Western Sydney University

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Computational Modeling of Retinal Neurons for Visual Prosthesis Research - Fundamental Approaches

The MARCS Institute,
School of Medicine