Biomedical subjects

Angus M Brown

Publications and source records attributed to Angus M Brown.

14 recordsLinked to original sources

PubMed2006

Fructose metabolism in the adult mouse optic nerve, a central white matter tract.

Our recent report that fructose supported the metabolism of some, but not all axons, in the adult mouse optic nerve prompted us to investigate in detail fructose metabolism in this tissue, a typical central white matter tract, as these data imply efficient fructose metabolism in the central nervous system (CNS). In artificial cerebrospinal fluid containing 10 mmol/L glucose or 20 mmol/L fructose, the stimulus-evoked compound action potential (CAP) recorded from the optic nerve consisted of three stable peaks. Replacing 10 mmol/L glucose with 10 mmol/L fructose, however, caused delayed loss of the 1st CAP peak (the 2nd and 3rd CAP peaks were unaffected). Glycogen-derived metabolic substrate(s) temporarily sustained the 1st CAP peak in 10 mmol/L fructose, as depletion of tissue glycogen by a prior period of aglycaemia or high-frequency CAP discharge rendered fructose incapable of supporting the 1st CAP peak. Enzyme assays showed the presence of both hexokinase and fructokinase (both of which can phosphorylate fructose) in the optic nerve. In contrast, only hexokinase was expressed in cerebral cortex. Hexokinase in optic nerve had low affinity and low capacity with fructose as substrate, whereas fructokinase displayed high affinity and high capacity for fructose. These findings suggest an explanation for the curious fact that the fast conducting axons comprising the 1st peak of the CAP are not supported in 10 mmol/L fructose medium; these axons probably do not express fructokinase, a requirement for efficient fructose metabolism.

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PubMed2006

A non-linear regression analysis program for describing electrophysiological data with multiple functions using Microsoft Excel.

The objective of this present study was to demonstrate a method for fitting complex electrophysiological data with multiple functions using the SOLVER add-in of the ubiquitous spreadsheet Microsoft Excel. SOLVER minimizes the difference between the sum of the squares of the data to be fit and the function(s) describing the data using an iterative generalized reduced gradient method. While it is a straightforward procedure to fit data with linear functions, and we have previously demonstrated a method of non-linear regression analysis of experimental data based upon a single function, it is more complex to fit data with multiple functions, usually requiring specialized expensive computer software. In this paper we describe an easily understood program for fitting experimentally acquired data, in this case the stimulus-evoked compound action potential from the mouse optic nerve, with multiple Gaussian functions. The program is flexible and can be applied to describe data with a wide variety of user-input functions.

Electronic Data Processing↗

PubMed2005

Fructose supports energy metabolism of some, but not all, axons in adult mouse optic nerve.

We used transmission electron microscopy (TEM) and electrophysiological techniques to characterize the morphology and stimulus-evoked compound action potential (CAP), respectively, of the adult mouse optic nerve (MON). Electrophysiological recordings demonstrated an identical CAP profile for each MON. An initial peak, smallest in area and presumably composed of the fastest-conducting axons displayed the lowest threshold for activation as expected for large axons. The second peak, the largest, was presumably composed of axons of intermediate diameter and conduction velocity, and the third peak was composed of the slowest and presumably smallest axons. In 10 mM fructose, the first CAP peak area was reduced by 78%, but the second and third peaks were unaffected. Histological analysis revealed a cross-sectional area of 33,346 microm2, containing 24,068 axons per MON. All axons were myelinated and axon diameter ranged from 0.09 to 2.58 microm, although 80 +/- 6% of the axons were <0.75 microm in diameter and only 0.6 +/- 0.3% of the axons were >2 microm in diameter. After bathing in fructose for 2 h 94 +/- 2% of normal appearing axons were <0.75 microm in diameter and none were >1.5 microm-all of the larger axons being grossly abnormal in structure. We conclude that fructose is unable to support function of the larger axons contributing to the first CAP peak, thus enabling us to identify a distinct population of axons that contributes to that peak.

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PubMed2005

Transfer of glycogen-derived lactate from astrocytes to axons via specific monocarboxylate transporters supports mouse optic nerve activity.

It is hypothesized that L-lactate derived from astrocyte glycogen sustains axon excitability in mouse optic nerve (MON). This theory was tested by using a competitive antagonist of L-lactate transport and immunocytochemistry to determine whether transport proteins are appropriately distributed in adult MON. L-lactate sustained the compound action potential (CAP), indicating that exogenous L-lactate was an effective energy substrate. During 60 min of aglycemia, the CAP persisted for 30 min, surviving on a glycogen-derived substrate (probably lactate), before failing. After failing, the CAP could be partially rescued by restoring 10 mM glucose or 20 mM L-lactate. Aglycemia in the presence of 20 mM D-lactate, a metabolically inert but transportable monocarboxylate, resulted in accelerated CAP decline compared with aglycemia alone, suggesting that D-lactate blocked the axonal uptake of glycogen-derived L-lactate, speeding the onset of energy failure and loss of the CAP. The CAP was maintained for up to 2 hr when exposed to 20% of normal bath glucose (i.e., 2 mM). To test whether glycogen-derived L-lactate "supplemented" available glucose (2 mM) in supporting metabolism, L-lactate uptake into axons was reduced by the competitive inhibitor D-lactate. Indeed, in the presence of 20 mM D-lactate, the CAP was lost more rapidly in MONs bathed in 2 mM glucose artificial cerebrospinal fluid. Immunocytochemical staining demonstrated cell-specific expression of monocarboxylate transporter (MCT) subtypes, localizing MCT2 predominantly to axons and MCT1 predominantly to astrocytes, supporting the idea that L-lactate is released from astrocytes and taken up by axons as an energy source for sustaining axon excitability.

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PubMed2005

A new software for carrying out one-way ANOVA post hoc tests.

The objective of this study was to develop a convenient, easy to use stand-alone program to statistically process experimental data. The method involves user input of data for analysis into a grid format, followed by one-way analysis of variance (ANOVA) of the data to determine the F ratio. If the value of the F ratio is greater than the appropriate critical F distribution at alpha=0.05, the null hypothesis is rejected, it is assumed that at least one of the group means is significantly different from the other group means, and it is valid to proceed with post hoc testing. A choice of four post hoc tests are available: Dunnett's, Fisher's, Scheffé's or Tukey's. The results of the post hoc test are displayed and can be printed for record keeping. The methodology developed in this present study should help scientists to carry out post hoc comparisons quickly and easily without the expense or time that commercial statistical packages require.

Analysis of Variance↗

PubMed2004

GFAP immunoreactivity and transcription in trigeminal and dental tissues of rats and transgenic GFP/GFAP mice.

Sensory mechanisms in teeth are not well understood and may involve pulpal-neural interactions. Tooth cells that proliferate in vitro have polyclonal immunoreactivity (IR) for glial fibrillary acidic protein (GFAP), growth-associated protein (GAP-43), and vimentin, plus glial-like ion channels. Here, we analyzed GFAP-IR patterns in dental and trigeminal tissues of rats, for comparison with green fluorescent protein (GFP) associated with GFAP transcription in transgenic mice, in order to better characterize glial-like cells in dental tissues. Astrocytes, ganglion satellite cells, and epineurial Schwann cells were demonstrated by anti-GFAP antibodies and GFP-GFAP, as expected. Odontoblasts did not stain by any of these methods and cannot be the glial-like cells. Fibroblasts and undifferentiated mesenchymal cells in pulp had polyclonal GFAP-IR and vimentin-IR, while nerve fibers reacted only with polyclonal antibody. Some Schwann cell subtypes in trigeminal nerve and oral mucosa were positive for GFP and for polyclonal anti-GFAP, but not for monoclonal antibody. In pulp almost all Schwann cells were unstained, but many Schwann cells in periodontal ligament had polyclonal GFAP-IR. These results show greater heterogeneity for Schwann cells than expected, and suggest that the glial-like pulp cells are fibroblasts and/or undifferentiated mesenchymal cells or stem cells. We also found that polyclonal GFAP revealed intermediate filaments in preterminal sensory nerve fibers, thereby providing a useful marker for that neural subregion. GFP transcription by some Schwann cell subtypes in oral mucosae and trigeminal nerve, but not trigeminal root was a novel finding that reveals more complexity in peripheral glia than previously recognized.

Animals↗

PubMed2004

Energy transfer from astrocytes to axons: the role of CNS glycogen.

We tested the hypothesis that astrocytic glycogen supports axon function under both pathological and physiological conditions. Functional activity of the rat (RON) or mouse optic nerve (MON), representative central white matter tracts, was assessed electrophysiologically as the area under the supramaximal compound action potential (CAP). During aglycaemia the CAP area of rodent optic nerve persisted for up to 30 min, after which the CAP rapidly failed. Glycogen content measured biochemically during the aglycaemic insult fell with a time course compatible with its rapid degradation in the absence of glucose. Pharmacological up-regulation of glycogen content prior to the aglycaemic insult with incubation in hyperglycaemic ambient glucose delayed CAP failure, whereas down-regulation of glycogen content induced by nor-adrenaline accelerated CAP failure. Inhibiting lactate transfer between astrocytes and axons during aglycaemia, where glycogen is the only utilisable energy reserve, resulted in accelerated CAP failure, implying that glycogen-derived lactate supports function when exogenous energy metabolites are withdrawn. Under normoglycaemic conditions glycogen content decreased during high frequency axon discharge, although CAP function was fully maintained. Both prior depletion of glycogen content, or blocking axonal lactate uptake rendered nerves incapable of fully supporting CAP function during high frequency firing in the presence of normoglycaemic glucose. These results indicated that during aglycaemia and increased metabolic demand, astrocytic glycogen was degraded to form lactate, which was used as a supplemental energy source when ambient normoglycaemic glucose was incapable of meeting immediate tissue energy demands.

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PubMed2004

Brain glycogen re-awakened.

The mammalian brain contains glycogen, which is located predominantly in astrocytes, but its function is unclear. A principal role for brain glycogen as an energy reserve, analogous to its role in the periphery, had been universally dismissed based on its relatively low concentration, an assumption apparently reinforced by the limited duration that the brain can function in the absence of glucose. However, during insulin-induced hypoglycaemia, where brain glucose availability is limited, glycogen content falls first in areas with the highest metabolic rate, suggesting that glycogen provides fuel to support brain function during pathological hypoglycaemia. General anaesthesia results in elevated brain glycogen suggesting quiescent neurones allow glycogen accumulation, and as long ago as the 1950s it was shown that brain glycogen accumulates during sleep, is mobilized upon waking, and that sleep deprivation results in region-specific decreases in brain glycogen, implying a supportive functional role for brain glycogen in the conscious, awake brain. Interest in brain glycogen has recently been re-awakened by the first continuous in vivo measurements using NMR spectroscopy, by the general acceptance of metabolic coupling between glia and neurones involving intercellular transfer of energy substrate, and by studies supporting a prominent physiological role for brain glycogen as a provider of supplemental energy substrate during periods of increased tissue energy demand, when ambient normoglycaemic glucose is unable to meet immediate energy requirements.

Animals↗

PubMed2003

Intracellular Ca2+ release and ischemic axon injury: the Trojan horse is back.

Ischemic injury of cells in the central nervous system is typically set in motion by influx of extracellular Ca(2+). In this issue of Neuron, Stys and colleagues propose that ischemic injury in spinal cord axons is partly the result of ryanodine receptor-mediated release of Ca(2+) from the endoplasmic reticulum (ER), a site of intracellular Ca(2+) storage.

Animals↗

PubMed2003

Glycogen regulation and functional role in mouse white matter.

CNS glycogen, contained predominantly in astrocytes, can be converted to a monocarboxylate and transported to axons as an energy source during aglycaemia. We analysed glycogen regulation and the role of glycogen in supporting neural activity in adult mouse optic nerve, a favourable white matter preparation. Axon function was quantified by measuring the compound action potential (CAP) area. During aglycaemia, axon function persisted for 20 min, then declined in conjunction with glycogen content. Lactate fully supported CAPs in the absence of glucose, but was unable to sustain glycogen content; thus, axon failure occurred rapidly when lactate was withdrawn. Glycogen content in the steady state was directly proportional to bath glucose concentration. Increasing [K+]o to 10 mM caused a rapid decrease in glycogen content. Latency to onset of CAP failure during aglycaemia was directly proportional to glycogen content and varied from about 2 to 30 min. Intense neural activity reduced glycogen content in the presence of 10 mM bath glucose and CAP area gradually declined. CAP area declined more rapidly during high frequency stimulation if monocarboxylate transport was inhibited. This suggested that astrocytic glycogen was broken down to a monocarboxylate(s) that was used by rapidly discharging axons. Likewise, depleting glycogen by brief periods of high frequency axon stimulation accelerated onset of CAP decline during aglycaemia. In summary, these experiments indicated that glycogen content was under dynamic control and that glycogen was used to support the energy needs of CNS axons during both physiological as well as pathological processes.

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PubMed2003

A modeling study predicts the presence of voltage gated Ca2+ channels on myelinated central axons.

The objective of this current study was to investigate whether voltage gated Ca(2+) channels are present on axons of the adult rat optic nerve (RON). Simulations of axonal excitability using a Hodgkin-Huxley based one-compartment model incorporating I(Na), I(K) and leak currents were used to predict conditions under which the potential contribution of a Ca(2+) current to an evoked action potential could be measured. Under control conditions the inclusion of a high threshold Ca(2+) current (I(Ca)) in the model had a negligible effect on the action potential. Reducing I(K), by decreasing the value of g(K), elongated the repolarizing phase of the action potential, increasing its duration. Subsequent incorporation of I(Ca) in the model revealed a significant I(Ca) contribution to the repolarizing phase of the action potential. The simulation thus suggests that Ca(2+) channels may be present on RON axons, but that pharmacological intervention is required to unmask their presence. Experiments based on the simulations revealed that there was no significant contribution of I(Ca) to the control action potential. However, as predicted by the simulation, reducing the repolarizing effect of I(K) by adding the K(+) channel blocker 4-AP revealed a Ca(2+) component on the repolarizing phase of the action potential that was blocked by the Ca(2+) channel inhibitor nifedipine.

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PubMed2003

Axon function persists during anoxia in mammalian white matter.

Axon function in the CNS has been reported to fail rapidly during anoxia, implying that there is no anaerobic capacity. This phenomenon was reassessed in rodent white matter using mouse or rat optic nerve. Axon function was semiquantitatively measured as area under the compound action potential. Mouse optic nerves exposed to anoxia (30-180 minutes) or cyanide (30-60 minutes) at 37 degrees C exhibited significant persistent function that was abolished by removing glucose. Reduction in compound action potential area increased with anoxia duration reaching a maximum of about 70% after 90 min. Rat optic nerves exposed to anoxia, in contrast to mouse optic nerves, showed rapid and complete loss of function. When artificial CSF glucose was increased from 10 to 30 mmol/L, rat optic nerves responded to anoxia in a similar manner to mouse optic nerves in 10-mmol/L glucose. The authors conclude that white matter is resistant to anoxia with a subset of axons able to subsist exclusively on anaerobically derived energy. Because the rat optic nerve is about twice the diameter of the mouse optic nerve, glucose diffusion into the rat optic nerve was inadequate during anoxia when artificial CSF glucose was 10 mmol/L but became adequate when artificial CSF glucose was 30 mmol/L. These observations have implications for white matter energy metabolism and susceptibility to injury during focal ischemia.

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PubMed2002

Neuroprotective effects of increased extracellular Ca(2+) during aglycemia in white matter.

We investigated the effects of extracellular [Ca(2+)] ([Ca(2+)](o)) on aglycemia-induced dysfunction and injury in adult rat optic nerves. Compound action potentials (CAPs) from adult rat optic nerve were recorded in vitro, and the area under the CAP was used to monitor nerve function before and after 1 h periods of aglycemia. In control artificial cerebrospinal fluid (ACSF) containing 2 mM Ca(2+), CAP function fell after 29.9 +/- 1.5 (SE) min and recovered to 48.8 +/- 3.9% following aglycemia. Reducing bath [Ca(2+)] during aglycemia progressively improved recovery. For example, in Ca(2+)-free ACSF, the CAP recovered to 99.1 +/- 3.8%. Paradoxically, increasing bath [Ca(2+)] also improved recovery from aglycemia. In 5 or 10 mM bath [Ca(2+)], CAP recovered to 78.8 +/- 9.2 or 91.6 +/- 5.2%, respectively. The latency to CAP failure during aglycemia increased as a function of bath [Ca(2+)] from 0 to 10 mM. Increasing bath [Mg(2+)] from 2 to 5 or 10 mM, with bath [Ca(2+)] held at 2 mM, increased latency to CAP failure with aglycemia and improved recovery from this insult. [Ca(2+)](o) recorded with calcium-sensitive microelectrodes in control ACSF, dropped reversibly during aglycemia from 1.54 +/- 0.03 to 0.45 +/- 0.04 mM. In the presence of higher ambient levels of bath [Ca(2+)] (i.e., 5 or 10 mM), the aglycemia-induced decrease in [Ca(2+)](o) declined, indicating that less Ca(2+) left the extracellular space to enter an intracellular compartment. These results indicate that the role of [Ca(2+)], and divalent cations in general, during aglycemia is complex. While extracellular Ca(2+) was required for irreversible aglycemic injury to occur, higher levels of [Ca(2+)] or [Mg(2+)] increased the latency to CAP failure and improved the extent of recovery, apparently by limiting Ca(2+) influx. These effects are theorized to be mediated by divalent cation screening.

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PubMedDate not supplied

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.

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