John C. G. Nicholson, Ph.D.

Enhanced Striatal Dopamine Transmission and Motor Performance with LRRK2 Overexpression in Mice Is Eliminated by Familial Parkinson's Disease Mutation G2019S
Li, Xianting; Patel, Jyoti C; Wang, Jing; Avshalumov, Marat V; Nicholson, Charles; Buxbaum, Joseph D; Elder, Gregory A; Rice, Margaret E; Yue, Zhenyu
2010 Feb 3;30(5):1788-1797, Journal of neuroscience
PARK8/LRRK2 (leucine-rich repeat kinase 2) was recently identified as a causative gene for autosomal dominant Parkinson's disease (PD), with LRRK2 mutation G2019S linked to the most frequent familial form of PD. Emerging in vitro evidence indicates that aberrant enzymatic activity of LRRK2 protein carrying this mutation can cause neurotoxicity. However, the physiological and pathophysiological functions of LRRK2 in vivo remain elusive. Here we characterize two bacterial artificial chromosome (BAC) transgenic mouse strains overexpressing LRRK2 wild-type (Wt) or mutant G2019S. Transgenic LRRK2-Wt mice had elevated striatal dopamine (DA) release with unaltered DA uptake or tissue content. Consistent with this result, LRRK2-Wt mice were hyperactive and showed enhanced performance in motor function tests. These results suggest a role for LRRK2 in striatal DA transmission and the consequent motor function. In contrast, LRRK2-G2019S mice showed an age-dependent decrease in striatal DA content, as well as decreased striatal DA release and uptake. Despite increased brain kinase activity, LRRK2-G2019S overexpression was not associated with loss of DAergic neurons in substantia nigra or degeneration of nigrostriatal terminals at 12 months. Our results thus reveal a pivotal role for LRRK2 in regulating striatal DA transmission and consequent control of motor function. The PD-associated mutation G2019S may exert pathogenic effects by impairing these functions of LRRK2. Our LRRK2 BAC transgenic mice, therefore, could provide a useful model for understanding early PD pathological events
— id: 106517, year: 2010, vol: 30, page: 1788, stat: Journal Article,

Calcium diffusion enhanced after cleavage of negatively charged components of brain extracellular matrix by chondroitinase ABC
Hrabetova, Sabina; Masri, Daniel; Tao, Lian; Xiao, Fanrong; Nicholson, Charles
2009 Aug 15;587(Pt 16):4029-4049, Journal of physiology
The concentration of extracellular calcium plays a critical role in synaptic transmission and neuronal excitability as well as other physiological processes. The time course and extent of local fluctuations in the concentration of this ion largely depend on its effective diffusion coefficient (D*) and it has been speculated that fixed negative charges on chondroitin sulphate proteoglycans (CSPGs) and other components of the extracellular matrix may influence calcium diffusion because it is a divalent cation. In this study we used ion-selective microelectrodes combined with pressure ejection or iontophoresis of ions from a micropipette to quantify diffusion characteristics of neocortex and hippocampus in rat brain slices. We show that D* for calcium is less than the value predicted from the behaviour of the monovalent cation tetramethylammonium (TMA), a commonly used diffusion probe, but D* for calcium increases in both brain regions after the slices are treated with chondroitinase ABC, an enzyme that predominantly cleaves chondroitin sulphate glycans. These results suggest that CSPGs do play a role in determining the local diffusion properties of calcium in brain tissue, most likely through electrostatic interactions mediating rapid equilibrium binding. In contrast, chondroitinase ABC does not affect either the TMA diffusion or the extracellular volume fraction, indicating that the enzyme does not alter the structure of the extracellular space and that the diffusion of small monovalent cations is not affected by CSPGs in the normal brain ionic milieu. Both calcium and CSPGs are known to have many distinct roles in brain physiology, including brain repair, and our study suggests they may be functionally coupled through calcium diffusion properties
— id: 101635, year: 2009, vol: 587, page: 4029, stat: Journal Article,

Characterizing molecular probes for diffusion measurements in the brain
Kaur, Gurjinder; Hrabetova, Sabina; Guilfoyle, David N; Nicholson, Charles; Hrabe, Jan
2008 Jun 30;171(2):218-225, Journal of neuroscience methods
Brain diffusion properties are at present most commonly evaluated by magnetic resonance (MR) diffusion imaging. MR cannot easily distinguish between the extracellular and intracellular signal components, but the older technique of real-time iontophoresis (RTI) detects exclusively extracellular diffusion. Interpretation of the MR results would therefore benefit from auxiliary RTI measurements. This requires a molecular probe detectable by both techniques. Our aim was to specify a minimum set of requirements that such a diffusion probe should fulfill and apply it to two candidate probes: the cation tetramethylammonium (TMA(+)), used routinely in the RTI experiments, and the anion hexafluoroantimonate ( [Formula: see text] ). Desirable characteristics of a molecular diffusion probe include predictable diffusion properties, stability, minimum interaction with cellular physiology, very slow penetration into the cells, and sufficiently strong and selective MR and RTI signals. These properties were evaluated using preparations of rat neocortical slices under normal and ischemic conditions, as well as solutions and agarose gel. While both molecules can be detected by MR and RTI, neither proved an ideal candidate. TMA(+) was very stable but it penetrated into the cells and accumulated there within tens of minutes. [Formula: see text] did not enter the cells as readily but it was not stable, particularly in ischemic tissue and at higher temperatures. Its presence also resulted in a decreased extracellular volume. These probe properties help to interpret previously published MR data on TMA(+) diffusion and might play a role in other diffusion experiments obtained with them
— id: 79402, year: 2008, vol: 171, page: 218, stat: Journal Article,

Diffusion in brain extracellular space
Sykova, Eva; Nicholson, Charles
2008 Oct;88(4):1277-1340, Physiological reviews
Diffusion in the extracellular space (ECS) of the brain is constrained by the volume fraction and the tortuosity and a modified diffusion equation represents the transport behavior of many molecules in the brain. Deviations from the equation reveal loss of molecules across the blood-brain barrier, through cellular uptake, binding, or other mechanisms. Early diffusion measurements used radiolabeled sucrose and other tracers. Presently, the real-time iontophoresis (RTI) method is employed for small ions and the integrative optical imaging (IOI) method for fluorescent macromolecules, including dextrans or proteins. Theoretical models and simulations of the ECS have explored the influence of ECS geometry, effects of dead-space microdomains, extracellular matrix, and interaction of macromolecules with ECS channels. Extensive experimental studies with the RTI method employing the cation tetramethylammonium (TMA) in normal brain tissue show that the volume fraction of the ECS typically is approximately 20% and the tortuosity is approximately 1.6 (i.e., free diffusion coefficient of TMA is reduced by 2.6), although there are regional variations. These parameters change during development and aging. Diffusion properties have been characterized in several interventions, including brain stimulation, osmotic challenge, and knockout of extracellular matrix components. Measurements have also been made during ischemia, in models of Alzheimer's and Parkinson's diseases, and in human gliomas. Overall, these studies improve our conception of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment. Knowledge of ECS diffusion properties is valuable in contexts ranging from understanding extrasynaptic volume transmission to the development of paradigms for drug delivery to the brain
— id: 96819, year: 2008, vol: 88, page: 1277, stat: Journal Article,

In vivo diffusion of lactoferrin in brain extracellular space is regulated by interactions with heparan sulfate
Thorne, Robert G; Lakkaraju, Aparna; Rodriguez-Boulan, Enrique; Nicholson, Charles
2008 Jun 17;105(24):8416-8421, Proceedings of the National Academy of Sciences of the United States of America
The intercellular spaces between neurons and glia contain an amorphous, negatively charged extracellular matrix (ECM) with the potential to shape and regulate the distribution of many diffusing ions, proteins and drugs. However, little evidence exists for direct regulation of extracellular diffusion by the ECM in living tissue. Here, we demonstrate macromolecule sequestration by an ECM component in vivo, using quantitative diffusion measurements from integrative optical imaging. Diffusion measurements in free solution, supported by confocal imaging and binding assays with cultured cells, were used to characterize the properties of a fluorescently labeled protein, lactoferrin (Lf), and its association with heparin and heparan sulfate in vitro. In vivo diffusion measurements were then performed through an open cranial window over rat somatosensory cortex to measure effective diffusion coefficients (D*) under different conditions, revealing that D* for Lf was reduced approximately 60% by binding to heparan sulfate proteoglycans, a prominent component of the ECM and cell surfaces in brain. Finally, we describe a method for quantifying heparan sulfate binding site density from data for Lf and the structurally similar protein transferrin, allowing us to predict a low micromolar concentration of these binding sites in neocortex, the first estimate in living tissue. Our results have significance for many tissues, because heparan sulfate is synthesized by almost every type of cell in the body. Quantifying ECM effects on diffusion will also aid in the modeling and design of drug delivery strategies for growth factors and viral vectors, some of which are likely to interact with heparan sulfate
— id: 80294, year: 2008, vol: 105, page: 8416, stat: Journal Article,

Diffusion of flexible random-coil dextran polymers measured in anisotropic brain extracellular space by integrative optical imaging
Xiao, Fanrong; Nicholson, Charles; Hrabe, Jan; Hrabetova, Sabina
2008 Aug;95(3):1382-1392, Biophysical journal
There are a limited number of methods available to quantify the extracellular diffusion of macromolecules in an anisotropic brain region, e.g., an area containing numerous aligned fibers where diffusion is faster along the fibers than across. We applied the integrative optical imaging method to measure diffusion of the fluorophore Alexa Fluor 488 (molecular weight (MW) 547) and fluorophore-labeled flexible random-coil dextran polymers (dex3, MW 3000; dex75, MW 75,000; dex282, MW 282,000; dex525, MW 525,000) in the extracellular space (ECS) of the anisotropic molecular layer of the isolated turtle cerebellum. For all molecules, two-dimensional images acquired an elliptical shape with major and minor axes oriented along and across, respectively, the unmyelinated parallel fibers. The effective diffusion coefficients, D*(major) and D*(minor), decreased with molecular size. The diffusion anisotropy ratio (DAR = D*(major)/D*(minor)) increased for Alexa Fluor 488 through dex75 but then unexpectedly reached a plateau. We argue that dex282 and dex525 approach the ECS width and deform to diffuse. In support of this concept, scaling theory shows the diffusion behavior of dex282 and dex525 to be consistent with transition to a reptation regime, and estimates the average ECS width at approximately 31 nm. These findings have implications for the interstitial transport of molecules and drugs, and for modeling neurotransmitter diffusion during ectopic release and spillover
— id: 94673, year: 2008, vol: 95, page: 1382, stat: Journal Article,

Aquaporin-4-deficient mice have increased extracellular space without tortuosity change
Yao, Xiaoming; Hrabetova, Sabina; Nicholson, Charles; Manley, Geoffrey T
2008 May 21;28(21):5460-5464, Journal of neuroscience
Aquaporin-4 (AQP4) is the major water channel expressed at fluid-tissue barriers throughout the brain and plays a crucial role in cerebral water balance. To assess whether these channels influence brain extracellular space (ECS) under resting physiological conditions, we used the established real-time iontophoresis method with tetramethylammonium (TMA(+)) to measure three diffusion parameters: ECS volume fraction (alpha), tortuosity (lambda), and TMA(+) loss (k'). In vivo measurements were performed in the somatosensory cortex of AQP4-deficient (AQP4(-/-)) mice and wild-type controls with matched age. Mice lacking AQP4 showed a 28% increase in alpha (0.23 +/- 0.007 vs 0.18 +/- 0.003) with no differences in lambda (1.62 +/- 0.04 vs 1.61 +/- 0.02) and k' (0.0045 +/- 0.0001 vs 0.0031 +/- 0.0009 s(-1)). Additional recordings in brain slices showed similarly elevated alpha in AQP4(-/-) mice, and no differences in lambda and k' between the two genotypes. This is the first direct comparison of ECS properties in adult mice lacking AQP4 water channels with wild-type animals and demonstrates a significant enlargement of the volume fraction but no difference in hindrance to TMA(+) diffusion, expressed as tortuosity. These findings provide direct evidence for involvement of AQP4 in modulation of the ECS volume fraction and provide a basis for future modeling of water and ion transport in the CNS
— id: 94672, year: 2008, vol: 28, page: 5460, stat: Journal Article,

Biophysical Properties of Brain Extracellular Space Explored with Ion-Selective Microelectrodes, Integrative Optical Imaging and Related Techniques
Hrabitova S; Nicholson C
Electrochemical methods for neuroscience : edited by Adrian C. Michael, Laura M. Borland Boca Raton : CRC Press/Taylor & Francis, 2007,
— id: 5910, year: 2007, vol: , page: ?, stat: Chapter,

In vivo diffusion analysis with quantum dots and dextrans predicts the width of brain extracellular space
Thorne, Robert G; Nicholson, Charles
2006 Apr 4;103(14):5567-5572, Proceedings of the National Academy of Sciences of the United States of America
Diffusion within the extracellular space (ECS) of the brain is necessary for chemical signaling and for neurons and glia to access nutrients and therapeutics; however, the width of the ECS in living tissue remains unknown. We used integrative optical imaging to show that dextrans and water-soluble quantum dots with Stokes-Einstein diameters as large as 35 nm diffuse within the ECS of adult rat neocortex in vivo. Modeling the ECS as fluid-filled 'pores' predicts a normal width of 38-64 nm, at least 2-fold greater than estimates from EM of fixed tissue. ECS width falls below 10 nm after terminal ischemia, a likely explanation for the small ECS visualized in electron micrographs. Our results will improve modeling of neurotransmitter spread after spillover and ectopic release and establish size limits for diffusion of drug delivery vectors such as viruses, liposomes, and nanoparticles in brain ECS
— id: 64461, year: 2006, vol: 103, page: 5567, stat: Journal Article,

Effects of bovine polymerized hemoglobin on coagulation in controlled hemorrhagic shock in swine
Arnaud, Francoise; Hammett, Mike; Asher, Ludmila; Philbin, Nora; Rice, Jennifer; Dong, Feng; Pearce, Bruce; Flournoy, William S; Nicholson, Carol; McCarron, Richard; Freilich, Daniel
2005 Aug;24(2):145-152, Shock
HBOC-201, a bovine polymerized hemoglobin, has been proposed as a novel oxygen-carrying resuscitative fluid for patients with hemorrhagic shock (HS). Herein, we evaluated the hemostatic effects of HBOC-201 in an animal model of HS. A 40% blood loss-controlled hemorrhage and soft tissue injury were performed in 24 invasively monitored Yucatan mini-pigs. Pigs were resuscitated with HBOC-201 (HBOC) or hydroxyethyl starch (HEX), or were not resuscitated (NON) based on cardiac parameters during a 4-h prehospital phase. Afterward, animals received simulated hospital care for 3 days with blood or saline transfusions. Hemostasis measurements included in vivo bleeding time (BT), thromboelastography (TEG), in vitro bleeding time (platelet function; PFA-CT), prothrombin time (PT), and partial thromboplastin time (PTT). Serum lactate was measured and lung sections were evaluated for microthrombi by electron microscopy. During the prehospital phase, BT remained unchanged in the HBOC group. TEG reaction time increased in HBOC pigs during the late prehospital phase and was greater than in NON or HEX pigs at 24 h (P = 0.03). TEG maximum amplitude was similar for the two fluid-resuscitated groups. PFA-CT increased in both resuscitated groups but less with HBOC (P = 0.02) in the prehospital phase; this effect was reversed by 24 h (P = 0.02). In the hospital phase, PT decreased (P < 0.02), whereas PTT increased above baseline (P < 0.01). Lactic acidosis in HBOC and HEX groups was similar. Aspartate aminotransferase was relatively elevated in the HBOC group at 24 h. Electron microscopy showed no evidence of platelet/fibrin clots or microthrombi in any of the animals. Twenty-four-hour group differences mainly reflected the fact that all HEX animals (8/8) received blood transfusions compared with only one HBOC animal (1/8). In swine with HS, HBOC resuscitation induced less thrombopathy than HEX during the prehospital phase. Mild delayed effects on platelet and clot formation during the hospital phase are transient and likely related to fewer blood transfusions. In swine with HS, HBOC resuscitation induced less thrombopathy than HEX during the prehospital phase but more thrombopathy in the hospital phase. The delayed effects on platelet and clot formation during the hospital phase are transient and may be related to the need for fewer blood transfusions
— id: 148771, year: 2005, vol: 24, page: 145, stat: Journal Article,

Optical current source density analysis in hippocampal organotypic culture shows that spreading depression occurs with uniquely reversing currents
Kunkler, Phillip E; Hulse, Raymond E; Schmitt, Michael W; Nicholson, Charles; Kraig, Richard P
2005 Apr 13;25(15):3952-3961, Journal of neuroscience
Spreading depression (SD) involves current flow through principal neurons, but the pattern of current flow over the expanse of susceptible tissues or individual principal neurons remains undefined. Accordingly, tissue and single cell maps made from digital imaging of voltage-sensitive dye changes in hippocampal organotypic cultures undergoing SD were processed via optical current source density analysis to reveal the currents associated with pyramidal neurons. Two distinctive current flow patterns were seen. The first was a trilaminar pattern (420 microm2) that developed with the onset of SD in CA3 pyramidal neurons, in which SD most often began. This initial pattern comprised a somatic current sink with current sources to either side in the dendrites that lasted for seconds extending into the first aspect of the classical "inverted saddle" interstitial direct current waveform of SD. Next, the somatic sink backpropagated at a speed of millimeters per minute into the proximal dendrites, resulting in a reversal of the initial current flow pattern to its second orientation, namely dendritic sinks associated with a somatic source. The latter persisted for the remainder of SD in CA3 and was the only pattern seen in CA1, in which SD was rarely initiated. This backpropagating SD current flow resembles that of activity-dependent synaptic activation. Retrograde and associative signaling via principal neuron current flow is a key means to affect tissue function, including synaptic activation and, by extension, perhaps SD. Such current-related postsynaptic signaling might not only help explain SD but also neuroprotection and migraine, two phenomena increasingly recognized as being related to SD.
— id: 62675, year: 2005, vol: 25, page: 3952, stat: Journal Article,

Drug utilization in the pediatric intensive care unit: monitoring prescribing trends and establishing prioritization of pharmacotherapeutic evaluation of critically ill children
Zuppa, Athena F; Adamson, Peter C; Mondick, John T; Davis, Lauren A; Maka, Deborah A; Narayan, Mahesh; Nicholson, Charles; Patel, Dimple; Collison, Kathryn R; Barrett, Jeffrey S
2005 Nov;45(11):1305-1312, Journal of clinical pharmacology
The primary objective of this study was to characterize the drug exposure for children hospitalized in the authors' institution's pediatric intensive care unit for the year 2002. Secondary objectives included the examination of drug utilization differences among various age criteria and the suitability of the most prevalent resources for pediatric dosing guidance. Many of the most commonly prescribed agents in the pediatric intensive care unit fall into the broad categories of pain management/sedation and anti-infectives. Based on the generally narrow windows afforded by each of these drug classes, it is obvious that more, well-defined investigations in critically ill children are warranted. The existing dosing guidance for many of these agents is neither generalizable nor sufficient to accommodate the diversity in pediatric intensive care unit patients, and the current drug monographs fall short of any practical dosing information
— id: 96820, year: 2005, vol: 45, page: 1305, stat: Journal Article,

Reproducibility of oligonucleotide arrays using small samples
McClintick, Jeanette N; Jerome, Ronald E; Nicholson, Charles R; Crabb, David W; Edenberg, Howard J
2003 ;4(1):4-4, BMC genomics
BACKGROUND: Low RNA yields from small tissue samples can limit the use of oligonucleotide microarrays (Affymetrix GeneChips). Methods using less cRNA for hybridization or amplifying the cRNA have been reported to reduce the number of transcripts detected, but the effect on realistic experiments designed to detect biological differences has not been analyzed. We systematically explore the effects of using different starting amounts of RNA on the ability to detect differential gene expression. RESULTS: The standard Affymetrix protocol can be used starting with only 2 micrograms of total RNA, with results equivalent to the recommended 10 micrograms. Biological variability is much greater than the technical variability introduced by this change. A simple amplification protocol described here can be used for samples as small as 0.1 micrograms of total RNA. This amplification protocol allows detection of a substantial fraction of the significant differences found using the standard protocol, despite an increase in variability and the 5' truncation of the transcripts, which prevents detection of a subset of genes. CONCLUSIONS: Biological differences in a typical experiment are much greater than differences resulting from technical manipulations in labeling and hybridization. The standard protocol works well with 2 micrograms of RNA, and with minor modifications could allow the use of samples as small as 1 micrograms. For smaller amounts of starting material, down to 0.1 micrograms RNA, differential gene expression can still be detected using the single cycle amplification protocol. Comparisons of groups of four arrays detect many more significant differences than comparisons of three arrays
— id: 96821, year: 2003, vol: 4, page: 4, stat: Journal Article,

Quantitative dual-probe microdialysis: mathematical model and analysis
Chen, Kevin C; Hoistad, Malin; Kehr, Jan; Fuxe, Kjell; Nicholson, Charles
2002 Apr;81(1):94-107, Journal of neurochemistry
Steady-state microdialysis is a widely used technique to monitor the concentration changes and distributions of substances in tissues. To obtain more information about brain tissue properties from microdialysis, a dual-probe approach was applied to infuse and sample the radiotracer, [3H]mannitol, simultaneously both in agar gel and in the rat striatum. Because the molecules released by one probe and collected by the other must diffuse through the interstitial space, the concentration profile exhibits dynamic behavior that permits the assessment of the diffusion characteristics in the brain extracellular space and the clearance characteristics. In this paper a mathematical model for dual-probe microdialysis was developed to study brain interstitial diffusion and clearance processes. Theoretical expressions for the spatial distribution of the infused tracer in the brain extracellular space and the temporal concentration at the probe outlet were derived. A fitting program was developed using the simplex algorithm, which finds local minima of the standard deviations between experiments and theory by adjusting the relevant parameters. The theoretical curves accurately fitted the experimental data and generated realistic diffusion parameters, implying that the mathematical model is capable of predicting the interstitial diffusion behavior of [3H]mannitol and that it will be a valuable quantitative tool in dual-probe microdialysis
— id: 96822, year: 2002, vol: 81, page: 94, stat: Journal Article,

Theory relating in vitro and in vivo microdialysis with one or two probes
Chen, Kevin C; Hoistad, Malin; Kehr, Jan; Fuxe, Kjell; Nicholson, Charles
2002 Apr;81(1):108-121, Journal of neurochemistry
In this paper, we further develop the general theory of microdialysis by extending the linear model of Bungay et al. to provide a theoretical basis for in vitro and in vivo microdialysis. Specifically, we considered the effect of active clearance processes on in vivo microdialysis, and thereby elaborated the theory of Benveniste et al. to endogenous compounds. We examined the use of steady state tissue diffusion resistance with negligible clearance processes to interpret microdialysis data. The influence of the tissue properties on the in vitro and in vivo recoveries in dual-probe microdialysis was analyzed and we simulated the effect of the operating parameters on dual probe microdialysis performance. We estimated that the minimum clearance rate constant detectable by microdialysis in a quasi-steady state is about 5.5 x 10(-5) s(-1). This minimum rate constant establishes a criterion, below which inhibition of the active clearance processes does not show detectable influences on the microdialysis extraction efficiency
— id: 96824, year: 2002, vol: 81, page: 108, stat: Journal Article,

Quantitative dual-probe microdialysis: evaluation of [3H]mannitol diffusion in agar and rat striatum
Hoistad, Malin; Chen, Kevin C; Nicholson, Charles; Fuxe, Kjell; Kehr, Jan
2002 Apr;81(1):80-93, Journal of neurochemistry
Dual-probe microdialysis was used to study interstitial diffusion in the rat brain. A radiolabelled tracer, (3H]mannitol, was continuously infused at different concentrations via a probe acutely implanted into the striatum of an anaesthetized male rat or into a dilute agar gel. Samples were collected by a second probe placed 1 mm away from the first, and the recovered [3H]mannitol was measured by liquid scintillation counting. In the striatum, the delivery of [3H]mannitol was counteracted by its removal from the extracellular space by passive uptake into cells and clearance into the microcirculation, causing the diffusion profile to approach quasi steady-state levels within 2 h. Diffusion data from brain and agar were analysed using a mathematical model. The apparent (effective) diffusion coefficient for [3H]mannitol was D* = 2.9 x 10(-6) cm2/s, the effective volume fraction alpha* = 0.30 and the clearance rate constant kappa= 2.3 x 10(-5)/s. A tortuosity, lambda = 1.81, and penetration distance r = 4.2 mm, were calculated. We conclude that, using dual-probe microdialysis, parameters reflecting geometric and dynamic tissue properties may be obtained using appropriate mathematical analysis. Quantitative dual-probe microdialysis will be valuable in characterizing interstitial diffusion and the clearance processes underpinning volume transmission in the brain
— id: 96823, year: 2002, vol: 81, page: 80, stat: Journal Article,

Independence of extracellular tortuosity and volume fraction during osmotic challenge in rat neocortex
Kume-Kick, June; Mazel, Tomas; Vorisek, Ivan; Hrabetova, Sabina; Tao, Lian; Nicholson, Charles
2002 Jul 15;542(Pt 2):515-527, Journal of physiology
The structural properties of brain extracellular space (ECS) are summarised by the tortuosity (lambda) and the volume fraction (alpha). To determine if these two parameters were independent, we varied the size of the ECS by changing the NaCl content to alter osmolality of bathing media for rat cortical slices. Values of lambda and alpha were extracted from diffusion measurements using the real-time ionophoretic method with tetramethylammonium (TMA+). In normal medium (305 mosmol kg(-1)), the average value of lambda was 1.69 and of alpha was 0.24. Reducing osmolality to 150 mosmol kg(-1), increased lambda to 1.86 and decreased alpha to 0.12. Increasing osmolality to 350 mosmol kg(-1), reduced lambda to about 1.67 where it remained unchanged even when osmolality increased further to 500 mosmol kg(-1). In contrast, alpha increased steadily to 0.42 as osmolality increased. Comparison with previously published experiments employing 3000 M(r) dextran to measure lambda, showed the same behaviour as for TMA+, including the same constant lambda in hypertonic media but with a steeper slope in the hypotonic solutions. These data show that lambda and alpha behave differently as the ECS geometry varies. When alpha decreases, lambda increases but when alpha increases, lambda rapidly attains a constant value. A previous model allowing cellular shape to alter during osmotic challenge can account qualitatively for the plateau behaviour of lambda
— id: 94675, year: 2002, vol: 542, page: 515, stat: Journal Article,

Poly[N-(2-hydroxypropyl)methacrylamide] polymers diffuse in brain extracellular space with same tortuosity as small molecules
Prokopova-Kubinova, S; Vargova, L; Tao, L; Ulbrich, K; Subr, V; Sykova, E; Nicholson, C
2001 Jan;80(1):542-548, Biophysical journal
Integrative optical imaging was used to show that long-chain synthetic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) polymers in a range of molecular weights from 7.8 to 1057 kDa were able to diffuse through the extracellular space in rat neocortical slices. Tortuosity (square root of ratio of diffusion coefficient in aqueous medium to that in brain) measured with such polymers averaged 1.57, a value similar to that obtained previously with tetramethylammonium, a small cation. When PHPMA was conjugated with bovine serum albumin (BSA) to make a bulky polymer with molecular weight 176 kDa, the tortuosity rose to 2.27, a value similar to that obtained previously with BSA alone and with 70-kDa dextran. The method of image analysis was justified with diffusion models involving spherical and nonspherical initial distributions of the molecules
— id: 148784, year: 2001, vol: 80, page: 542, stat: Journal Article,

Ascorbate inhibits edema in brain slices
Brahma B; Forman RE; Stewart EE; Nicholson C; Rice ME
2000 Mar;74(3):1263-1270, Journal of neurochemistry
Ascorbate is an essential antioxidant in the CNS, localized predominantly in neuronal cytosol. Slices of mammalian brain rapidly lose ascorbate, however, when incubated in ascorbate-free media; brain slices also take up water and swell. Here we investigated water gain in coronal slices of rat forebrain incubated with and without ascorbate for 1-3 h at 34 degrees C. Slices progressively gained water in ascorbate-free media, with a significant 12% water increase after 3 h at 34 degrees C, compared with the water content of slices after a 1-h recovery period at 24 degrees C, immediately following slice preparation. Inclusion of 400 micro M ascorbate in the medium led to an increase in tissue ascorbate content and prevented water gain at 34 degrees C. By contrast, water gain was not inhibited by isoascorbate or thiourea, which are antioxidants that are not accumulated in brain cells. The oxidant H2O2 enhanced water gain, whereas a cocktail of NMDA and non-NMDA receptor blockers inhibited edema formation to the same extent as ascorbate. These data demonstrate that brain edema, linked to glutamate-receptor activation, can result from intracellular oxidative stress and that this can be prevented by ascorbate
— id: 8545, year: 2000, vol: 74, page: 1263, stat: Journal Article,

Changes in brain cell shape create residual extracellular space volume and explain tortuosity behavior during osmotic challenge
Chen KC; Nicholson C
2000 Jul 18;97(15):8306-8311, Proceedings of the National Academy of Sciences of the United States of America
Diffusion of molecules in brain extracellular space is constrained by two macroscopic parameters, tortuosity factor lambda and volume fraction alpha. Recent studies in brain slices show that when osmolarity is reduced, lambda increases while alpha decreases. In contrast, with increased osmolarity, alpha increases, but lambda attains a plateau. Using homogenization theory and a variety of lattice models, we found that the plateau behavior of lambda can be explained if the shape of brain cells changes nonuniformly during the shrinking or swelling induced by osmotic challenge. The nonuniform cellular shrinkage creates residual extracellular space that temporarily traps diffusing molecules, thus impeding the macroscopic diffusion. The paper also discusses the definition of tortuosity and its independence of the measurement frame of reference
— id: 11604, year: 2000, vol: 97, page: 8306, stat: Journal Article,

Spatial buffering of potassium ions in brain extracellular space
Chen KC; Nicholson C
2000 Jun;78(6):2776-2797, Biophysical journal
It has long been assumed that one important mechanism for the dissipation of local potassium gradients in the brain extracellular space is the so-called spatial buffer, generally associated with glial cells. To date, however, there has been no analytical description of the characteristic patterns of K(+) clearance mediated by such a mechanism. This study reanalyzed a mathematical model of Gardner-Medwin (1983, J. Physiol. (Lond.). 335:393-426) that had previously been solved numerically. Under suitable approximations, the transient solutions for the potassium concentrations and the corresponding membrane potentials of glial cells in a finite, parallel domain were derived. The analytic results were substantiated by numerical simulations of a detailed two-compartment model. This simulation explored the dependence of spatial buffer current and extracellular K(+) on the distribution of inward rectifier K(+) channels in the glial endfoot and nonendfoot membranes, the glial geometric length, and the effect of passive KCl uptake. Regarding the glial cells as an equivalent leaky cable, the analyses indicated that a maximum endfoot current occurs when the glial geometric length is equal to the corresponding electrotonic space constant. Consequently, a long glial process is unsuitable for spatial buffering, unless the axial space constant can match the length of the process. Finally, this study discussed whether the spatial buffer mechanism is able to efficiently transport K(+) over distances of more than several glial space constants
— id: 11682, year: 2000, vol: 78, page: 2776, stat: Journal Article,

Perspectives on spreading depression
Martins-Ferreira, H; Nedergaard, M; Nicholson, C
2000 Apr;32(1):215-234, Brain research. Brain research reviews
Spreading depression (SD) consists of a transient suppression of all neuronal activity that spreads slowly across regions of gray matter. The paper is divided into three parts. Martins-Ferreira describes 30 years of research on SD in the isolated retina. Much of this work has relied on the prominent intrinsic optical signals that accompany SD in the retina. By inducing SD to propagate in circles with a velocity of 3.7 mm min(-1), it is possible to investigate the finely balanced electrochemical equilibrium that maintains the traveling wave. SD is accompanied by a slow negative extracellular voltage and ion movements that are greatest in the inner plexiform layer of the retina. Nedergaard discusses the role of astrocytes in SD propagation. Astrocytes mediate slowly moving waves of intracellular Ca(2+) increase, for which gap junctions are essential. SD is accompanied by entry of Ca(2+) into cells and fails when gap junctions are blocked. SD, however, is blocked by glutamate receptor antagonists but glial Ca(2+) waves are not. Astrocytic Ca(2+) waves are probably involved in the initiation of SD but other factors, including K(+), glutamate and purinergic receptors, are necessary for sustained propagation. Nicholson describes studies on the different preparations that helped clarify the role of extracellular space in SD. It has long been known that extracellular K(+) reaches levels of 50 mM or more during SD. Studies with ion-selective microelectrodes showed that extracellular Na(+) and Cl(-) fall by as much as 100 mM during SD, and water leaves the extracellular space. Further work showed that extracellular Ca(2+) falls 10-fold during SD and significant changes in extracellular pH and ascorbate occur. These studies imply that large perturbations of the extracellular milieu occur during SD and are an essential part of the interlocking cascade of events that produce this still mysterious phenomenon
— id: 148798, year: 2000, vol: 32, page: 215, stat: Journal Article,

Hiss Martins-Ferreira
Nicholson C
2000 Apr;32(1):6-8, Brain research. Brain research reviews
— id: 11772, year: 2000, vol: 32, page: 6, stat: Journal Article,

Signals that go with the flow
Nicholson C
1999 Apr;22(4):143-145, Trends in neurosciences
— id: 6092, year: 1999, vol: 22, page: 143, stat: Journal Article,

Extracellular space structure revealed by diffusion analysis [see comments]
Nicholson C; Sykova E
1998 May;21(5):207-215, Trends in neurosciences
The structure of brain extracellular space resembles foam. Diffusing molecules execute random movements that cause their collision with membranes and affect their concentration distribution. By measuring this distribution, the volume fraction (alpha) and the tortuosity (lambda) can be estimated. The volume fraction indicates the relative amount of extracellular space and tortuosity is a measure of hindrance of cellular obstructions. Diffusion measurements with molecules <500 Mr show that alpha approximately 0.2 and lambda approximately 1.6, although some brain regions are anisotropic. Molecules > or =3000 Mr show more hindrance, but molecules of 70000 Mr can move through the extracellular space. During stimulation, and in pathophysiological states, alpha and lambda change, for example in severe ischemia alpha = 0.04 and lambda = 2.2. These data support the feasibility of extrasynaptic or volume transmission in the extracellular space
— id: 7709, year: 1998, vol: 21, page: 207, stat: Journal Article,

A tortuous and viscous route to understanding diffusion in the brain - Reply
Nicholson, C; Sykova, E
1998 NOV ;21(11):470-470, Trends in neurosciences
— id: 53678, year: 1998, vol: 21, page: 470, stat: Journal Article,

Critical volume of rat cortex and extracellular threshold concentration for a pentylenetetrazol-induced epileptic focus
Schwindt, W; Nicholson, C; Lehmenkuhler, A
1997 Apr 4;753(1):86-97, Brain research
The initiation of focal interictal epileptiform activity (FIEA) has been shown to depend on the activation of a sufficiently large volume of brain tissue. We estimated the size of this 'critical volume' for the convulsant pentylenetetrazol (PTZ) by analyzing the diffusion following its microinjection into rat motor cortex. PTZ concentration was monitored 100-200 microm away from the injection site with a PTZ-sensitive microelectrode. Diffusion analysis in 0.3% agar yielded the free diffusion coefficient D (8.50 +/- 0.15 X 10(-6) cm2 x s(-1) at 37 degrees C, median +/- S.E.M.). In brain tissue, diffusion was modified by extracellular volume fraction (alpha), tortuosity (lambda = (D/ADC)1/2; ADC = apparent diffusion coefficient) and non-specific uptake (k'). Using a value of 0.2 for alpha from previous studies, we found values of lambda = 1.61 +/- 0.01, k' = 3.37 +/- 0.15 X 10(-3) s(-1) and an injected volume U of 5.16 +/- 0.45 X 10(-10) l for pulses without FIEA, and lambda = 1.95 +/- 0.06, k' = 6.24 +/- 1.73 X 10(-3) s(-1) and U = 7.40 +/- 0.66 X 10(-10) l for pulses with FIEA. From the calculated concentration distribution of PTZ during FIEA we estimated a threshold concentration of about 1.77 mM PTZ and a volume with a radius of about 219 microm in which this concentration had to be exceeded. Since this critical volume was comparable in size to foci elicited by penicillin or electric stimuli in previous studies, it is concluded that it is determined by intrinsic tissue properties rather than by the convulsive agent being used
— id: 133233, year: 1997, vol: 753, page: 86, stat: Journal Article,

Water compartmentalization and extracellular tortuosity after osmotic changes in cerebellum of Trachemys scripta
Krizaj D; Rice ME; Wardle RA; Nicholson C
1996 May 1;492(Pt 3):887-896, Journal of physiology
1. Water compartmentalization in the turtle cerebellum subject to media of different osmolalities was quantified by combining extracellular diffusion analysis with wet weight and dry weight measurements. The diffusion analysis also determined the tortuosity of the extracellular space. 2. Isolated cerebella were immersed in normal, oxygenated physiological saline (302 mosmol kg-1), hypotonic saline (238 mosmol kg-1) and a series of hypertonic salines (up to 668 mosmol kg-1). The osmolality was varied by altering the NaCl content. 3. Extracellular volume fraction and tortuosity of the granular layer of the cerebellum were determined from measurements of ionophoretically induced diffusion profiles of tetramethylammonium, using ion-selective microelectrodes. The volume fraction was 0.22 in normal saline, 0.12 in hypotonic medium and 0.60 in the most hypertonic medium. Tortuosity was 1.70 in the normal saline, 1.79 in the hypotonic and 1.50 in the most hypertonic saline. 4. The water content, defined as (wet weight-dry weight)/wet weight, of a typical isolated cerebellum (including granular, Purkinje cell and molecular layers) was 82.9%. It increased to 85.2% in hypotonic saline and decreased to 80.1% in the most hypertonic saline. 5. Measurements of extracellular volume fraction and water content were combined to show that hypotonic solutions caused water to move from the extracellular to the intracellular compartment while hypertonic solutions caused water to move from the intracellular to extracellular compartment, with only a relatively small changes in total water in both cases. 6. These results suggest the use of the isolated turtle cerebellum as a model system for studying light scattering or diffusion-weighted magnetic resonance imaging
— id: 12609, year: 1996, vol: 492, page: 887, stat: Journal Article,

Diffusion of albumins in rat cortical slices and relevance to volume transmission
Tao L; Nicholson C
1996 Dec;75(3):839-847, Neuroscience
The apparent diffusion coefficient, D*, was measured in rat cortical slices and compared to the free diffusion coefficient, D, for three negatively charged proteins, lactalbumin (mol. wt = 14,500), ovalbumin (45,000) and bovine serum albumin (66,000). The temporal evolution of the spatial distribution of albumin molecules labeled with the Texas Red fluorophore was determined using integrative optical imaging at intervals after a brief pressure injection from a micropipette in slices of adult rat cerebral cortex and dilute agarose gel. Diffusion coefficients were obtained by fitting appropriate equations to the data. In slices at 34 degrees C, the values of D* (10(-7) cm2/s, mean +/- S.E.M.) for lactalbumin, ovalbumin and bovine serum albumin were 2.37 +/- 0.10, 1.60 +/- 0.08 and 1.63 +/- 0.07, respectively. In agarose gel, values of D (10(-7) cm2/s) were 11.87 +/- 0.20, 10.02 +/- 0.25 and 8.29 +/- 0.17, respectively. From these data the tortuosity factors, (D/D*)0.5, were calculated, with 2.24 obtained for lactalbumin, 2.50 for ovalbumin and 2.26 for bovine serum albumin. Previous optical measurements using dextrans with mol. wts of 40,000 and 70,000 gave tortuosities of 2.16 and 2.25, but in contrast previous determinations with ion-selective microelectrodes using the small cation tetramethylammonium (mol. wt = 74.1) give tortuosities of about 1.6. The results show that proteins as large as bovine serum albumin diffuse through brain extracellular space but are more hindered than smaller molecules. A simple model compared the differences in diffusion properties of bovine serum albumin, dopamine and nitric oxide in brain tissue and discussed the implications for volume transmission of chemical information between cells. The results are also relevant to the behavior of diffusible factors in brain development and the delivery of therapeutic agents
— id: 8212, year: 1996, vol: 75, page: 839, stat: Journal Article,

Long distance pathways of diffusion for dextran along fibre bundles in brain. Relevance for volume transmission
Bjelke, B; England, R; Nicholson, C; Rice, M E; Lindberg, J; Zoli, M; Agnati, L F; Fuxe, K
1995 May 9;6(7):1005-1009, Neuroreport
Texas Red-labelled dextran with a mol. wt of 3000 g mol-1, a marker for the extracellular space, was injected unilaterally into the neostriatum of adult rats (0.3-30 micrograms microliter-1) and its distribution evaluated 1 min to 5 h later. Diffusion in the neuropil was observed with clearance starting after 30 min. After 10-15 min strong labelling along the myelinated fibre bundles was observed in the entire neostriatum. After about 20 min the labelling along the fibres reached into the corpus callosum and the overlaying deep layers of the cerebral cortex. A marked cellular uptake and accumulation of labelled dextran was found in putative perivascular pericytes. Thus, in the living brain preferential extracellular fluid pathways for diffusion exist, especially along fibre bundles, which allow the exchange of chemical signals between two distant regions. These may represent extracellular fluid pathways for volume transmission
— id: 145559, year: 1995, vol: 6, page: 1005, stat: Journal Article,

Interaction between diffusion and Michaelis-Menten uptake of dopamine after iontophoresis in striatum
Nicholson C
1995 May;68(5):1699-1715, Biophysical journal
A quantitative description of the behavior of a neurotransmitter in the brain extracellular microenvironment requires an understanding of the relative importance of diffusion versus uptake processes. This paper models the behavior of dopamine released from a small iontophoresis electrode and its voltammetric detection by a carbon fiber sensor 100 microns away as a basis for developing a new paradigm for measuring dopamine kinetics in intact rat neostriatum. The diffusion equation incorporating uptake, characterized by a maximum velocity Vmax and a Michaelis-Menten constant Km, was transformed to an integral equation and solved numerically for the dopamine concentration, C. Analytical solutions were derived for limiting cases of a steady-state free-boundary problem when C >> Km and the linear time-dependent problem when C << Km. These solutions were compared with complete numerical solutions, both for normal uptake (Vmax = 0.2 or 0.8 microM s-1; Km = 0.15 microM), and in the presence of the uptake blocker nomifensine (Km = 6 microM). The results suggest that an experimental strategy for the quantitative analysis of dopamine, and other compounds, in living tissue is to fit a family of concentration versus time curves generated with different iontophoretic current strengths and recorded with a microsensor, to the numerical solution of the diffusion-uptake equation
— id: 6703, year: 1995, vol: 68, page: 1699, stat: Journal Article,

Extracellular potassium, volume fraction, and tortuosity in rat hippocampal CA1, CA3, and cortical slices during ischemia
Perez-Pinzon MA; Tao L; Nicholson C
1995 Aug;74(2):565-573, Journal of neurophysiology
1. An in vitro slice model of ischemia was used to study changes in extracellular potassium concentration and diffusion properties in the stratum pyramidale of CA1 and CA3 regions of the hippocampus and in the cortex of the rat. Slices were submerged in artificial cerebrospinal fluid, and ischemia was induced by removing oxygen and glucose until anoxic depolarization occurred. 2. Extracellular potassium concentration was measured with a valinomycin-based ion-selective microelectrode. The bathing medium contained 5 mM potassium, and in vitro ischemia caused the potassium concentration to rise to 45 mM in CA1, 12 mM in CA3, and 32 mM in cortex. 3. Extracellular volume fraction and tortuosity were determined during normoxic conditions and in vitro ischemia by measuring the diffusion of tetramethylammonium. This cation was iontophoretically released into the extracellular space and its concentration as a function of time determined with an ion-selective microelectrode approximately 100 microns away from the source. 4. During normoxia the volume fraction was 0.14, 0.20, and 0.18, and tortuosity was 1.50, 1.57, and 1.62 in CA1, CA3, and cortex, respectively. These data confirm that the volume fraction of CA1 is smaller than in the two other regions. 5. During ischemia the volume fraction decreased to 0.05, 0.17, and 0.09 in CA1, CA3, and cortex, respectively. Only in CA3 did the tortuosity change significantly by increasing to 1.75. Because of limitations in the time resolution of the diffusion method, the changes in volume fraction and tortuosity during the anoxic depolarization phase of ischemia may have been underestimated.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 6857, year: 1995, vol: 74, page: 565, stat: Journal Article,

The three-dimensional point spread functions of a microscope objective in image and object space
Tao L; Nicholson C
1995 Jun;178(Pt 3):267-271, Journal of microscopy
The three-dimensional point spread function (3-D PSF) of an optical system in image space is distinguished from the 3-D PSF in object space and the relation between the two 3-D PSFs is derived. By using this relation one 3-D PSF can be easily obtained from the other. The 3-D PSFs are given in a single integral expression, which can be computed numerically. The results of this study can be used in 3-D image processing for microscopy and have been applied to the analysis of the diffusion of fluorescent molecules in a 3-D porous medium
— id: 6869, year: 1995, vol: 178, page: 267, stat: Journal Article,

Origin of the apparent tissue conductivity in the molecular and granular layers of the in vitro turtle cerebellum and the interpretation of current source-density analysis
Okada, Y C; Huang, J C; Rice, M E; Tranchina, D; Nicholson, C
1994 Aug;72(2):742-753, Journal of neurophysiology
1. We determined the origin of the apparent tissue conductivity (sigma 2) of the turtle cerebellum in vitro. 2. Application of a current with a known current density (J) along the longitudinal axis of a conductivity cell produced an electric field in the cerebellum suspended in the cell. The measured electric field (E) perpendicular to the cerebellar surface indicated a significant inhomogeneity in sigma a (= J/E) with a major discontinuity between the molecular layer (0.25 +/- 0.05 S/m, mean +/- SD) and granular layers (0.15 +/- 0.03 S/m) (n = 39). 3. This inhomogeneity was more pronounced after anoxic depolarization. The value of sigma a decreased to 0.11 +/- 0.03 and 0.040 +/- 0.008 S/m in the molecular and granular layers, respectively. The ratio of sigma a S in the two layers increased from 1.67 in the normoxic condition to 2.75 after anoxic depolarization. 4. This difference in sigma a across the two layers was present within the range of frequencies (DC to 10 kHz) studied where the phase of sigma a was small (less than +/- 2 degrees) and therefore sigma a was ohmic. 5. The inhomogeneity in sigma a was in part due to an inhomogeneity in the extracellular conductivity (sigma e) as determined from the extracellular diffusion of ionophoresed tetramethylammonium. Like sigma a, the value of sigma e was also higher in the molecular layer (0.165 S/m) than in the granular layer (0.097 S/m). The inhomogeneity in sigma e was due to a smaller tortuosity and a larger extracellular volume fraction in the molecular layer compared with the granular layer. 6. sigma a was, however, consistently higher, by approximately 50%, than sigma e. A core conductor model of the cerebellum indicated that these discrepancies between sigma a and sigma e were attributable to additional conductivity produced by a passage of the longitudinal applied current through the intracellular space of Purkinje cells and ependymal glial cells, with the glial compartment playing the dominant role. Cells with a long process and a short space constant such as the ependymal glia evidently enhance the effective 'extracellular' conductivity by serving as intracellular conduits for the applied current. The result implies that the effective sigma e may be larger than sigma e for neuronally generated currents in the turtle cerebellum because the space constant for Purkinje cells is several times greater than that for the ependymal glia and consequently Purkinje cell-generated currents travel over a long distance relative to the space constant of glial cells.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 145560, year: 1994, vol: 72, page: 742, stat: Journal Article,

Direct monitoring of dopamine and 5-HT release in substantia nigra and ventral tegmental area in vitro
Rice ME; Richards CD; Nedergaard S; Hounsgaard J; Nicholson C; Greenfield SA
1994 ;100(3):395-406, Experimental brain research
Fast-scan cyclic voltammetry with carbon fibre microelectrodes was used to detect endogenous dopamine (DA) and 5-hydroxytryptamine (5-HT) release from three distinct regions of guinea-pig mid-brain in vitro: rostral and caudal substantia nigra (SN) and the ventral tegmental area (VTA). Previous electrophysiological studies have demonstrated that cells of the caudal SN and the VTA have similar characteristics, whereas cells in the rostral SN have distinctly different properties. In the present study, we confirmed that each region has tyrosine hydroxylase-positive neurons and determined, using high-performance liquid chromatography, that DA levels were similar in rostral and caudal SN, but lower in SN than in VTA. In each region, application of veratrine, which was shown by intracellular recordings to have a reversible depolarising action, evoked a signal attributable to DA and distinguishable from that of 5-HT. Release signals were monitored every 250 ms with a spatial resolution of less than 50 microns.l DA release was calcium-dependent and was not detectable in a catecholamine-poor area such as the cerebellum, or in mid-brain tissue pre-treated with reserpine. Within the normal mid-brain, the amount of DA released was correlated with tissue content in that it was higher in the VTA than in either region of SN. It is concluded that DA released from somato-dendritic parts of mid-brain neurons exhibits site-specific variation. This is the first report of direct monitoring of DA and 5-HT release from these regions with in situ electrodes and demonstrates the utility of fast-scan cyclic voltammetry to investigate the mechanisms and possible non-classical functions of somato-dendritic DA release
— id: 6727, year: 1994, vol: 100, page: 395, stat: Journal Article,

Superfusion of verapamil on the cerebral cortex does not suppress epileptic discharges due to restricted diffusion (rats, in vivo)
Kohling, R; Lehmenkukhler, A; Nicholson, C; Speckmann, E J
1993 Oct 29;626(1-2):149-155, Brain research
The organic calcium channel blocker verapamil has been demonstrated to block epileptic activity in various experimental models both in vitro and in vivo. The drug, however, does not pass the blood-brain barrier, so that both the oral route and intravenous administration of the drug are ruled out for antiepileptic treatment. The present investigations analyzed the effects of verapamil applied epicortically in experimental models of interictal penicillin-induced and ictal pentylenetetrazol-induced epileptic activity in rats. Such epicortical application of verapamil was ineffective in suppressing either interictal or ictal epileptic activity. To test whether this lack of effect was due to poor penetration of the substance into the cortical tissue, the diffusion characteristics of verapamil were studied in agar and in gray matter by pressure microejection and an appropriate verapamil-selective microelectrode. The diffusion could be described fully by a diffusion coefficient D (5.08 x 10(-6) cm2 x s-1), tortuosity lambda (1.51) and concentration-dependent uptake, k' (2.23 x 10(-3) s-1). Using these values, the depth-dependent concentration gradient resulting from superfusion of the substance was calculated for agar and brain. In concentration measurements done in brain tissue, however, verapamil could not be detected in cortical layers deeper than 150 microns, which did not agree with the theoretical prediction. This observation may indicate a diffusion barrier at the interface between superfusing fluid and tissue. The results indicate that epicortical administration of verapamil is not efficacious in treatment of epilepsy
— id: 148773, year: 1993, vol: 626, page: 149, stat: Journal Article,

Extracellular space parameters in the rat neocortex and subcortical white matter during postnatal development determined by diffusion analysis
Lehmenkuhler, A; Sykova, E; Svoboda, J; Zilles, K; Nicholson, C
1993 Jul;55(2):339-351, Neuroscience
Extracellular space volume fraction, tortuosity and nonspecific uptake of tetramethylammonium--three diffusion parameters of brain tissue--were measured in gray matter of the somatosensory neocortex and subcortical white matter of the rat during postnatal development. The three parameters were determined from concentration-time profiles of tetramethylammonium in postnatal days 2-120 in vivo. Tetramethylammonium concentration was measured with ion-selective microelectrodes positioned 130-200 microns from an iontophoretic source. Data were correlated with cytoarchitectonic structure and average thickness of the regions in 0-90-day-old rats using rapidly frozen tissue. Extracellular space volume fraction was largest in the newborn rats and diminished with age. In two-to three-day-old animals, volume fraction (mean +/- S.E.) was 0.36 +/- 0.04 in layers III and IV, 0.38 +/- 0.02 in layer V, 0.41 +/- 0.01 in layer VI and 0.46 +/- 0.01 in white matter. The earliest decrease in volume fraction was found in layers V and VI at postnatal days 6-7 followed by a decrease in layer III and IV at postnatal days 8-9 and in white matter at postnatal days 10-11. A further dramatic reduction in volume fraction occurred in all cortical layers and especially in the white matter between postnatal days 10 and 21. There was no further decrease in volume fraction between postnatal day 21 and adults (90-120 days old). The adult volume fraction values were: layer II, 0.19 +/- 0.002; III, 0.20 +/- 0.004; IV, 0.21 +/- 0.003; V, 0.22 +/- 0.003; VI, 0.23 +/- 0.007; white matter, 0.20 +/- 0.008. Values of tortuosity ranged between 1.51 and 1.65, nonspecific cellular uptake varied from 3.3 x 10(-3)/s to 6.3 x 10(-3)/s. The variations in each parameter were not statistically significant at any age. These data represent the first characterization of diffusion parameters in a developing brain. They confirm previous histological indications of a relatively large extracellular volume fraction during early postnatal development. The constancy of the tortuosity shows that diffusion of small molecules is no more hindered in the developing brain than in the adult. The large extracellular space volume fraction of the neonatal brain could significantly dilute ions, metabolites and neuroactive substances released from cells, relative to release in adults, and may be a factor in preventing anoxia, seizure and spreading depression in young animals. The diffusion characteristics could also play an important role in the developmental process itself
— id: 148796, year: 1993, vol: 55, page: 339, stat: Journal Article,

Diffusion analysis of valproate and trans-2-en-valproate in agar and in cerebral cortex of the rat
Lucke, A; Lehmenkuhler, A; Altrup, U; Nicholson, C; Reith, H; Schmitz-Moormann, C; Speckmann, E J
1993 Dec 24;631(2):187-190, Brain research
The diffusion of valproate (VPA) and trans-2-en-valproate were studied in agar gel and in the cerebral cortex of the rat using pressure microejection and VPA-selective microelectrodes. From the agar measurements a free diffusion coefficient for VPA of 6.52 x 10(-6) cm2.s-1 and for trans-2-en-VPA of 5.25 x 10(-6) cm2.s-1 for 37 degrees C was determined. The tortuosity value in the cortex was 1.92 for VPA and 1.67 for trans-2-en-VPA. The tortuosity values suggest that VPA and trans-2-en-VPA diffuse mainly in the extracellular space of the brain
— id: 148772, year: 1993, vol: 631, page: 187, stat: Journal Article,

Ion-selective microelectrodes and diffusion measurements as tools to explore the brain cell microenvironment
Nicholson C
1993 Jul;48(3):199-213, Journal of neuroscience methods
The construction and application of liquid-membrane ion-selective microelectrodes (ISM) are described. Recommendations are provided for the selection of appropriate cocktails containing neutral carriers to form the liquid membrane to sense K+, Ca2+, H+ and Na+. The use of charged carriers to sense Cl- and the cation tetramethylammonium (TMA+) is discussed. A detailed protocol is given for constructing double-barreled electrodes (ion-sensor and reference barrel) with tips of 1 micron diameter or more for extracellular ion measurements. The primary results obtained with ISMs in the brain cell microenvironment are briefly surveyed. The theoretical basis for measuring diffusion properties of extracellular space is described. Such measurements enable the estimation of volume fraction (proportion of tissue that is extracellular space) and tortuosity (hindrance of diffusion due to cellular obstructions). A method is given for using TMA+ ISMs in combination with iontophoresis or pressure ejection of TMA+ from a nearby micropipette to measure diffusion properties
— id: 6471, year: 1993, vol: 48, page: 199, stat: Journal Article,

Diffusion properties of brain tissue measured with electrode methods and prospects for optical analysis
Nicholson C; Tao L
1993 ;333:213-223, Advances in experimental medicine & biology
— id: 13320, year: 1993, vol: 333, page: 213, stat: Journal Article,

Hindered diffusion of high molecular weight compounds in brain extracellular microenvironment measured with integrative optical imaging
Nicholson C; Tao L
1993 Dec;65(6):2277-2290, Biophysical journal
This paper describes the theory of an integrative optical imaging system and its application to the analysis of the diffusion of 3-, 10-, 40-, and 70-kDa fluorescent dextran molecules in agarose gel and brain extracellular microenvironment. The method uses a precisely defined source of fluorescent molecules pressure ejected from a micropipette, and a detailed theory of the intensity contributions from out-of-focus molecules in a three-dimensional medium to a two-dimensional image. Dextrans tagged with either tetramethylrhodamine or Texas Red were ejected into 0.3% agarose gel or rat cortical slices maintained in a perfused chamber at 34 degrees C and imaged using a compound epifluorescent microscope with a 10 x water-immersion objective. About 20 images were taken at 2-10-s intervals, recorded with a cooled CCD camera, then transferred to a 486 PC for quantitative analysis. The diffusion coefficient in agarose gel, D, and the apparent diffusion coefficient, D*, in brain tissue were determined by fitting an integral expression relating the measured two-dimensional image intensity to the theoretical three-dimensional dextran concentration. The measurements in dilute agarose gel provided a reference value of D and validated the method. Values of the tortuosity, lambda = (D/D*)1/2, for the 3- and 10-kDa dextrans were 1.70 and 1.63, respectively, which were consistent with previous values derived from tetramethylammonium measurements in cortex. Tortuosities for the 40- and 70-kDa dextrans had significantly larger values of 2.16 and 2.25, respectively. This suggests that the extracellular space may have local constrictions that hinder the diffusion of molecules above a critical size that lies in the range of many neurotrophic compounds
— id: 6470, year: 1993, vol: 65, page: 2277, stat: Journal Article,

Anisotropic and heterogeneous diffusion in the turtle cerebellum: implications for volume transmission
Rice ME; Okada YC; Nicholson C
1993 Nov;70(5):2035-2044, Journal of neurophysiology
1. Measurements of extracellular diffusion properties were made in three orthogonal axes of the molecular and granular layers of the isolated turtle cerebellum with the use of iontophoresis of tetramethylammonium (TMA+) combined with ion-selective microelectrodes. 2. Diffusion in the extracellular space of the molecular layer was anisotropic, that is, there was a different value for the tortuosity factor, lambda i, associated with each axis of that layer. The x- and y-axes lay in the plane parallel to the pial surface of this lissencephalic cerebellum with the x-axis in the direction of the parallel fibers. The z-axis was perpendicular this plane. The tortuosity values were lambda x = 1.44 +/- 0.01, lambda y = 1.95 +/- 0.02, and lambda z = 1.58 +/- 0.01 (mean +/- SE). By contrast, the granular layer was isotropic with a single tortuosity value, lambda Gr = 1.77 +/- 0.01. 3. These data confirm the applicability of appropriately extended Fickian equations to describe diffusion in anisotropic porous media, including brain tissue. 4. Heterogeneity between the molecular and granular layer was revealed by a striking difference in extracellular volume fraction, alpha, for each layer. In the molecular layer alpha = 0.31 +/- 0.01, whereas in the granular layer alpha = 0.22 +/- 0.01. 5. Volume fraction and tortuosity affected the time course and amplitude of extracellular TMA+ concentration after iontophoresis. This was modeled by the use of the average parameters determined experimentally, and the nonspherical pattern of diffusion in the molecular layer was compared with the spherical distribution in the granular layer and agarose gel by computing isoconcentration ellipsoids. 6. One functional consequence of these results was demonstrated by measuring local changes in [K+]o and [Ca2+]o after microiontophoresis of a cerebellar transmitter, glutamate. The ratios of ion shifts in the x- and y-axes in the granular layer were close to unity, with a ratio of 1.04 +/- 0.08 for the rise in [K+]o and 1.03 +/- 0.17 for the decrease in [Ca2+]o. In contrast, ion shifts in the molecular layer had an x:y ratio of 1.44 +/- 0.14 for the rise in [K+]o and 2.10 +/- 0.42 for the decrease in [Ca2+]o. 7. These data demonstrate that the structure of cellular aggregates can channel the migration of substances in the extracellular microenvironment, and this could be a mechanism for volume transmission of chemical signals. For example, the preferred diffusion direction of glutamate along the parallel fibers would help constrain an incoming excitatory stimulus to stay 'on-beam.'
— id: 6495, year: 1993, vol: 70, page: 2035, stat: Journal Article,

Extracellular dopamine concentration in the retina of the clawed frog, Xenopus laevis
Witkovsky P; Nicholson C; Rice ME; Bohmaker K; Meller E
1993 Jun 15;90(12):5667-5671, Proceedings of the National Academy of Sciences of the United States of America
Dopamine reaches targets in the outer retina of the clawed frog (Xenopus laevis) by diffusion from a network of dopaminergic cells and processes located predominantly at the junction of inner nuclear and inner plexiform layers. We obtained values for the steady-state release, uptake, and extracellular concentration of dopamine in the retina by a combination of HPLC (with electrochemical detection), scintillation spectroscopy, and fast-scan cyclic voltammetry. Vitreal concentrations of dopamine varied from 564 +/- 109 nM in light-adapted eyes near the time of subjective dawn to 156 +/- 12 nM in dark-adapted eyes. The data are consistent with a simple model for steady-state dopamine diffusion from an appropriately sited thin-sheet source. This model was used to generate a profile of extracellular dopamine concentration as a function of retinal depth. The model predicted an increase in the dopamine concentration from the vitreous to the layer of dopaminergic cells, remaining constant from that layer to the distal tips of the photoreceptors. This prediction was borne out by comparing fast-scan voltammetric measures of dopamine at the distal tips of the receptors with the vitreal concentrations determined by HPLC using electrochemical detection
— id: 13131, year: 1993, vol: 90, page: 5667, stat: Journal Article,

Quantitative analysis of extracellular space using the method of TMA+ iontophoresis and the issue of TMA+ uptake
Nicholson C
1992 ;70 Suppl:S314-S322, Canadian journal of physiology & pharmacology
The tetramethylammonium (TMA+) method for measuring the volume fraction and tortuosity of brain extracellular space is presented in detail. The temporal and spatial distribution of TMA+ in the extracellular space following iontophoresis or pressure microinjection is described by suitable equations and illustrated with graphs. By fitting the equations to the concentration versus time data obtained from measurements with ion-selective micropipettes, the volume fraction and tortuosity can be measured. In addition, the concentration-dependent uptake of TMA+ can be estimated from the given equations. The final section of the paper derives simple numerical estimates of TMA+ loss from the extracellular space by this mechanism
— id: 13805, year: 1992, vol: 70 Suppl, page: S314, stat: Journal Article,

Extracellular volume decreases while cell volume is maintained by ion uptake in rat brain during acute hypernatremia
Cserr HF; DePasquale M; Nicholson C; Patlak CS; Pettigrew KD; Rice ME
1991 Oct;442:277-295, Journal of physiology
1. Regulation of brain extracellular and intracellular water content, regarded as volume, and electrolytes in response to 90 min of hypernatremia has been studied in the cerebral cortex of rats under urethane anaesthetic. 2. Total tissue electrolytes and water were partitioned between extracellular and intracellular compartments based on measurements made in two series of experiments. In one, tissue samples were collected and analysed for total water, Na+, K+ and Cl-. In the other, tissue extracellular volume fraction, [Na+] and [K+] were measured in situ using ion-selective microelectrodes. 3. Osmotically induced water loss from cerebral cortex was less than that predicted for ideal osmotic behaviour, revealing a degree of volume regulation, and this regulation was associated with net tissue uptake of Na+, Cl- and K+. 4. Total water content was 3.77 g H2O (g dry weight)-1 in control cortex and this decreased by 7% after 30 min of hypernatremia and then remained relatively stable at this value. Control extracellular water content, based on an extracellular volume fraction of 0.18, was 0.88 g H2O (g dry weight)-1. Control intracellular water content, estimated as the difference between total and extracellular water contents, was 2.89 g H2O (g dry weight)-1. After 30 min of hypernatremia, extracellular water content decreased by an average of 27% but intracellular water did not change. This indicates selective regulation of cell volume. By 90 min the extracellular water content had decreased by 47% and the loss in extracellular water content appeared to be accompanied by a roughly equivalent increase in intracellular water content. The intracellular volume increase, however, was not statistically significant. The tortuosity of the extracellular space averaged 1.57 and increased to 1.65 during the hypernatremia. 5. Brain extracellular fluid and plasma [Na+] were roughly equal in control tissue. Both increased by 30 mu equiv (g H2O)-1 as a result of the hypernatremia, although extracellular [Na+] lagged behind the plasma value during much of the first 60 min of hypernatremia. Extracellular [K+] was homeostatically regulated at 3 mu equiv (g H2O)-1 independent of changes in plasma electrolytes. 6. Estimates of extracellular and intracellular ion content (mu equiv (g dry weight)-1) indicate that extracellular Na+, Cl- and K+ content decreased during hypernatremia, by 32, 21 and 42% respectively, whereas intracellular ion content increased by 100, 169 and 5% respectively. 7. It is concluded that during acute hypernatremia the extracellular space decreases in volume through the loss of water and electrolytes while the intracellular compartment maintains its water content and gains electrolytes.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 65259, year: 1991, vol: 442, page: 277, stat: Journal Article,

Threshold extracellular concentration distribution of penicillin for generation of epileptic focus measured by diffusion analysis
Lehmenkuhler, A; Nicholson, C; Speckmann, E J
1991 Oct 11;561(2):292-298, Brain research
The tissue volume required to produce a penicillin-induced interictal discharge in the local EEG was estimated. A pair of microelectrodes were lowered into the motor cortex of anaesthetised and artificially ventilated rats. One double-barrelled electrode was used to release tetramethylammonium (TMA+) by iontophoresis or to pressure eject a solution containing penicillin (PEN-) and TMA+ concentration. The extracellular distribution of PEN- was defined using diffusion analysis of the TMA+. From these data the spatial distribution of PEN- was estimated at the times of first interictal spikes in the EEG. The critical mass of active nerve cells was calculated from the threshold concentration of PEN- needed to elicit paroxysmal depolarisation shifts in neocortical slices and found to lie within a tissue sphere with a radius of ca. 150 microns
— id: 148774, year: 1991, vol: 561, page: 292, stat: Journal Article,

Multimodal characterization of population responses evoked by applied electric field in vitro: extracellular potential, magnetic evoked field, transmembrane potential, and current-source density analysis
Lopez L; Chan CY; Okada YC; Nicholson C
1991 Jul;11(7):1998-2010, Journal of neuroscience
An external electric field applied parallel to longitudinal axis of neurons selectively depolarizes either end and thereby activates voltage-sensitive conductance changes in a large population of neurons. Here, we characterized such population responses in the in vitro turtle cerebellum. The responses were recorded and analyzed using a multimodal approach: the magnetic evoked field was measured using a Superconducting Quantum Interference Device (SQ
— id: 13966, year: 1991, vol: 11, page: 1998, stat: Journal Article,

Diffusion characteristics and extracellular volume fraction during normoxia and hypoxia in slices of rat neostriatum
Rice ME; Nicholson C
1991 Feb;65(2):264-272, Journal of neurophysiology
1. Diffusion properties of submerged, superfused slices from the rat neostriatum were measured by quantitative analysis of concentration-time profiles of tetramethylammonium (TMA+) introduced by iontophoresis. TMA+ was sensed at an ion-selective microelectrode (ISM) positioned 100-150 microns from the source pipette. Slice viability was assessed from the extracellular field potentials evoked by intrastriatal electrical stimulation. 2. Under normoxic conditions the extracellular volume fraction (alpha) was 0.21 (range 0.18-0.24), and the tortuosity (lambda) was 1.54, in slices with good field potentials. In slices with poor field potentials, alpha was 0.09-0.16. Extraction of correct alpha and lambda in the slice required evaluation of nonspecific uptake, k', which was 1 x 10(-2) s-1. 3. Slices were made hypoxic by superfusing physiological saline equilibrated with 95% N2-5% CO2 for 10-30 min. Synaptic components of field potentials were inhibited after 3-4 min in hypoxic media. In some experiments extracellular K+ concentration [( K+]o) was monitored with ISMs. During hypoxia, [K+]o rose from an average baseline of 5.1 mM to 7-10 mM. After reoxygenation, [K+]o transiently fell below the original level. 4. The average value for alpha during hypoxia was 0.13 (a 38% decrease), which was significantly different from control (P less than 0.001) and increased progressively during hypoxic exposure. In contrast, tortuosity and k' were unchanged by this treatment. 5. These data represent the first characterization of the diffusion properties of the rat striatal slice and of changes in extracellular volume fraction during hypoxia in a brain slice preparation.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 14132, year: 1991, vol: 65, page: 264, stat: Journal Article,

Distortion of magnetic evoked fields and surface potentials by conductivity differences at boundaries in brain tissue
Huang, J C; Nicholson, C; Okada, Y C
1990 Jun;57(6):1155-1166, Biophysical journal
We investigated the conditions under which inhomogeneity in electrical conductivity may significantly modify the magnetic evoked field (MEF) due to primary currents (i.e., neuronal currents) in the brain. In the case of an isolated turtle cerebellum immersed in a large bath of physiological saline, our theoretical analysis showed the cerebellar surface to significantly enhance the MEF due to a primary current, by a factor of as much as two, for experimentally determined values of the conductivities of the cerebellar tissue and saline. A further parametric investigation of the conductivity effect revealed that conductivity boundaries may significantly modify the MEF due to neuronal currents located within 1 mm of a conductivity boundary, as would be the case for active neurons near an edema, an anoxic fringe such as might occur during stroke, or a ventricle in the human head. For a stationary neural source, conductivity boundaries may modify the magnitude of its MEF without affecting its temporal waveform. However, this boundary effect was found to be small for a model geometry locally approximating cortical sources in a sulcus or a fissure, where the boundary effects from adjacent sulcal walls tend to cancel each other
— id: 148768, year: 1990, vol: 57, page: 1155, stat: Journal Article,

Glutamate- and aspartate-induced extracellular potassium and calcium shifts and their relation to those of kainate, quisqualate and N-methyl-D-aspartate in the isolated turtle cerebellum
Rice, M E; Nicholson, C
1990 ;38(2):295-310, Neuroscience
Ion-selective microelectrodes can be used to evaluate the characteristics and laminar distribution of excitatory amino acid agonist-induced K+ and Ca2+ shifts in the extracellular environment of brain cells. This report describes the pattern of K+ increases and Ca2+ decreases elicited by glutamate and aspartate at 100 microns intervals in the isolated turtle cerebellum. These responses were compared to ion shifts evoked by kainate, quisqualate and N-methyl-D-aspartate. Glutamate and aspartate produced indistinguishable laminar patterns of ion shifts, with the greatest [K+]o and [Ca2+]o shifts in the granular layer. The average maximum granular and molecular layer increases in [K+]o were, respectively, 130% and 24% larger than the increase in the Purkinje cell layer. Kainate-induced increases in [K+]o also followed this granular greater than molecular greater than Purkinje cell layer pattern; however, the corresponding [Ca2+]o decreases were smaller and more variable. Quisqualate-evoked ion shifts in the molecular layer closely mimicked the shape of glutamate- and aspartate-induced responses. In the granular layer, however, quisqualate caused little ion change during iontophoresis followed by large [K+]o and [Ca+]o shifts after the end of the pulse. The minimal ion shifts induced during quisqualate application in the granular layer gave this agonist the distinction of being the only agent tested to have its greatest direct effect in the molecular layer. N-Methyl-D-aspartate caused large, two-phase [K+]o and [Ca2+]o shifts in the granular layer, only small [K+]o rises in the Purkinje cell and ventral molecular layers, and no response in the dorsal molecular layer. The lack of similarity between glutamate- and aspartate-induced ion shifts in the granular layer and those of any one agonist demonstrate the mixed agonist action of glutamate and aspartate in the cerebellum. These studies provide new information about the dynamics of excitatory amino acid receptor activation that is complementary to autoradiographic receptor mapping data and to single cell electrophysiological studies
— id: 145561, year: 1990, vol: 38, page: 295, stat: Journal Article,

Changes in dietary patterns over a 6-year period in an elderly population
Garry, P J; Rhyne, R L; Halioua, L; Nicholson, C
1989 ;561:104-112, Annals of the New York Academy of Sciences
We have not employed a rigid statistical interpretation of the data in this report because, as Botwinick and Arenberg pointed out, the time span of a study (if short enough) and the number of independent observations (if small enough) can affect statistical significance. In the present study, the time span was only 6 years, and the males, as well as the females, were grouped into a single age cohort. These limitations do not allow us to assess true time effects. Nevertheless, we attributed the differences noted between the cross-sectional and longitudinal slopes to a time effect, that is, to a secular trend, over the span of the study. This conclusion was based on the following line of reasoning: First, we assumed that there were no significant cohort effects in the population considered in the present study. This assumption was supported by results from the Baltimore Longitudinal Study of Aging, where no cohort effects were found. (In the Baltimore study, 180 males were followed over a period of 15 years, and the age range covered 40 years.) Second, we documented evidence that the participants in the present study actively limited their cholesterol and fat intake because of their keen awareness of an association between cholesterol intake and coronary heart disease as reported in the lay press over the past 10 years. In the healthy male population considered in the present study, we determined that there was a decrease in daily energy intake of approximately 12 kcal/year. We based this determination on the average of the cross-sectional analysis as we attributed much of the negative effect noted in the longitudinal analysis, as stated above, to a secular trend. Aging had a much lower negative effect on energy intake in the female population, being approximately 4 kcal/year. The decrease in protein intake with age was higher in males (1.1 g/day/year) than in females (0.6 g/day/year). This finding parallels the large negative effect of aging in energy intake noted for males compared with females. The same sex difference was noted for carbohydrate intake between males and females (-0.4 and -0.2 g/day/year, respectively). The cross-sectional findings for fat intake are unremarkable for the males (no change) as well as for the females (only -0.1 g/day/year).(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 148799, year: 1989, vol: 561, page: 104, stat: Journal Article,

Volume and ion regulation in both intracellular and extracellular compartments determined by diffusion, gravimetric and ion analysis
Nicholson, C; Cserr, H F; Rice, M E; DePasquale, M; Patlak, C S
1989 ;582:24-24, Acta physiologica Scandinavica. Supplementum
— id: 145563, year: 1989, vol: 582, page: 24, stat: Journal Article,

Measurement of nanomolar dopamine diffusion using low-noise perfluorinated ionomer coated carbon fiber microelectrodes and high-speed cyclic voltammetry
Rice, M E; Nicholson, C
1989 Sep 1;61(17):1805-1810, Analytical chemistry
Several improvements in the fabrication and use of carbon fiber voltammetric microelectrodes (CFVMs) are described. These procedures did not involve oxidative treatment, but resulted in sensitivities and selectivities approaching those of treated CFVMs, without the inherent slow response times associated with the latter electrodes. To accomplish this we reduced CFVM noise by (1) improving the adhesive seal between the 8 microns o.d. carbon fiber and the glass insulation using vacuum, (2) snapping rather than cutting or beveling the fiber to be flush with the glass, and (3) using a concentrated electrolyte solution to make electrical contact with the fiber. System noise was reduced by digital smoothing and signal averaging. Selectivity of the CFVMs for dopamine over ascorbate was enhanced to better than 2000:1 by coating with Naflon, a perfluorinated cation exchange polymer, using a low (+0.5 V vs Ag/AgCl) electroplating potential. This low voltage also prevented electrode surface oxidation. To demonstrate the performance of our CFVMs, we used them in conjunction with high-speed cyclic voltammetry to accurately measure the diffusion coefficient of iontophoretically released dopamine at concentrations as low as 35 nM over distances of less than 200 microns in agarose gel
— id: 145562, year: 1989, vol: 61, page: 1805, stat: Journal Article,

Effects of electric fields on transmembrane potential and excitability of turtle cerebellar Purkinje cells in vitro
Chan CY; Hounsgaard J; Nicholson C
1988 Aug;402:751-771, Journal of physiology
1. Transmembrane potential (TMP) responses of Purkinje cells (PCs) in isolated turtle cerebellum to externally applied quasi-steady-state electric fields aligned with the dendritic axis were continuously measured using simultaneous intracellular and extracellular recording. TMP was obtained by subtraction of extracellular voltage fields from intracellular potential recorded at the same depth in the cerebellum. 2. The applied field changed the TMP with the polarity and amplitude dependent on the location on the PC membrane. This response at a given location increased linearly with external field up to a threshold level, beyond which active responses appeared. 3. The basic effect on TMP consisted of depolarization in the half of the dendrite towards which the fields were directed, and hyperpolarization in the other half. A pooled TMP depth-profile shows a steady increase in polarization from the middle of the molecular layer towards each end. This profile correlates with that predicted from previously proposed cable models, giving them empirical support for the first time. 4. Active responses were triggered by the field-induced depolarization. Tetrodotoxin (TTX)-sensitive action potentials arose with the primary depolarization in the somatic region. Notched, Ca2+-dependent action potentials arose with primary depolarization in the distal and mid-dendritic regions. 5. A TTX-sensitive voltage plateau was triggered by TMP-depolarization in the proximal region. It in turn activated Na+-spike trains. The frequency of spiking was proportional to the external field. At around 160 spikes/s, the Na+ spikes inactivated, and the TMP level rose to a more depolarized plateau. This latter plateau was also TTX-sensitive. 6. During depolarization of the distal dendritic region, sometimes a Ca2+-dependent plateau was observed. It appears to be associated with a small conductance increase. 7. Field-induced hyperpolarization suppressed local spiking and voltage plateaux, but remote Ca2+ spikes with reduced amplitude appeared in recordings from the proximal region. Similarly, in the distal region, low-amplitude, remote Na+ spikes and a Na+ plateau were observed superimposed on the hyperpolarizing baseline. The Na+ plateau apparently did not contribute to shunting of membrane currents in the distal dendrite. 8. The phase characteristics of the action potentials correlate with the modulation pattern noted in our extracellular study (Chan & Nicholson, 1986). Thus, the extracellular units ('giant spikes') were probably Na+ spikes activated in the soma and spread distally. Occasionally Ca2+ spikes, with a higher threshold, might also be activated to give dual-phase response.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 11006, year: 1988, vol: 402, page: 751, stat: Journal Article,

Quisqualate, kainate and NMDA can initiate spreading depression in the turtle cerebellum
Lauritzen M; Rice ME; Okada Y; Nicholson C
1988 Dec 20;475(2):317-327, Brain research
This study evaluated the role of excitatory amino acid (EAA) receptor activation in spreading depression (SD), using the in vitro turtle cerebellum as a model system. SD was triggered by electrical stimulation or by elevated K+ after the cerebellum had been conditioned for at least 30 min with physiological saline in which most of the chloride had been replaced by propionate. SD was recognized as a transient (1-3 min) negative shift of extracellular potential accompanied by depression of evoked potentials (15-30 min) and an increase of extracellular K+ up to 60 mM, which spread across the cerebellum at rates of 1-7 mm/min. SD usually commenced in the granular layer, which apparently contains the 3 major EAA receptor subtypes, quisqualate, kainate and N-methyl-D-aspartate (NMDA), then subsequently spread to the molecular layer, which is largely free of NMDA receptors. Glutamate, aspartate, NMDA, kainate and quisqualate all triggered SD. Kynurenic acid and 2-aminophosphonovaleric acid (APV) inhibited SD under certain conditions further suggesting involvement of EAA receptors. The initiation of SD was blocked by high Mg2+ and facilitated in low extracellular Mg2+, which also eliminated the delay in molecular layer SD onset. Our data suggest that no one EAA receptor subtype is singly responsible for SD
— id: 10860, year: 1988, vol: 475, page: 317, stat: Journal Article,

Diffusion of penicillin in agar and cerebral cortex of the rat
Lehmenkuhler, A; Kersting, U; Nicholson, C
1988 Mar 15;444(1):181-183, Brain research
The diffusion of penicillin was studied in agar gel and the cerebral cortex of the rat using pressure microinjection and ion-selective microelectrodes selective to penicillin. From the agar measurements a free diffusion coefficient for penicillin of 3.52 +/- 0.08 (mean +/- S.E.M.) X 10(-6) cm2.s-1 for 37 degrees C was determined. The tortuosity value in the cortex was 1.62 +/- 0.03 (mean +/- S.E.M.) at the same temperature implying an apparent diffusion coefficient of 1.34 +/- 0.07 (mean +/- S.E.M.) x 10(-6) cm2.s-1. This tortuosity value means that penicillin diffuses in the cortex in a similar manner to other extracellular substances. These diffusion values clarify previous estimates and permit accurate evaluation of epilepsy models based on the application of penicillin
— id: 148775, year: 1988, vol: 444, page: 181, stat: Journal Article,

Issues involved in the transmission of chemical signals through the brain extracellular space
Nicholson C
1988 89;26(2-3):69-80, Acta morphologica Neerlando-Scandinavica
Two classes of substances exist within the extracellular space: energetic and informational. Examples of the former are glucose, dissolved oxygen and CO2 while the latter include excitatory amino acids, cathecholamines and opiates. The simple ions Na+ and Cl- are generally associated with energetic processes while extracellular K+ and Ca2+ tend to be informational in function. Local release of an informational substance brings about a concentration gradient that causes the substance to be dispersed in the extracellular space by diffusion. This process is modified relative to a free aqueous medium by the constraints of volume fraction, tortuosity and uptake. Volume fraction is defined simply as the fraction of a brain region that is extracellular. If a given quantity of substance is released into a region with a reduced volume fraction then the substance will reach a higher concentration than it would in a free medium. Tortuosity is related to the increase in the path length of the random walk of a diffusing particle due to the necessity to navigate around cellular obstructions. Tortuosity manifests itself as a decrease in the diffusion coefficient. Uptake represents the movement of a substance from the extracellular space to the intracellular. Since initially a concentration gradient exists in this direction and all membranes have some permeability some concentration-dependent uptake always occurs. In addition there exist specific carrier-mediated uptake processes for some substances such as amino acids or catecholamines. In some regions the dispersal process can be dominated by uptake rather than diffusion. While volume fraction, tortuosity and uptake have all been demonstrated by a technique based on the use of radiolabels and other methods, these classical techniques have limited spatial and temporal resolution. The advent of methods based on micro-injection of substances by iontophoresis or pressure and subsequent detection with ion-selective microelectrodes (ISMs) or voltammetric microsensors (VMs) has opened a new window onto the dynamic local behavior of the extracellular space. In the last decade our laboratory and others have studied the migration of the test substances tetramethylammonium, tetraethylammonium, AsF6- and alpha naphthalene sulfonate, the endogenous ions K+ and Ca2+, the epileptogenic agent penicillin and the neurotransmitter dopamine. These studies have been carried out on the cerebellum and some other regions in a variety of species that include rat, turtle, skate and an intervertebrate, the cuttlefish.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 11271, year: 1988, vol: 26, page: 69, stat: Journal Article,

Magnetic field associated with spreading depression: a model for the detection of migraine
Okada YC; Lauritzen M; Nicholson C
1988 Feb 23;442(1):185-190, Brain research
Slow variations of the magnetic field were recorded in real time during spreading depression (SD) in the isolated turtle cerebellum. The magnetic signal lasted for 2-10 min with the largest amplitude in the first minute. The field strength was of sufficient magnitude to be measured unaveraged at 2-4 cm from the tissue. The directions and time course of the magnetic signal indicated that cerebellar SD is accompanied by current normal to the cerebellar surface. The observations reported here are of clinical interest due to the potential involvement of SD in various neurological disorders, notably head trauma and migraine
— id: 11181, year: 1988, vol: 442, page: 185, stat: Journal Article,

Magnetic evoked field associated with transcortical currents in turtle cerebellum
Okada YC; Nicholson C
1988 May;53(5):723-731, Biophysical journal
The neural basis of magnetic evoked fields of the brain was studied with an isolated turtle cerebellum as a model preparation. The turtle cerebellum is a nearly flat tissue with neural processes arranged along three orthogonal axes of symmetry. According to theoretical results, this geometry should enable us to selectively measure the magnetic field due to a subpopulation of nerve cells whose longitudinal axes are perpendicular to the cerebellar surface, by simply placing the cerebellum vertically in the bath so that these cells are horizontal and by measuring the field along the rostrocaudal axis perpendicular to the longitudinal axis of these nerve cells. To elicit neural activity in these cells the dorsal midline was electrically stimulated with a bipolar electrode. Consistent with our expectations, the one-dimensional profile of the field normal to bath surface (Bz) was antisymmetrical along the rostrocaudal axis, implying that the underlying currents were transcortical. Also, the Bz field at a field extremum varied as a cosine of the orientation of the cerebellum when it was rotated about its rostrocaudal axis with the amplitude being zero when the cerebellum was horizontal. The Bz field was dipolar as judged by statistically excellent fits of the dipolar field to the one-dimensional field profile and to the distance function relating the field magnitude at an extremum to measuring distance. This was the case even for the initial component thought to be due to antidromic action currents invading the soma and dendrites of Purkinje cells. We also showed that the dipolar term of the source could be localized within 1 mm of the actual source location in most cases
— id: 11098, year: 1988, vol: 53, page: 723, stat: Journal Article,

Behavior of extracellular K+ and pH in skate (Raja erinacea) cerebellum
Rice ME; Nicholson C
1988 Oct 4;461(2):328-334, Brain research
Ion-selective microelectrodes were used to measure extracellular K+ concentrations ([K+]o) and extracellular pH (pHo) in skate cerebellum under resting and stimulated conditions. Consistent with earlier ion analysis of elasmobranch cerebrospinal fluid (CSF), [K+]o was 3.6 +/- 0.1 mM. During parallel fiber activation, [K+]o increased to an upper limit of 12-14 mM with an approximately linear dependence on stimulation frequency (1-20 Hz). Post-stimulus undershoots of 0.1-0.6 mM were seen throughout an animal temperature range of 13-18 degrees C. When stimulation produced spreading depression (SD), [K+]o first increased to about 10 mM, then rose more rapidly to about 30 mM. These observations indicate a K+ ceiling of 10-12 mM in elasmobranchs. This ceiling is the same as that seen in mammals, despite marked differences in CSF composition and osmolality between mammalian and elasmobranch species. Extracellular pH (resting pHo was 7.1-7.3) was also altered during parallel fiber stimulation. An initial alkaline shift and subsequent extracellular acidification were characteristic of the response. These pHo transients were similar to those reported in other preparations, although the alkaline shift was enhanced. This may be attributed to the relatively low buffering capacity of elasmobranch CSF and to summation with a generally smaller acid shift
— id: 10933, year: 1988, vol: 461, page: 328, stat: Journal Article,

Magnetic localization of neuronal activity in the human brain
Yamamoto T; Williamson SJ; Kaufman L; Nicholson C; Llinas R
1988 Nov;85(22):8732-8736, Proceedings of the National Academy of Sciences of the United States of America
The performance of a cryogenic system that monitors the extracranial magnetic field simultaneously at 14 positions over the scalp has been evaluated to determine the accuracy with which neuronal activity can be located within the human brain. Initially, measurements were implemented on two model systems, a lucite sphere filled with saline and a model skull. With a magnetic field strength similar to that of a human brain, the measurement and analysis procedures demonstrated a position accuracy better than 3 mm, for a current dipole 3 cm beneath the surface. Subsequently, measurements of the magnetic field pattern appearing 100 ms after the onset of an auditory tone-burst stimulus were obtained in three human subjects. The location of the current dipole representing intracellular ionic current in active neurons of the brain was determined, with 3-mm accuracy, to be within the cortex forming the floor of the Sylvian fissure of the individual subjects, corresponding closely to the Heschl gyrus as determined from magnetic resonance images. With the sensors placed at appropriate positions, the locations of neuronal sources for different tone frequencies could be obtained without moving the recording instrument. Adaptation of activity in human auditory cortex was shown to reveal long-term features with a paradigm that compared response amplitudes for three tones randomly presented
— id: 9932, year: 1988, vol: 85, page: 8732, stat: Journal Article,

Calcium diffusion in the brain cell microenvironment
Nicholson, C; Rice, M E
1987 May;65(5):1086-1091, Canadian journal of physiology & pharmacology
A review of some of the literature on Ca2+ diffusion in free media and a variety of nervous tissues is presented. In the majority of tissue studies the apparent diffusion coefficient of Ca2+ is three to nine times smaller than that in a free aqueous medium. The methodology of using pressure microejection and Ca2+ ion-selective microelectrodes to measure Ca2+ diffusion is discussed. Our ongoing studies of Ca2+ diffusion in the cerebral cortex of the rat, using these methods, also confirm that Ca2+ diffusion is mainly influenced by the tortuosity of the tissue rather than other factors such as binding to extracellular charge sites or uptake
— id: 145565, year: 1987, vol: 65, page: 1086, stat: Journal Article,

Magnetic field associated with neural activities in an isolated cerebellum
Okada, Y C; Lauritzen, M; Nicholson, C
1987 May 26;412(1):151-155, Brain research
A superconducting sensor was used to measure the magnetic field evoked from the isolated cerebellum of the turtle by brief electrical stimulations of the dorsal surface. The field was generated by neuronal activities, since its amplitude was reduced when Mn2+ was applied and completely abolished with tetrodotoxin. In normal bathing medium, the field amplitude at a distance of 17 mm was as much as 1 pT, demonstrating that the magnetic technique can be used to remotely monitor neural activities in regions of 10 mm3 or even less
— id: 148769, year: 1987, vol: 412, page: 151, stat: Journal Article,

MEG source models and physiology
Okada, Y; Lauritzen, M; Nicholson, C
1987 Jan;32(1):43-51, Physics in medicine & biology
We report in vitro experiments on the source(s) of the magnetic fields produced by the brain. Theoretical arguments suggest that the dominant sources should be dipolar and oriented parallel to the scalp. Using an isolated turtle cerebellum as a model, we find that the fields produced following dorsal stimulation are attributable to current flow perpendicular to the cerebellum surface, suggesting Purkinje cell sources. We also discuss observations of longer lasting fields associated with spreading depression induced in the cerebellum
— id: 148770, year: 1987, vol: 32, page: 43, stat: Journal Article,

Interstitial ascorbate in turtle brain is modulated by release and extracellular volume change
Rice, M E; Nicholson, C
1987 Oct;49(4):1096-1104, Journal of neurochemistry
The isolated turtle cerebellum was used as a model system to study effects of depolarizing conditions on interstitial ascorbic acid concentration. The depolarizing stimulus was Leao's spreading depression, which is characterized by transient negative extracellular potentials, high potassium levels (20-60 mM), and local depression of neuronal activity. Interstitial concentrations of ascorbate (200-400 microM) and other electroactive species were monitored voltammetrically, using graphite fiber microelectrodes. Total tissue ascorbate (1,810 nmol/g tissue wet weight) was similar to mammalian levels and was several orders of magnitude higher than catecholamine and indoleamine content. During spreading depression, a large (up to 200 microM) increase in concentration of interstitial electroactive species was monitored. Use of Nafion- and ascorbate oxidase-coated electrodes and uricase confirmed that ascorbate was the only substance detected. Simultaneous monitoring of ascorbate, extracellular potential, and extracellular volume (using tetramethylammonium and ion-selective microelectrodes) indicated that (a) the ascorbate increase began with the decrease in extracellular volume during spreading depression, and (b) much of the increase was the result of extracellular volume decrease. In sucrose-substituted medium, in which volume changes are eliminated, a 50 microM increase in interstitial ascorbate, caused by release from intracellular stores, was also seen. The ascorbate concentration increase was prolonged in sucrose medium, suggesting that an uptake process involving sodium may further regulate interstitial ascorbate concentration
— id: 145564, year: 1987, vol: 49, page: 1096, stat: Journal Article,

Modulation by applied electric fields of Purkinje and stellate cell activity in the isolated turtle cerebellum
Chan, C Y; Nicholson, C
1986 Feb;371:89-114, Journal of physiology
Quasi steady-state electric fields were applied across the isolated turtle cerebellum to study the relationship between applied field, neuronal morphology and the modulation of the neuronal spike firing pattern. Spiking elements were identified electrophysiologically using extracellular recording methods and by subsequent horseradish peroxidase injection, which revealed their dendritic morphology and orientation. The electric field was precisely defined by measuring the voltage gradients induced in the cerebellum by 40 s constant-current pulses. The field was constant in the vertical (dorso-ventral) axis and zero in the horizontal plane, in agreement with theory. Neurones were modulated by applying a sinusoidal field at frequencies between 0.05 and 1.0 Hz. Modulated cells exhibited an increase in firing frequency and fell into one of four classes, depending on the direction of the field that produced the modulation. Thus neurones were excited by: ventricle-directed fields (V modulation), pia-directed fields (P modulation), both of the above (V/P modulation) or showed no consistent modulation (non-modulation). Most Purkinje somata and primary dendrites (nineteen out of twenty-eight) and most Purkinje dendrites (eighteen out of thirty), were V modulated with maximum rate proportional to the peak field intensity. The dendrites of these cells were consistently oriented toward the pia. Among the stellate cells, the lower molecular layer stellates, with dendrites extending predominantly towards the pia, were mostly (nineteen out of thirty-two) V modulated. The mid-molecular layer stellates, which showed much variability in dendritic orientation, were distributed among all four of the modulation classes. The upper molecular layer stellates, with a mostly horizontal dendritic alignment, were mainly (nine out of sixteen) non-modulated. All groups of spiking elements showed a correlation between patterns of modulation by applied fields and dendritic orientation, which suggests the degree of differential polarization of the extended cable elements of the neurone by the applied field as the basic mechanism for field-induced excitation or inhibition. The threshold for modulation among all neurones was 15-20 mV/mm, which is similar to the fields that modulate other nervous tissues. This suggests that many neurones can be modulated by fields of the order of 10-20 mV/mm
— id: 148790, year: 1986, vol: 371, page: 89, stat: Journal Article,

Dynamics of the neuronal microenvironment
Nicholson, C
1986 ;481:43-45, Annals of the New York Academy of Sciences
— id: 148800, year: 1986, vol: 481, page: 43, stat: Journal Article,

The migration of substances in the neuronal microenvironment
Nicholson, C; Rice, M E
1986 ;481:55-71, Annals of the New York Academy of Sciences
— id: 145566, year: 1986, vol: 481, page: 55, stat: Journal Article,

A model for the polarization of neurons by extrinsically applied electric fields
Tranchina, D; Nicholson, C
1986 Dec;50(6):1139-1156, Biophysical journal
A model is presented for the subthreshold polarization of a neuron by an applied electric field. It gives insight into how morphological features of a neuron affect its polarizability. The neuronal model consists of one or more extensively branched dendritic trees, a lumped somatic impedance, and a myelinated axon with nodes of Ranvier. The dendritic trees branch according to the 3/2-power rule of Rall, so that each tree has an equivalent cylinder representation. Equations for the membrane potential at the soma and at the nodes of Ranvier, given an arbitrary specified external potential, are derived. The solutions determine the contributions made by the dendritic tree and the axon to the net polarization at the soma. In the case of a spatially constant electric field, both the magnitude and sign of the polarization depend on simple combinations of parameters describing the neuron. One important combination is given by the ratio of internal resistances for longitudinal current spread along the dendritic tree trunk and along the axon. A second is given by the ratio between the DC space constant for the dendritic tree trunk and the distance between nodes of Ranvier in the axon. A third is given by the product of the electric field and the space constant for the trunk of the dendritic tree. When a neuron with a straight axon is subjected to a constant field, the membrane potential decays exponentially with distance from the soma. Thus, the soma seems to be a likely site for action potential initiation when the field is strong enough to elicit suprathreshold polarization. In a simple example, the way in which orientation of the various parts of the neuron affects its polarization is examined. When an axon with a bend is subjected to a spatially constant field, polarization is focused at the bend, and this is another likely site for action potential initiation
— id: 148785, year: 1986, vol: 50, page: 1139, stat: Journal Article,

Organization of the filum terminale in the frog
Chesler, M; Nicholson, C
1985 Sep 22;239(4):431-444, Journal of comparative neurology
The histological organization of the filum terminale of the spinal cord in Rana catesbeiana and Rana pipiens was characterized to determine if this region possessed an organized neuropil or whether it was merely a glial remnant that persisted after absorption of the larval tail. The excised filum was maintained in vitro. Intracellular electrophysiological recording was performed with horseradish peroxidase injection. Tyrosine hydroxylase and serotonin distribution were revealed by immunocytochemical methods. Astroglia were the dominant cell type and displayed an elaborate variety of forms. The mean membrane potential was logarithmically related to the extracellular potassium concentration but displayed a sub-Nernstian slope. Oligodendroglia were also seen, as well as ependyma that extended from the central canal to the pial surface. Neuronal activity was revealed by occasional intracellular penetration of elements that displayed spontaneous excitatory postsynaptic or action potentials. The major evidence for the presence of neurons was the demonstration of tyrosine hydroxylase (TH) immunoreactivity in a large population of cerebrospinal fluid-contacting neurons that abutted the ventral half of the central canal. The axons of these cells entered a ventral bundle and ascended the cord; some fibers left this tract and apparently terminated on large arcuate neurons within the filum. Serotoninergic fibers were primarily confined to a subpial location at the dorsal midline. We conclude that the filum terminale of the frog has a sparse but functional neuropil that is organized around the central canal and supported by a profusion of elaborate glial forms
— id: 148801, year: 1985, vol: 239, page: 431, stat: Journal Article,

Regulation of intracellular pH in vertebrate central neurons
Chesler, M; Nicholson, C
1985 Jan 28;325(1-2):313-316, Brain research
The regulation of intracellular pH (pHi) was investigated in reticulospinal neurons of the lamprey using ion-selective microelectrodes. Steady-state pHi in 23 mM HCO-3-buffered Ringer was 7.44 +/- 0.03 with a membrane potential of 54 +/- 4 mV (mean +/- S.E.M., n = 6). In nominally HCO-3-free solutions, pHi recovery from acid loading was blocked by 10(-3)M amiloride. Recovery was stimulated by transition to HCO-3-containing solutions. Results suggest that pHi regulation in lamprey reticulospinal neurons is mediated by a Na+-H+ exchanger. The presence of a distinct, HCO-3-dependent pHi regulatory mechanism is postulated
— id: 148777, year: 1985, vol: 325, page: 313, stat: Journal Article,

Diffusion from an injected volume of a substance in brain tissue with arbitrary volume fraction and tortuosity
Nicholson, C
1985 May 6;333(2):325-329, Brain research
When a substance is pressure-injected from a micropipette into the extracellular space of the brain it may either form a cavity or it may infiltrate the extracellular space. In either case subsequent diffusion is governed by the volume fraction and tortuosity of the brain tissue as well as the diffusion coefficient of the substance itself. Appropriate equations, solutions and approximations to these problems are discussed. The results are relevant to the interpretation of studies on neuropharmacology and in situ electrochemistry
— id: 148776, year: 1985, vol: 333, page: 325, stat: Journal Article,

Comparative neurophysiology of spreading depression in the cerebellum
Nicholson, C
1984 Dec;56(4):481-494, Anais da Academia Brasileira de Ciencias
— id: 148802, year: 1984, vol: 56, page: 481, stat: Journal Article,

Changes of extracellular potassium activity induced by electric current through brain tissue in the rat
Gardner-Medwin, A R; Nicholson, C
1983 Feb;335:375-392, Journal of physiology
Ion-selective micro-electrodes have been used to measure K+ and Ca2+ activity changes in extracellular space beneath the surface of the neocortex and cerebellar cortex during current flow across the tissue surface in anaesthetized rats. Inward currents produced decreases of [K+]o and outward currents produced increases, with insignificant changes in [Ca2+]o. Changes of [K+]o were largest just under the surface of the tissue, but were detectable down to depths of ca. 1 mm. With appropriate sitting of electrodes in the cerebellar cortex, currents of 22 microA mm-2 for 400 sec produced changes averaging -42% for inward current and +66% for outward current. The [K+]o changes near the surface were most rapid immediately after the onset of current and more gradual after some tens of seconds. Deeper within the tissue the rate of change was more uniform and after the end of stimulation the return to base line was slower. The amplitude, depth dependence and time course of the [K+]o changes were in reasonable agreement with the results calculated for a model in which K+ moves partly through extracellular space but primarily through membranes and cytoplasm within the tissue. The [K+]o changes were not attributable to variations in neuronal activity, although unit activity could be modified by current, since alternating currents failed to produce [K+]o changes and neither 0.1 mM-tetrodotoxin nor 5 mM-Mn2+ abolished the changes. The [K+]o changes were not abolished by topically applied ouabain (4 X 10(-4) M), 2,4-dinitrophenol (20 mM) or iodoacetate (10 mM), or by asphyxiation. Consequently the [K+]o changes are not dependent on metabolism. The data suggest that there is a selective mechanism for passive K+ transport in an electrochemical gradient within brain tissue that results in higher K+ fluxes than could be supported by ionic mobility in the extracellular fluid. This mechanism exists not only at the surface but within the brain parenchyma and may involve current flow through glial cells
— id: 148792, year: 1983, vol: 335, page: 375, stat: Journal Article,

Potassium accumulation around individual purkinje cells in cerebellar slices from the guinea-pig
Hounsgaard, J; Nicholson, C
1983 Jul;340:359-388, Journal of physiology
K+-selective micropipettes were used to measure external K+ concentration [( K+]o) in the immediate vicinity of Purkinje cells in slices from guinea-pig cerebellum. The cells were either spontaneously active or were polarized via a separate intracellular micro-electrode. The level of [K+]o rose by 1-3 mM around the soma and dendrites of Purkinje cells during spike activity. The increases in [K+]o were usually greater during Ca2+-mediated spikes than during Na+-mediated spikes. This was even true at the soma where the Ca2+ spike only invaded electrotonically from the dendrites, in contrast to the Na+ spikes which were generated at the soma. No [K+]o changes were seen in the vicinity of Purkinje cells when the cells were hyperpolarized with current passage nor was any [K+]o change seen during subthreshold depolarizations. In glial cells, however, a hyperpolarizing current reduced [K+]o while a depolarizing current increased [K+]o in a symmetrical manner. When Ba2+ was substituted for Ca2+ in the bathing Ringer solution, prolonged plateau-potential spikes could be evoked from Purkinje cells. These spikes were accompanied by large [K+]o elevations but the plateau potentials outlasted the [K+]o elevations. These experiments suggest that large [K+]o increases can occur in the absence of Ca2+-mediated K+ conductances. Substitution of Mn2+ for Ca2+ in the Ringer solution removed some of the [K+]o increases at the Purkinje cell soma. Addition of tetrodotoxin to normal Ringer solution also reduced, but did not abolish the [K+]o increases at the soma. These experiments confirmed that both Na+ and Ca2+ spikes were involved in the [K+]o change. The diffusion characteristics of the slices were determined by an ionophoretic method using tetramethylammonium and ion-selective micropipettes. The extracellular volume fraction of the slice averaged 0.28 while the tortuosity averaged 1.84. These values were close to those found previously in the intact rat cerebellum. These data were used to make quantitative estimates of the expected [K+]o accumulation in the vicinity of a single cell (see Appendix). Such estimates showed reasonable agreement with the measured values. Our data show that quite large increases in [K+]o may occur around single Purkinje cells. Such increases have previously only been evident during the activation of cell populations in mammalian preparations. The present results are probably due to the superior recording conditions of the slice. Implications for intercellular communication are discussed
— id: 148791, year: 1983, vol: 340, page: 359, stat: Journal Article,

Alkaline and acid transients in cerebellar microenvironment
Kraig, R P; Ferreira-Filho, C R; Nicholson, C
1983 Mar;49(3):831-850, Journal of neurophysiology
1. Extracellular pH (pHo) was measured in the cerebellar cortex of the rat using a recently developed liquid membrane ion-selective micropipette (ISM). pHo was determined during stimulus-evoked neuronal activity, elevated extracellular potassium concentration, [K+]o, spreading depression (SD), and complete ischemia. In many experiments [K+]o was simultaneously determined. 2. A train of local surface stimuli (LOC) produced an initial alkaline shift in pHo from a base line of 7.20-7.30 to 7.25-7.35. This was followed by a long-lasting acid phase that reached a plateau of 7.05-7.15 after 64 s of stimulation. pHo decrease was related to stimulus frequency, intensity, and duration. 3. Superfusion with Ringer solution containing manganese ions rapidly abolished parallel fiber-induced Purkinje cell synaptic depolarization together with the alkaline shifts while enhancing the acid shifts. 4. Superfusion of the cerebellar cortex with Ringer solution containing increasingly elevated [K+] progressively lowered pHo to a plateau of 6.95-7.05. The acidification occurred in the presence of ouabain but was reversed on return to the normal [K+]o or with the addition of the glycolytic blocker, fluoride. Stimulus-evoked alkaline shifts were enhanced by K+-Ringer superfusion. These experiments suggested that the acid shift was due to the metabolic production of an anion, possibly lactate. 5. Elevation of [K+]o above 8-12 mM often produced oscillation in pHo and [K+]o with a period of about 40 s. Sometimes these oscillations ended in a spontaneous SD or SD could be evoked by stimulation. Under these conditions of raised [K+]o, the SD consisted of a very pronounced alkaline transient followed by a small, long-lasting acid shift. When SD was induced by conditioning the cerebellum with proprionate or lowered NaCl, the alkaline phase was reduced and the acid enhanced. 6. Complete ischemia began with a progressive decrease of pHo and rise in [K+]o. When [K+]o reached 12 mM, a second more rapid rise in [K+]o to 40 mM or more occurred. This was correlated with 0.1-0.2 pHo transient increase similar to that seen during SD. pHo eventually reached a plateau of 6.60-6.80, close to neutrality. 7. Superfusion with Ringer solution containing acetazolamide immediately altered pHo homeostasis by increasing base-line pHo by about 0.10 and enhanced the induced pHo changes. These results suggest that carbonic anhydrase (CA) is important for acute buffering of the brain extracellular microenvironment. 8. The above results were interpreted in terms of changes in extracellular strong ion concentration differences ( [SID]o), extracellular concentration of total weak acid ( [Atot]o) and partial pressure of CO2 (Pco2) in the brain microenvironment. The results indicate that neuronal activity produces changes in many of the constituents of the microenvironment
— id: 148786, year: 1983, vol: 49, page: 831, stat: Journal Article,

Tetrodotoxin resistant propagation and extracellular sodium changes during spreading depression in rat cerebellum
Tobiasz, C; Nicholson, C
1982 Jun 10;241(2):329-333, Brain research
Spreading depression (SD) was elicited in the benzoate-conditioned rat cerebellum by microinjection of KC1. Median SD propagation velocity was 9.2 mm . min-1. [Na+]0 decreases, median value 77.5 mM, were measured with ion-selective micropipettes. Superfusion of the cerebellum with 10-5 M tetrodotoxin did not modify the propagation velocity or decrease the [Na+]0 change. This confirms that classical voltage-mediated Na+ conductances are not involved in SD
— id: 148778, year: 1982, vol: 241, page: 329, stat: Journal Article,

Ion diffusion modified by tortuosity and volume fraction in the extracellular microenvironment of the rat cerebellum
Nicholson, C; Phillips, J M
1981 Dec;321:225-257, Journal of physiology
1. The validity of the macroscopic laws of ion diffusion was critically examined within the microenvironment of the extracellular space in the rat cerebellum using ion-selective micropipettes and ionophoretic point sources. 2. The concepts of volume averaging, volume fraction (alpha) and tortuosity (lambda) were defined and shown to be theoretically appropriate for quantifying diffusion in a complex medium such as the brain. 3. Diffusion studies were made with the cations tetramethylammonium and tetraethylammonium and the anions alpha-naphthalene sulphonate and hexafluoro-arsenate, all of which remained essentially extracellular during the measurements. Diffusion parameters were measured for a period of 50s and over distances of the order of 0.1 mm. 4. Measurements of the diffusion coefficients of the ions in agar gel gave values that were very close to those derivable from the literature, thus confirming the validity of the method. 5. Measurements in the cerebellum did not reveal any systematic influences of ionophoretic current strength, electrode separation, anisotropy, inhomogeneity, charge discrimination or uptake, within the limits tested. 6. The pooled data from measurements with all the ions gave alpha = 0.21 +/- 0.02 (mean +/- S.E. of mean) and lambda = 1.55 +/- 0.05 (mean +/- S.E. of mean). 7. These results show that the extracellular space occupies about 20% of the rat cerebellum and that the diffusion coefficient for small monovalent extracellular ions is reduced by a factor of 2.4 (i.e. lambda 2) without regard to charge sign. The over-all effect of this is to increase the apparent strength of any ionic source in the cerebellum by a factor of lambda 2/alpha, about 12-fold in the present case, and to modify the time course of diffusion. 8. These conclusions confirm that the laws of macroscopic diffusion are closely obeyed in the cerebellum for small ions in the extracellular space, provided that volume fraction and tortuosity are explicitly taken into account. It is likely that these conclusions are generally applicable to other brain regions and other diffusing substances
— id: 148793, year: 1981, vol: 321, page: 225, stat: Journal Article,

Identification of the Low Molecular Weight Copper Protein from Copper-intoxicated Mung Bean Plants
Nicholson C; Stein J; Wilson KA
1980 Aug;66(2):272-275, Plant physiology
Mung bean plants (Wilczek) accumulate increasingly greater amounts of buffer-extractable copper in both their shoots and roots when grown in liquid medium containing greater than 2 micrograms per milliliter copper (31.4 micromolar) as cupric sulfate. This increase in soluble copper is accompanied by an increase in the relative amount of low molecular weight (7,000 to 20,000) macromolecular-bound copper and a decrease in the relative amount of high molecular weight (greater than 20,000) copper. The major low molecular weight copper protein has been isolated from copper-intoxicated mung bean plants by a combination of ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. It was identified as mung bean plastocyanin on the basis of its molecular weight, optical behavior, and amino acid composition. No evidence was found for a low molecular weight copper-binding protein corresponding to mammalian thionein or chelatin
— id: 96825, year: 1980, vol: 66, page: 272, stat: Journal Article,

Diffusion of anions and cations in the extracellular micro-environment of the brain [proceedings]
Nicholson, C; Phillips, J M
1979 Nov;296:66P-66P, Journal of physiology
— id: 148794, year: 1979, vol: 296, page: 66P, stat: Journal Article,

Diffusion from an iontophoretic point source in the brain: role of tortuosity and volume fraction
Nicholson, C; Phillips, J M; Gardner-Medwin, A R
1979 Jun 29;169(3):580-584, Brain research
— id: 148780, year: 1979, vol: 169, page: 580, stat: Journal Article,

Anion permeability in spreading depression investigated with ion-sensitive microelectrodes
Phillips, J M; Nicholson, C
1979 Sep 21;173(3):567-571, Brain research
— id: 148779, year: 1979, vol: 173, page: 567, stat: Journal Article,

Measurements of extracellular potassium and calcium concentration during passage of current across the surface of the brain [proceedings]
Gardner-Medwin, A R; Nicholson, C
1978 Feb;275:66P-67P, Journal of physiology
— id: 148795, year: 1978, vol: 275, page: 66P, stat: Journal Article,

Extracellular ionic variations during spreading depression
Kraig, R P; Nicholson, C
1978 ;3(11):1045-1059, Neuroscience
— id: 148797, year: 1978, vol: 3, page: 1045, stat: Journal Article,

Calcium and potassium changes in extracellular microenvironment of cat cerebellar cortex
Nicholson, C; ten Bruggencate, G; Stockle, H; Steinberg, R
1978 Jul;41(4):1026-1039, Journal of neurophysiology
1. Local stimulus-evoked changes in concentration of extracellular calcium ions, [Ca2+]0, and potassium ions, [K+[0, were measured in the cerebellar cortex of the cat using paired ion-selected micropipettes. 2. Repetitive stimulation of 30 s duration decreased [Ca2+]0 from a base line of 1.2 mM to as low as 0.8 mM and increased [K+]0 from 3 mM to as much as 8 mM. The magnitude of the changes was directly related to stimulus frequency. Laminar analysis showed that the greatest ion changes occurred at the level of maximum parallel fiber-Purkinje cell dendrite stimulation, but that the [Ca2+]0 changes were more localized than the [K+]0 changes. 3. Combining real-time current-source density measurement with [K+]0 determination and local manganese application, showed that the Mn blocked parallel fiber-Purkinje cell synaptic transmission, but that much of the [K+]0 changes persisted. Thus, a large part of the [K+]0 flux most probably originated in the parallel fibers. In contrast, [Ca2+]0 changes were abolished by the Mn, indicating that the decrease in this ion was probably associated with synaptic transmission or dendritic events. 4. In a few cases, spreading depression occurred in the cat cerebellar cortex. This could be accompanied by decreases in [Ca2+]0 to as low as 0.12 mM and increases in [K+]0 in excess of 48 mM. 5. These results show that significant changes in [Ca2+]0 and [K+]0 occur during cerebellar stimulation and indicate possible origins of the ion fluxes in terms of neuronal elements. This work also shows that the cerebellar cortex of the cat can support spreading depression. The present results, together with those of earlier studies on [Ca2+]0 and [K+]0 changes in the presence of aminopyridine in the cat cerebellum, suggest that synaptic or dendritic electroresponsive properties may play a role in the observed [Ca2+]0 and [K+]0 changes
— id: 148787, year: 1978, vol: 41, page: 1026, stat: Journal Article,

Reversal properties of climbing fiber potential in cat Purkinje cells: an example of a distributed synapse
Llinas R; Nicholson C
1976 Mar;39(2):311-323, Journal of neurophysiology
1. The electrophysiological properties of the EPSP generated in Purkinje cells by the activation of CFs were studies in the cat cerebellar cortex. 2. CF-EPSPs were evoked by electrical stimulation of the cerebellar white matter and recorded intracellularly from the soma of the Purkinje cells. 3. Current was injected into the Purkinje cells via the recording micropipette using a bridge amplifer in order to study the reversal properties of the EPSP. 4. The CF-EPSP reversal was biphasic with the early portion reversing first. 5. The reversed EPSP waveform was not a mirror image of the EPSP, but displayed a briefer time course. 6. A four-compartment computer stimulation showed that the reversal properities of the CF-EPSP were explicable in terms of a distributed synapse on a cable. 7. The biphasic reversal and asymmetry were shown to be due to the spatially nonuniform potential distribution created by the somatic current injection, which predominantly reversed the proximal part of the distributed synapse. Delayed rectification may also have contributed to the reversal asymmetry. 8. The advantages of a distributed synapse over a point synapse are discussed and the reversal properties of the CF-EPSP compared to those of the Ia-evoked EPSP in motoneurons.
— id: 9987, year: 1976, vol: 39, page: 311, stat: Journal Article,

Large potassium signals and slow potentials evoked during aminopyridine or barium superfusion in cat cerebellum
Nicholson, C; Bruggencate, G T; Senekowitsch, R
1976 Sep 3;113(3):606-610, Brain research
— id: 148781, year: 1976, vol: 113, page: 606, stat: Journal Article,

Experimental optimization of current source-density technique for anuran cerebellum
Freeman, J A; Nicholson, C
1975 Mar;38(2):369-382, Journal of neurophysiology
This paper represents a systematic, semirigorous attempt to optimize the technique of current source-density (CSD) analysis experimentally. We compared different spatial differentiation formulas in terms of accuracy, aliasing, and smoothing, and provide experimental and theoretical rationale for their use. Sources of error have also been investigated. Expressions were derived to enable one to estimate the relative magnitude of errors due to electrical noise, uncertainty in tip position of recording electrodes, and error in the conductivity tensor. Corresponding experiments illlustrating the validity of such estimates are also presented. Methods to determine the optimum interelectrode spacing are given, based on computations of spatial energy-density spectra in the anuran cerebellum. The application of the technique of CSD analysis developed in this, and the accompanying paper, to the vestibulocerebellar input in the toad cerebellum provided significantly better temporal and spatial resolution of neuronal events than conventional field-potential analysis. Considerations germane to the optimum application of this technique to other neural structures are also discussed
— id: 148789, year: 1975, vol: 38, page: 369, stat: Journal Article,

Calcium role in depolarization-secretion coupling: an aequorin study in squid giant synapse
Llinas R; Nicholson C
1975 Jan;72(1):187-190, Proceedings of the National Academy of Sciences of the United States of America
Aequorin, a protein that emits light in the presence of calcium, was injected in the presynaptic terminal of the squid giant synapse. This injection was preceded by intracellular tetraethylammonium administration, which prolonged the duration of the presynaptic action potential. After this procedure light emission was evoked by single presynaptic spikes capable of releasing synaptic transmitter. In a second set of experiments, presynaptic tetraethylammonium injection was followed by the administration of tetrodotoxin extracellularly, which abolished the presynaptic action potential. Under these conditions artificial depolarization of the presynaptic terminal triggered the release of synaptic transmitter, in a graded manner. However, as previously reported by other authors, membrane potential steps to an internal positive value of approximately plus 90 mV (the suppression potential) produced a blockage of transmitter release for the duration of the imposed potential. Synaptic transmission recurred, nevertheless, as the current injection was terminated. A similar set of experiments, performed after the intracellular injection of aequorin in the presynaptic fiber, demonstrated that the aequorin light response was evoked by membrane potential steps capable of releasing synaptic transmitter. If the membrane potential was made positive to the 'suppression' level, no light response was evoked but the light emission appeared, as did transmitter release, at the end of the current pulse. These experiments demonstrate that release of transmitter is directly correlated with intracellular calcium concentration and that the suppression potential is compatible with the existence of a calcium equilibrium potential at the presynaptic terminal.
— id: 9993, year: 1975, vol: 72, page: 187, stat: Journal Article,

Real time current source-density analysis using multi-electrode array in cat cerebellum
Nicholson C; Llinas R
1975 Dec 19;100(2):418-424, Brain research
— id: 9989, year: 1975, vol: 100, page: 418, stat: Journal Article,

Theory of current source-density analysis and determination of conductivity tensor for anuran cerebellum
Nicholson, C; Freeman, J A
1975 Mar;38(2):356-368, Journal of neurophysiology
The theoretical basis of current source-density (CSD) analysis in the central nervous system is described. Equations relating CSD, the current flow vector, and the extracellular field potential are given. It is shown that the CSD provides superior resolution of neuronal events when compared to conventional field-potential analysis. Expressions for the CSD in rectangular Cartesian coordinates are derived, including the general case of anisotropic, inhomogeneous conductive tissue, and a coordinate system rotated with respect to the principal axes (APPENDIX). The minimum number of spatial dimensions for accurate CSD analysis is discussed. The conductivity tensor was experimentally measured in frog and toad cerebella. All three principal components of the tensor were evaluated and their spatial gradients determined to be negligible. It was also shown that the conductivity was independent of potential. Thus the anuran cerebellum is anisotropic, homogeneous, and ohmic. On the basis of these results the appropriate mathematical expression for the CSD was selected
— id: 148788, year: 1975, vol: 38, page: 356, stat: Journal Article,

Chloride and potassium changes measured during spreading depression in catfish cerebellum
Nicholson, C; Kraig, R P
1975 Oct 17;96(2):384-389, Brain research
— id: 148782, year: 1975, vol: 96, page: 384, stat: Journal Article,

Equilibrium potential for the postsynaptic response in the squid giant synapse
Llinas R; Joyner RW; Nicholson C
1974 Nov;64(5):519-535, Journal of general physiology
— id: 9995, year: 1974, vol: 64, page: 519, stat: Journal Article,

Intracellular staining in neurobiology
Kater, Stanley B; Nicholson, Charles
New York, Springer-Verlag, 1973,
— id: 2365, year: 1973, vol: , page: , stat: ,

Calcium transient in presynaptic terminal of squid giant synapse: detection with aequorin
Llinas R; Blinks JR; Nicholson C
1972 Jun 9;176(39):1127-1129, Science
— id: 10008, year: 1972, vol: 176, page: 1127, stat: Journal Article,

Electrophysiological properties of dendrites and somata in alligator Purkinje cells
Llinas, R; Nicholson, C
1971 Jul;34(4):532-551, Journal of neurophysiology
— id: 10016, year: 1971, vol: 34, page: 532, stat: Journal Article,

Field potentials in the alligator cerebellum and theory of their relationship to Purkinje cell dendritic spikes
Nicholson, C; Llinas, R
1971 Jul;34(4):509-531, Journal of neurophysiology
— id: 10017, year: 1971, vol: 34, page: 509, stat: Journal Article,

Dendritic spikes versus cable properties
Llinas R; Nicholson C; Freeman JA; Hillman DE
1969 Jan 3;163(862):97-97, Science
— id: 10028, year: 1969, vol: 163, page: 97, stat: Journal Article,

Preferred centripetal conduction of dendritic spikes in alligator Purkinje cells
Llinas R; Nicholson C; Precht W
1969 Jan 10;163(863):184-187, Science
— id: 10027, year: 1969, vol: 163, page: 184, stat: Journal Article,

Inhibition of Purkinje cells in the cerebellum of elasmobranch fishes
Nicholson C; Llinas R
1969 Feb;12(2):477-481, Brain research
— id: 10026, year: 1969, vol: 12, page: 477, stat: Journal Article,

Dendritic spikes and their inhibition in alligator Purkinje cells
Llinas R; Nicholson C; Freeman JA; Hillman DE
1968 Jun 7;160(832):1132-1135, Science
— id: 10030, year: 1968, vol: 160, page: 1132, stat: Journal Article,