Jonathan A. Coles

Potassium activity in photoreceptors, glial cells and extracellular space in the drone retina: changes during photostimulation.

1. A double‐barrelled potassium‐sensitive micro‐electrode was developed that was fine enough to record intracellular electrical potentials and potassium activities (aK) in the drone retina. 2. aK was measured in the photoreceptor cells, in the pigment (glial) cells, and in the extracellular space, in the superfused, cut, retina. The effect of photostimulation was studied: 20 msec light flashes, intense enough to evoke receptor potentials of maximum amplitude were presented, 1/sec, in a train lasting about 2 min. 3. In photoreceptors with membrane potentials greater than or equal to 50 mV aK in the dark was 79 mM, S.D. = 27 mM, n = 11. During photostimulation aK fell by 21.5 +/‐ 9.5 mM with a half‐time of 30 +/‐ 22 sec. (A tentative conversion from activities to free concentrations can be made by taking the activity coefficient as 0.70 its value in the Ringer solution). 4. In pigment cells with membrane potentials greater than or equal to 50 mV, aK in the dark was 52 mM, S.D. = 13 mM, n = 11. During photostimulation aK increased by 14 +/‐ 5 mM. 5. In the extracellular space aK increased during photostimulation with a mean half‐time of less than 1.3 sec to a maximum (mean value 14 mM, S.D. = 8.4 mM, n = 22), and then fell to a plateau. 6. It is estimated from the anatomy that the photoreceptors occupy approximately 38% of the total volume of the retina, the pigment cells 57%, and extracellular space 5%. Hence, it seems possible that during photostimulation nearly all the net loss of potassium from the photoreceptors is temporarily stored in the pigment cells. 7. Recordings were made in the extracellular space of the intact animal by passing the electrode through a hole in the cornea. The mean aK in the dark was 7.7 mM, S.E. = 0.4 mM, n = 22. In the superfused retina, aK in the dark was 6.3 mM, S.E. = 0.7 mM, n = 22, even though aK in the Ringer solution was 2.2 mM. Increasing the aK of the Ringer solution to 7.0 mM had no apparent effect on aK in the extracellular space at depths greater than 20 micron. 8. In the intact animal the amplitude and time course of the change in extracellular aK evoked by the standard pattern of photostimulation were within the range observed in the superfused preparation.

Modification of potassium movement through the retina of the drone (Apis mellifera male) by glial uptake.

Intracellular recordings were made in photoreceptors and glial cells (outer pigment cells) of the superfused cut head of the honey‐bee drone (Apis mellifera male). When the [K+] in the superfusate was abruptly increased from 3.2 mM to 17.9 mM both photoreceptors and glial cells depolarized. The time course of the depolarization of the photoreceptors was slower with increasing depth from the surface. Half time of depolarization was plotted against depth: this graph was compatible with the arrival of K+ being exclusively by diffusion through the extracellular clefts. However, as we then showed, this interpretation is inadequate. The time course of depolarization of the glial cells was almost the same at all depths. This indicates that they are electrically coupled. Consequently, current‐mediated K+ flux (spatial buffering) through glial cells will contribute to the transport of K+ through the tissue: K+ ions enter the glial syncytium in the region of high external potassium concentration, [K+]0, and an equivalent quantity of K+ ions leave in regions of low [K+]0. Intracellular K+ activity (aiK) was measured with double‐barrelled K+‐sensitive micro‐electrodes in slices of retina superfused on both faces. When [K+] in the superfusate was increased from 7.5 mM to 17.9 mM an increase in aiK was observed in glial cells at all depths in the slice (initial rate 1.7 mM min‐1, S.E. of the mean = 0.2 mM min‐1), but there was little increase in the photoreceptors (0.3 +/‐ 0.2 mM min‐1). The increase in aiK in glial cells near the centre of the slice could not have been caused by spatial buffering; it presumably resulted from net uptake. We conclude that when [K+] is increased at the surface of this tissue, the build up of K+ in the extracellular clefts depends on extracellular diffusion, spatial buffering and net uptake. The latter two processes, which have opposing effects, involve about 10 times as much K+ as the first. This is in rough agreement with less direct experiments on mammalian brain (Gardner‐Medwin, 1977, 1983b).

Uptake of locally applied deoxyglucose, glucose and lactate by axons and Schwann cells of rat vagus nerve

We asked whether, in a steady state, neurons and glial cells both take up glucose sufficient for their energy requirements, or whether glial cells take up a disproportionate amount and transfer metabolic substrate to neurons. A desheathed rat vagus nerve was held crossways in a laminar flow perfusion chamber and stimulated at 2 Hz. 14C‐labelled substrate was applied from a micropipette for 5 min over a < 0.6 mm band of the surface of the nerve. After 10‐55 min incubation, the nerve was lyophilized and the longitudinal distribution of radioactivity measured. When the weakly metabolizable analogue of glucose, 2‐deoxy‐[U‐14C]d‐glucose (*DG), was applied, the profiles of the radioactivity broadened with time, reaching distances several times the mean length of the Schwann cells (0.32 mm; most of the Schwann cells are non‐myelinating). The profiles were well fitted by curves calculated for diffusion in a single compartment, the mean diffusion coefficient being 463 ± 34 μm2 s−1 (±s.e.m., n= 16). Applications of *DG were repeated in the presence of the gap junction blocker, carbenoxolone (100 μm). The profiles were now narrower and better fitted with two compartments. One compartment had a coefficient not significantly different from that in the absence of the gap junction blocker (axons), the other compartment had a coefficient of 204 ± 24 μm2 s−1, n= 4. Addition of the gap junction blocker 18‐α‐glycyrrhetinic acid, or blocking electrical activity with TTX, also reduced longitudinal diffusion. Ascribing the compartment in which diffusion was reduced by these treatments to non‐myelinating Schwann cells, we conclude that 78.0 ± 3.6 % (n= 9) of the uptake of *DG was into Schwann cells. This suggests that there was transfer of metabolic substrate from Schwann cells to axons. Local application of [14C]glucose or [14C]lactate led to variable labelling along the length of the nerve, but with both substrates narrow peaks were often present at the application site; these were greatly reduced by subsequent treatment with amylase, a glycogen‐degrading enzyme.

Cellular pathways of energy metabolism in the brain: is glucose used by neurons or astrocytes?

Most techniques presently available to measure cerebral activity in humans and animals, i.e. positron emission tomography (PET), autoradiography, and functional magnetic resonance imaging, do not record the activity of neurons directly. Furthermore, they do not allow the investigator to discriminate which cell type is using glucose, the predominant fuel provided to the brain by the blood. Here, we review the experimental approaches aimed at determining the percentage of glucose that is taken up by neurons and by astrocytes. This review is integrated in an overview of the current concepts on compartmentation and substrate trafficking between astrocytes and neurons. In the brain in vivo, about half of the glucose leaving the capillaries crosses the extracellular space and directly enters neurons. The other half is taken up by astrocytes. Calculations suggest that neurons consume more energy than do astrocytes, implying that astrocytes transfer an intermediate substrate to neurons. Experimental approaches in vitro on the honeybee drone retina and on the isolated vagus nerve also point to a continuous transfer of intermediate metabolites from glial cells to neurons in these tissues. Solid direct evidence of such transfer in the mammalian brain in vivo is still lacking. PET using [18F]fluorodeoxyglucose reflects in part glucose uptake by astrocytes but does not indicate to which step the glucose taken up is metabolized within this cell type. Finally, the sequence of metabolic changes occurring during a transient increase of electrical activity in specific regions of the brain remains to be clarified.

Silanization of glass in the making of ion-sensitive microelectrodes

The silanization of glass, particularly Pyrex, was studied using reaction conditions that might be applied in the fabrication of ion-sensitive microelectrodes of the liquid-membrane type. The efficacy was tested by measuring the hydrophobicity (contact angle) or electrical resistivity of the treated surface. Aminosilanes, such as trimethyl-(dimethylamino)-silane are better than chlorosilanes, the optimum temperature is 250-330 degrees C, and the reaction comes near to completion in 5 min. Silanization of glass that is newly exposed (as in the pulling of a micropipette) is greatly improved if the surface is treated with acid. There is considerable variation from one kind of glass to another. A recipe for making double-barrelled ion-sensitive microelectrodes is given.

Effects of injections of calcium and EGTA into the outer segments of retinal rods of Bufo marinus.

1. Intracellular recordings were made from the outer segments of rods in the isolated, superfused retina of Bufo marinus. Cells were impaled under observation with a compound microscope fitted with an infra‐red image converter. Changes of membrane voltage and some concomitant changes of input resistance were measured in response to light, membrane polarization and iontophoretic injections.

Effects of adapting lights on the time course of the receptor potential of the anuran retinal rod.

1. The intracellular receptor potential of the retinal rod cell was recorded in the unperfused, isolated retina of Rana catesbiana and in the perfused, isolated retina of Bufo marinus. Qualitatively, the responses from the two preparations were similar. 2. The rate at which the receptor potential returned to the dark level at the termination of a pulse of light (Voff) was measured at a fixed potential chosen to be about 0–6 of the way from the dark level to the peak of the response. 3. When the light intensity was such that less than about 10‐minus 5 of the photopigment was bleached per second, Voff increased as the duration of the pulse was increased, reaching a maximum in 50–100 s. 4. When a brief test flash was presented at various intervals after an adapting pulse lasting about 50 s, Voff for the test flash was greater than the value in the dark adapted state for times up to about 80 s after the adapting pulse. 5. It has been hypothesized that in the vertebrate rod light causes release from the disk sacs of particles which block conducting channels in the surface membrane (Yoshikami & Hagins, 1971, 1973). A modification is proposed in which the blocking particles are converted to an inactive state can be increased by light adaptation. 6. This modified hypothesis will account qualitatively for the further observations that (a) during the response to illumination lasting several seconds the membrane potential recovers part of the way to the dark level and (b) if a second light pulse is superimposed on this background illumination then after the superimposed pulse the depolarization is increased.

Serial in vivo spectroscopic nuclear magnetic resonance imaging of lactate and extracellular pH in rat gliomas shows redistribution of protons away from sites of glycolysis

The acidity of the tumor microenvironment aids tumor growth, and mechanisms causing it are targets for potential therapies. We have imaged extracellular pH (pHe) in C6 cell gliomas in rat brain using 1H magnetic resonance spectroscopy in vivo. We used a new probe molecule, ISUCA [(+/-)2-(imidazol-1-yl)succinic acid], and fast imaging techniques, with spiral acquisition in k-space. We obtained a map of metabolites [136 ms echo time (TE)] and then infused ISUCA in a femoral vein (25 mmol/kg body weight over 110 min) and obtained two consecutive images of pHe within the tumor (40 ms TE, each acquisition taking 25 min). pHe (where ISUCA was present) ranged from 6.5 to 7.5 in voxels of 0.75 microL and did not change detectably when [ISUCA] increased. Infusion of glucose (0.2 mmol/kg.min) decreased tumor pHe by, on average, 0.150 (SE, 0.007; P < 0.0001, 524 voxels in four rats) and increased the mean area of measurable lactate peaks by 54.4 +/- 3.4% (P < 0.0001, 287 voxels). However, voxel-by-voxel analysis showed that, both before and during glucose infusion, the distributions of lactate and extracellular acidity were very different. In tumor voxels where both could be measured, the glucose-induced increase in lactate showed no spatial correlation with the decrease in pHe. We suggest that, although glycolysis is the main source of protons, distributed sites of proton influx and efflux cause pHe to be acidic at sites remote from lactate production.

Changes in sodium activity during light stimulation in photoreceptors, glia and extracellular space in drone retina.

Ion‐selective micro‐electrodes were used to measure Na+ activity, aNa, in the two types of cell, photoreceptors and glial cells, and in the extracellular space, in superfused slices of the retina of the honey‐bee drone, Apis mellifera male. Movements of Na+ were induced by light stimulation, or by increasing [K+] in the superfusate. In the dark, aNa in the photoreceptors was 10 mM (S.E. of the mean = 1 mM); in the glial cells it was higher: 37 +/‐ 2 mM. We estimate that in this preparation about 2/3 of the free Na+ in the tissue is in the glial cells. Stimulation with a train of light flashes, 1 s‐1 for 90 s caused aNa in the photoreceptors to increase by 16 +/‐ 2 mM. K+ activity, aK, decreased by 21 +/‐ 3 mM. During the standard train of light flashes, aNa in glial cells decreased by only 1.5 +/‐ 0.3 mM, much less than the increase in aK (7 +/‐ 2 mM). One possible interpretation of this result is that most of the increase in aK is due to K+ uptake by a mechanism other than Na+‐K+ exchange. In extracellular fluid, stimulation caused aNa to fall to a relatively steady value in about 10 s. Unlike aK, there was no tendency for aNa to return to the base line during the remainder of the 90 s stimulation. The fall in aNa was 14 +/‐ 1 mM: a greater fall is prevented by extracellular electric currents and a decrease in extracellular volume. When [K+] in the superfusate was increased from 7.5 to 18 mM, aNa decreased in the glial cells but not in the photoreceptors. In this tissue, stimulation causes changes in aNa in the neurones that might be large enough to modify the biochemistry of the cells. But in the glia, the fractional changes are small.

Extracellular K+ in the supraoptic nucleus of the rat during reflex bursting activity by oxytocin neurones.

1. We have investigated changes in extracellular potassium concentration [K+]o in the supraoptic nucleus of lactating rats and in particular those that occur during the intense burst of firing by the oxytocin neurones involved in the milk ejection reflex. 2. Double‐barrelled K(+)‐selective microelectrodes containing a highly selective sensor based on valinomycin were lowered through the exposed cortex towards the supraoptic nucleus (SON) of female rats anaesthetized with urethane. The mean resting [K+]o in the hypothalami of five rats was 2.4 mM, S.D. = 0.3 mM. 3. Where the reference barrel recorded extracellular action potentials from an oxytocin cell, the reflex burst of firing (4 s, typical maximum 50 Hz) was accompanied by a mean increase in [K+]o (delta[K+]o) of 0.22 mM (S.E.M. = 0.02 mM, fifty‐seven bursts in eight cells in seven rats). The rise in [K+]o did not begin more than 0.1 s before the onset of the burst, and began to fall from its maximum during the burst. Slow field potentials, indicative of spatial buffering of K+, were undetectable (less than 50 microV). When the electrode was advanced in steps, the amplitudes of both delta[K+]o and the action potential declined steeply to about 10% over a distance of 20 microns: K+ from oxytocin cells appears to be prevented from dispersing freely through the extracellular space of the SON. 4. When the electrode recorded action potentials from a vasopressin cell, delta[K+]o during an oxytocin cell burst was very small: 0.021 mM (S.E.M. = 0.005 mM). At other sites in the SON, where antidromic stimulation evoked a field potential but no action potential, delta[K+]o was 0.047 +/‐ 0.005 mM. We conclude that the reason oxytocin bursts do not affect vasopressin cells is that [K+]o rises very little around vasopressin cells. A fortiori, since the increases in [K+]o were very small except where action potentials from oxytocin cells were recorded, they can make no significant contribution to synchronizing the onsets of bursts in oxytocin cells that are not contiguous. 5. A standard antidromic stimulation from the pituitary stalk, at 40 Hz for 4 s, which stimulated both oxytocin neurones and vasopressin neurones, caused a delta[K+]o of 0.17‐1.8 mM, the variation being mainly from rat to rat. The larger delta[K+]o values were accompanied by slow negative potentials of up to 1.5 mV, there was a gradient in delta[K+]o decreasing towards the pia at the inferior limit of the SON, and there was a slow increase in [K+] in the subarachnoid space.(ABSTRACT TRUNCATED AT 400 WORDS)