Bruce R. Ransom

New roles for astrocytes: Redefining the functional architecture of the brain

Astrocytes have traditionally been considered ancillary, satellite cells of the nervous system. However, work over the past decade has revealed that they interact with the vasculature to form a gliovascular network that might organize not only the structural architecture of the brain but also its communication pathways, activation, thresholds and plasticity. The net effect is that astroglia demarcate gray matter regions, both cortical and subcortical, into functional compartments whose internal activation thresholds and external outputs are regulated by single glial cells. The array of these astrocyte-delimited microdomains along the capillary microvasculature allows the formation of higher-order gliovascular units, which serve to match local neural activity and blood flow while regulating neuronal firing thresholds through coordinative glial signaling. By these means, astrocytes might establish the functional as well as the structural architecture of the adult brain.

Uniquely hominid features of adult human astrocytes.

Defining the microanatomic differences between the human brain and that of other mammals is key to understanding its unique computational power. Although much effort has been devoted to comparative studies of neurons, astrocytes have received far less attention. We report here that protoplasmic astrocytes in human neocortex are 2.6-fold larger in diameter and extend 10-fold more GFAP (glial fibrillary acidic protein)-positive primary processes than their rodent counterparts. In cortical slices prepared from acutely resected surgical tissue, protoplasmic astrocytes propagate Ca2+ waves with a speed of 36 μm/s, approximately fourfold faster than rodent. Human astrocytes also transiently increase cystosolic Ca2+ in response to glutamatergic and purinergic receptor agonists. The human neocortex also harbors several anatomically defined subclasses of astrocytes not represented in rodents. These include a population of astrocytes that reside in layers 5–6 and extend long fibers characterized by regularly spaced varicosities. Another specialized type of astrocyte, the interlaminar astrocyte, abundantly populates the superficial cortical layers and extends long processes without varicosities to cortical layers 3 and 4. Human fibrous astrocytes resemble their rodent counterpart but are larger in diameter. Thus, human cortical astrocytes are both larger, and structurally both more complex and more diverse, than those of rodents. On this basis, we posit that this astrocytic complexity has permitted the increased functional competence of the adult human brain.

Astrocyte glycogen and brain energy metabolism

The brain contains glycogen but at low concentration compared with liver and muscle. In the adult brain, glycogen is found predominately in astrocytes. Astrocyte glycogen content is modulated by a number of factors including some neurotransmitters and ambient glucose concentration. Compelling evidence indicates that astrocyte glycogen breaks down during hypoglycemia to lactate that is transferred to adjacent neurons or axons where it is used aerobically as fuel. In the case of CNS white matter, this source of energy can extend axon function for 20 min or longer. Likewise, during periods of intense neural activity when energy demand exceeds glucose supply, astrocyte glycogen is degraded to lactate, a portion of which is transferred to axons for fuel. Astrocyte glycogen, therefore, offers some protection against hypoglycemic neural injury and ensures that neurons and axons can maintain their function during very intense periods of activation. These emerging principles about the roles of astrocyte glycogen contradict the long held belief that this metabolic pool has little or no functional significance.

Functional connexin “hemichannels”: A critical appraisal

“Hemichannels” are defined as the halves of gap junction channels (also termed connexons) that are contributed by one cell; “hemichannels” are considered to be functional if they are open in nonjunctional membranes in the absence of pairing with partners from adjacent cells. Several recent reviews have summarized the blossoming literature regarding functional “hemichannels”, in some cases encyclopedically. However, most of these previous reviews have been written with the assumption that all data reporting “hemichannel” involvement really have studied phenomena in which connexons actually form the permeability or conductance pathway. In this review, we have taken a slightly different approach. We review the concept of “hemichannels”, summarize properties that might be expected of half gap junctions and evaluate the extent to which the properties of presumptive “hemichannels” match expectations. Then we consider functions attributed to hemichannels, provide an overview of other channel types that might fulfill similar roles and provide sets of criteria that might be applied to verify involvement of connexin hemichannels in cell and tissue function. One firm conclusion is reached. The study of hemichannels is technically challenging and fraught with opportunities for misinterpretation, so that future studies must apply rigorous standards for detection of hemichannel expression and function. At the same time there are reasons to expect surprises, including the possibility that some time honored techniques for studying gap junctions may prove unsuitable for detecting hemichannels. We advise hemichannel researchers to proceed with caution and an open mind.

Pentobarbitone pharmacology of mammalian central neurones grown in tissue culture.

1. The effects of the barbiturate anaesthetic pentobarbitone on the membrane properties and amino acid pharmacology of mammalian C.N.S. neurones grown in tissue culture were studied using intracellular recording coupled with bath application, extracellular ionophoresis, or focal diffusion. 2. The addition of an anaesthetic concentration of pentobarbitone to the bathing medium abolished all spontaneous synaptic activity, but did not render individual cells electrically inexcitable nor prevent evoked synaptic acitivity. 3. Focal ionophoresis of pentobarbitone or diffusion from blunt micropipettes reversibly increased membrane conductance, effectively dampening excitability without directly affecting individual action potential characteristics. 4. Pentobarbitone‐induced membrane conductance was reversibly blocked by picrotoxin. The inversion potential of the pentobarbitone voltage response depended on Cl‐ ion gradients and was similar to that of GABA. 5. Pentobarbitone reversibly enhanced the conductance increase produced by GABA with a variable slowing of response kinetics, shifting GABA dose‐response curves to the left. Responses to glycine and beta‐alanine were not affected. 6. Higher ionophoretic currents of pentobarbitone, which measurably increased membrane conductance, attenuated and markedly slowed GABA responses. Similar effects on GABA responses were observed by superimposing GABA pulses on low level GABA currents. 7. Pentobarbitone, in the absence of an increase in membrane conductance, reversibly depressed depolarizing responses to glutamate without changing response kinetics. Slower responses to acetylcholine which were associated with an apparent decrease in membrane conductance were not affected by the drug. 8. Analysis of double‐reciprocal plot data suggested a non‐competitive type of antagonism between pentobarbitone and glutamate. Pentobarbitone depression of glutamate was not affected by picrotoxin. 9. Both GABA and glutamate responses appeared to be equally sensitive to pentobarbitone. Specific interaction of the drug with amino acid receptor‐coupled events is indicated by the requirement for pentobarbitone pipette placement close to the amino acid response site. 10. The results suggest that pentobarbitone depresses neuronal excitability by (1) directly activating post‐synaptic GABA‐receptor coupled Cl‐ conductance, (2) potentiating post‐synaptic GABA‐induced conductance events, probably at the level of the GABA receptor, and (3) depressing post‐synaptic glutamate‐induced excitation, probably at the level of the conductance mechanism.

Amino acid pharmacology of mammalian central neurones grown in tissue culture.

1. Spinal and cerebellar‐brainstem areas of fetal mouse were dissociated and grown in tissue culture until large enough to permit stable intracellular recording. 2. The tissue‐cultured neurones, growing as a monolayer and accessible under direct vision using phase contrast optics, allowed precise placement of intracellular recording and extracellular ionophoretic pipettes. 3. Ionophoresis of GABA and glutamate revealed a non‐uniform distribution of responses over the cell surface, with a lack of spatial coincidence in sensitivity between the two. GABA inhibited and glutamate excited all cells tested. 4. GABA responses evoked at the cell body and on nearby process membrane were almost uniformly hyperpolarizing, while those at some peripheral process membrane were either hyperpolarizing, depolarizing or a combination of both events. All responses were associated with an increase in membrane slope conductance. 5. Membrane polarization showed that all hyperpolarizing events extrapolated to about the same inversion potential, which averaged about 9 mV more negative than resting potential (n = 95 cells). The depolarizing phases of responses evoked at peripheral membranes extrapolated to about 0 mV (n = 5 cells). 6. The hyperpolarization and increase in membrane conductance of GABA responses at the cell body were dependent on Cl‐ ions and the inversion potential of the response was dependent on the Cl‐ ion concentration gradient. The inversion potentials of GABA, glycine and beta‐alanine responses were identical. 7. When matched in magnitude for evoked conductance increase, glycine responses decayed more rapidly than GABA. Glycine and beta‐alanine voltage responses both decayed faster than GABA responses of comparable size. 8. In about half the cells tested sustained or rapidly repeated application of GABA and glycine transformed hyperpolarizing responses into depolarizations which were associated with a maintained conductance increase. Results from conditioning‐test experiments with pairs of GABA and glycine responses suggest that the reversal of response polarity is due to a rapid redistribution of Cl‐ ions. 9. The limiting slope of log‐log dose‐response curves for GABA‐induced conductance averaged about 2, while those for glutamate‐induced depolarizations averaged about 1. The results suggest that two molecules of GABA and one molecule of glutamate participate in the respective post‐synaptic responses. 10. The observation indicate that mammalian C.N.S. tissue grown in culture is a suitable model to study C.N.S. membrane pharmacology with increasing precision.

Loss of Astrocytic Domain Organization in the Epileptic Brain

Gliosis is a pathological hallmark of posttraumatic epileptic foci, but little is known about these reactive astrocytes beyond their high glial fibrillary acidic protein (GFAP) expression. Using diolistic labeling, we show that cortical astrocytes lost their nonoverlapping domain organization in three mouse models of epilepsy: posttraumatic injury, genetic susceptibility, and systemic kainate exposure. Neighboring astrocytes in epileptic mice showed a 10-fold increase in overlap of processes. Concurrently, spine density was increased on dendrites of excitatory neurons. Suppression of seizures by the common antiepileptic, valproate, reduced the overlap of astrocytic processes. Astrocytic domain organization was also preserved in APP transgenic mice expressing a mutant variant of human amyloid precursor protein despite a marked upregulation of GFAP. Our data suggest that loss of astrocytic domains was not universally associated with gliosis, but restricted to seizure pathologies. Reorganization of astrocytes may, in concert with dendritic sprouting and new synapse formation, form the structural basis for recurrent excitation in the epileptic brain.

Glycogen regulation and functional role in mouse white matter

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

Compound action potential of nerve recorded by suction electrode: a theoretical and experimental analysis

Suction electrodes are widely used for recording compound action potentials (CAPs) from peripheral nerves or central tracts. Unfortunately, the recordings obtained with suction electrodes often vary over time, making quantitative measurement of CAP amplitude difficult. We developed an equivalent electrical model which predicts that the magnitude of a recorded potential will be linearly related to the resistance of the electrode with a nerve inserted. Mathematical procedures were developed that allow correction of virtually all variability inherent in this type of recording; this variability may arise from resistance drift, variable stimulus artifact, or potentials generated as a result of the current of injury. The validity of the theoretical analysis was confirmed experimentally using rat optic nerves. The magnitude of the CAP and electrode resistance varied spontaneously by as much as 100% over time, due to changes in electrode resistance and size of the stimulus artifact. Because the CAP was linearly related to resistance, it was therefore best quantified by the slope computed from this relationship. The stimulus artifact, unlike the CAP itself, was shown to be independent of recording electrode resistance and therefore only resulted in a variable offset to the area vs resistance linear relationship; the slope of this relationship was unaffected. In the absence of stimulation, a steady negative DC potential was recorded from the optic nerve, which was greatest immediately after dissection, and was also a linear function of electrode resistance. In contrast to CAP amplitude, the latencies of the component peaks within the CAP were not significantly altered by changes in electrode resistance. The experimental results confirmed the validity of the electrical model and demonstrated that suction electrodes can be a very reliable and quantitative recording method if the signals are properly corrected.

Astrocytes convert the parkinsonism inducing neurotoxin, MPTP, to its active metabolite, MPP+

The ability of astrocytes to convert 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) to its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) was directly tested. Cultured astrocytes rapidly converted MPTP (25 micrograms/ml) to MPP+; after 6 h MPP+ concentrations reached 1.5 micrograms/ml, within the toxic range for neurons. MPTP (above 10 micrograms/ml) reduced glial density after 5 days of exposure. This toxic effect was blocked by pargyline, a monoamine oxidase inhibitor; pargyline also reduced the conversion of MPTP to MPP+ by 85%. When neurons were added to astrocyte cultures, MPTP conversion to MPP+ was not enhanced. Astrocytes appear critical in converting MPTP to MPP+, and are damaged by chronic exposure to MPTP.