Mitchell Chesler

Axon-Glia Interactions and the Domain Organization of Myelinated Axons Requires Neurexin IV/Caspr/Paranodin

Myelinated fibers are organized into distinct domains that are necessary for saltatory conduction. These domains include the nodes of Ranvier and the flanking paranodal regions where glial cells closely appose and form specialized septate-like junctions with axons. These junctions contain a Drosophila Neurexin IV-related protein, Caspr/Paranodin (NCP1). Mice that lack NCP1 exhibit tremor, ataxia, and significant motor paresis. In the absence of NCP1, normal paranodal junctions fail to form, and the organization of the paranodal loops is disrupted. Contactin is undetectable in the paranodes, and K(+) channels are displaced from the juxtaparanodal into the paranodal domains. Loss of NCP1 also results in a severe decrease in peripheral nerve conduction velocity. These results show a critical role for NCP1 in the delineation of specific axonal domains and the axon-glia interactions required for normal saltatory conduction.

Rapid astrocyte death induced by transient hypoxia, acidosis, and extracellular ion shifts

Death of astrocytes requires hours to days in injury models that use hypoxia, acidosis, or calcium paradox protocols. These methods do not incorporate the shifts in extracellular K+, Na+, Cl−, and Ca2+ that accompany acute brain insults. We studied astrocyte survival after exposure to hypoxic, acidic, ion‐shifted Ringer (HAIR), with respective [Ca2+], [K+], [Na+], [Cl−], and [HCO  −3 ] of 0.13, 65, 51, 75, and 13 mM (15% CO2/85% N2, pH 6.6). Intracellular pH (pHi) was monitored with the fluorescent dye BCECF. Cell death was indicated by a steep fall in the pH‐insensitive, 440‐nm‐induced fluorescence (F440) and was confirmed by propidium iodide staining. After 15–40‐min HAIR exposure, reperfusion with standard Ringer caused death of most cultured (and acutely dissociated) astrocytes within 20 min. Cell death was not prevented if low Ca2+ was maintained during reperfusion. Survival fell with increased HAIR duration, elevated temperature, or absence of external glucose. Comparable durations of hypoxia, acidosis, or ion shifts alone did not lead to acute cell death, while modest loss was noted when acidosis was paired with either hypoxia or ion shifts. Severe cell loss required the triad of hypoxia, acidosis, and ion shifts. Intracellular pH was significantly higher in HAIR media, compared with solutions of low pH alone or with low pH plus hypoxia. These results indicate that astrocytes can be killed rapidly by changes in the extracellular microenvironment that occur in settings of traumatic and ischemic brain injury. GLIA 34:134–142, 2001.

Endogenous H+ modulation of NMDA receptor-mediated EPSCs revealed by carbonic anhydrase inhibition in rat hippocampus.

1. The occurrence of extracellular alkaline transients during excitatory synaptic transmission suggests that the NMDA receptor H(+)‐modulatory site may have a physiological role. Here we amplify these pH shifts using benzolamide (a carbonic anhydrase inhibitor) and describe concomitant effects on EPSCs in whole‐cell clamped CA1 neurones in rat hippocampal slices. 2. In CO2‐HCO3(‐)‐buffered media, benzolamide increased the time to 50% decay (t50) of the EPSCs by 78 +/‐ 14% (P < 0.01, n = 10). This occurred simultaneously with amplification of the extracellular alkaline shift (154 +/‐ 14%). 3. In CO2‐HCO3(‐)‐buffered media containing DL‐2‐amino‐5‐phosphonovalerate (APV), the EPSC t50 was unaltered by benzolamide, while the extracellular alkaline shifts were increased (111 +/‐ 23%, n = 8). 4. In Hepes‐buffered media, neither the EPSC t50 nor the extracellular alkaline shift was altered by benzolamide (n = 9). 5. These data demonstrate that NMDA receptor activity is dependent on the buffering kinetics of the brain extracellular space. The results suggest that endogenous pH shifts can modulate NMDA receptor function in a physiologically relevant time frame.

Failure and function of intracellular pH regulation in acute hypoxic-ischemic injury of astrocytes

Astrocytes can die rapidly following ischemic and traumatic injury to the CNS. Brain acid–base status has featured prominently in theories of acute astrocyte injury. Failure of astrocyte pH regulation can lead to cell loss under conditions of severe acidosis. By contrast, the function of astrocyte pH regulatory mechanisms appears to be necessary for acute cell death following the simulation of transient ischemia and reperfusion. Severe lactic acidosis, and the failure of astrocytes to regulate intracellular pH (pHi) have been emphasized in brain ischemia under hyperglycemic conditions. Direct measurements of astrocyte pHi after cardiac arrest demonstrated a mean pHi of 5.3 in hyperglycemic rats. In addition, both in vivo and in vitro studies of astrocytes have shown similar pH levels to be cytotoxic. Whereas astrocytes exposed to hypoxia alone may require 12–24 h to die, acidosis has been found to exacerbate and speed hypoxic loss of these cells. Recently, astrocyte cultures were exposed to hypoxic, acidic media in which the large ionic perturbations characteristic of brain ischemia were simulated. Upon return to normal saline (“reperfusion”), the majority of cells died. This injury was dependent on external Ca2+ and was prevented by inhibition of reversed Na+‐Ca2+ exchange, blockade of Na+‐H+ exchange, or by low pH of the reperfusion saline. These data suggested that cytotoxic elevation of [Ca2+]i occurred during reperfusion due to a sequence of activated Na+‐H+ exchange, cytosolic Na+ loading, and resultant reversal of Na+‐Ca2+ exchange. The significance of this reperfusion model to ischemic astrocyte injury in vivo is discussed.

Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains.

Mapping the conditions of spider silk proteins along the silk gland, and combining with molecular studies, reveals a pH controlled switch between lock and trigger forms, providing insights into spider silk formation.

Role of Na+-H+ and Na+-Ca2+ exchange in hypoxia-related acute astrocyte death

Cultured astrocytes do not succumb to hypoxia/zero glucose for up to 24 h, yet astrocyte death following injury can occur within 1 h. It was previously demonstrated that astrocyte loss can occur quickly when the gaseous and interstitial ionic changes of transient brain ischemia are simulated: After a 20–40‐min exposure to hypoxic, acidic, ion‐shifted Ringer (HAIR), most cells died within 30 min after return to normal saline (i.e., “reperfusion”). Astrocyte death required external Ca2+ and was blocked by KB‐R7943, an inhibitor of reversed Na+‐Ca2+ exchange, suggesting that injury was triggered by a rise in [Ca2+]i. In the present study, we confirmed the elevation of [Ca2+]i during reperfusion and studied the role of Na+‐Ca2+ and Na+‐H+ exchange in this process. Upon reperfusion, elevation of [Ca2+]i was detectable by Fura‐2 and was blocked by KB‐R7943. The low‐affinity Ca2+ indicator Fura‐FF indicated a mean [Ca2+]i rise to 4.8 ± 0.4 μM. Loading astrocytes with Fura‐2 provided significant protection from injury, presumably due to the high affinity of the dye for Ca2+. Injury was prevented by the Na+‐H+ exchange inhibitors ethyl isopropyl amiloride or HOE‐694, and the rise of [Ca2+]i at the onset of reperfusion was blocked by HOE‐694. Acidic reperfusion media was also protective. These data are consistent with Na+ loading via Na+‐H+ exchange, fostering reversal of Na+‐Ca2+ exchange and cytotoxic elevation of [Ca2+]i. The results indicate that mechanisms involved in pH regulation may play a role in the fate of astrocytes following acute CNS injuries.

Preemptive Regulation of Intracellular pH in Hippocampal Neurons by a Dual Mechanism of Depolarization Induced Alkalinization

Numerous studies have documented the mechanisms that regulate intracellular pH (pHi) in hippocampal neurons in response to an acid load. Here, we studied the response of pHi to depolarization in cultured hippocampal neurons. Elevation of external K+ (6–30 mm) elicited an acid transient followed by a large net alkaline shift. Similar responses were observed in acutely dissociated hippocampal neurons. In Ca2+-free media, the acid response was curtailed and the alkaline shift enhanced. DIDS blocked the alkaline response and revealed a prolonged underlying acidification that was highly dependent on Ca2+ entry. Similar alkaline responses could be elicited by AMPA, indicating that this rise in pHi was a depolarization-induced alkalinization (DIA). The DIA was found to consist of Cl−-dependent and Cl−-independent components, each accounting for approximately one-half of the peak amplitude. The Cl−-independent component was postulated to arise from operation of the electrogenic Na+–HCO3− cotransporter NBCe1. Quantitative PCR and single-cell multiplex reverse transcription-PCR demonstrated message for NBCe1 in our hippocampal neurons. In neurons cultured from Slc4a4 knock-out (KO) mice, the DIA was reduced by approximately one-half compared with wild type, suggesting that NBCe1 was responsible for the Cl−-independent DIA. In Slc4a4 KO neurons, the remaining DIA was virtually abolished in Cl−-free media. These data demonstrate that DIA of hippocampal neurons occurs via NBCe1, and a parallel DIDS-sensitive, Cl−-dependent mechanism. Our results indicate that, by activating net acid extrusion in response to depolarization, hippocampal neurons can preempt a large, prolonged, Ca2+-dependent acidosis.

GABAA receptors modulate axonal conduction in dorsal columns of neonatal rat spinal cord

gamma-Aminobutyric acid (GABA) can influence conduction in a number of axonal preparations from the peripheral and central nervous system. In the spinal cord, the excitability of primary afferent terminals has long been known to be affected by GABA. Whether conduction in the long fiber tracts of the spinal cord can be similarly modulated is unknown. Since GABA causes a pronounced depression of excitability in preparations of unmyelinated axons, and myelination is incomplete in the neonatal rat, we tested whether GABA can modulate conduction in the dorsal columns of 10-17-day-old rats. Experiments were performed in vitro, on isolated dorsal column segments (n = 18). The extracellular compound action potential evoked by submaximal stimuli was recorded with a glass micropipette positioned 0.5-2.0 mm from a stimulating electrode. At concentrations of 10(-4) - 10(-3) M, GABA decreased excitability, reversibly depressing the compound action potential amplitude, and increasing the latency by 47 +/- 11% and 22 +/- 9% (mean +/- S.E.M., n = 5, 10(-3) M), respectively. These effects were blocked by picrotoxin and mimicked by isoguvacine (10(-4) M), which decreased the compound action potential amplitude by 44 +/- 10% and increased the latency by 9 +/- 4% (n = 5). Lower concentrations of these agents caused a modest increase in excitability. At 10(-5) M, GABA increased the compound action potential amplitude by 14 +/- 2% and decreased the latency by 3 +/- 2% (n = 5). Our results demonstrate that functional GABAA receptors are present in neonatal dorsal columns.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous Alkaline Transients Boost Postsynaptic NMDA Receptor Responses in Hippocampal CA1 Pyramidal Neurons

In hippocampus, activation of the Schaffer collaterals generates an extracellular alkaline transient both in vitro and in vivo. This pH change may provide relief of the H+ block of NMDA receptors (NMDARs) and thereby increase excitability. To test this hypothesis, we augmented extracellular buffering in mouse hippocampal slices by adding 2 μm bovine type II carbonic anhydrase to the superfusate. With addition of enzyme, the alkaline transient elicited by a 10 pulse, 100 Hz stimulus train was reduced by 33%. At a holding potential (VH) of −30 mV, the enzyme decreased the half-time of decay and charge transfer of EPSCs by 32 and 39%, respectively, but had no effect at a VH of −80 mV. In current clamp, a 10 pulse, 100 Hz stimulus train gave rise to an NMDAR-dependent afterdepolarization (ADP). Exogenous enzyme curtailed the ADP half-width and voltage integral by 20 and 25%, respectively. Similar reduction of the ADP was noted with a brief 12 Hz stimulus train. The effect persisted in the presence of GABAergic antagonists or the L-type Ca2+ channel blocker methoxyverapamil hydrochloride but was absent in the presence of the carbonic anhydrase inhibitor benzolamide or when the exogenous enzyme was heat inactivated. The effects of the enzyme in voltage and current clamp were noted in 0 Mg2+ media but were abolished when (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate was included in the patch pipette. These results provide strong evidence that endogenous alkaline transients are sufficiently large in the vicinity of the synapse to augment NMDAR responses.

Extracellular alkalinization evoked by GABA and its relationship to activity‐dependent pH shifts in turtle cerebellum.

1. The effect of gamma‐aminobutyric acid (GABA) on extracellular pH (pHo) was investigated in the turtle cerebellum, in vitro, using double‐barrelled, H(+)‐selective microelectrodes. Responses evoked by GABA were compared with pHo shifts evoked by repetitive stimulation of the parallel fibres. 2. In media buffered with 35 mM‐HCO3‐ and 5% CO2, superfusion of GABA (1 mM) elicited an abrupt alkaline shift in the molecular layer, which averaged 0.05 +/‐ 0.02 pH units (+/‐ S.D., range 0.02‐0.12 pH units). pHo often recovered in the continued presence of GABA, and displayed a rebound acidification upon wash‐out. 3. The GABA‐evoked alkaline shift was blocked by picrotoxin and was mimicked by the GABAA agonists isoguvacine and muscimol. The GABAB agonist baclofen did not elicit an alkaline shift. Alkaline shifts evoked by stimulation of the parallel fibres were unaffected by picrotoxin. 4. In nominally HCO3(‐)‐free solutions, buffered with 35 mM‐HEPES, superfusion of GABA caused either no pHo change or a slow acid shift. In contrast, the alkaline shift evoked by stimulation of the parallel fibres became enhanced in HEPES‐buffered media. 5. The alkaline shift evoked by GABA was accompanied by an increase in extracellular K+ ([K+]o) which averaged 1.7 mM above baseline. Experimental elevation of [K+]o to a comparable level always caused a pure acid shift in the extracellular space. 6. The GABA‐evoked alkaline shift persisted when synaptic transmission was blocked using 4 mM‐kynurenic acid or saline prepared with nominally zero Ca2+ and 10 mM‐Mg2+. The alkaline shift evoked by repetitive stimulation of the parallel fibres was completely abolished in these media. 7. Although the GABA‐evoked alkaline shift was blocked in nominally HCO3(‐)‐free media, substitution of 35 mM‐formate for HCO3‐ restored the GABA response. Superfusion of 1 mM‐GABA in formate saline produced an alkaline shift of 0.040 +/‐ 0.034 pH units. 8. These results indicate that gating of GABAA channels in the vertebrate CNS gives rise to an HCO3‐ efflux which can significantly increase the pH of the brain microenvironment. However, this mechanism cannot account for the extracellular alkalinization caused by parallel fibre stimulation. Extracellular alkaline shifts capable of modulating local synaptic operations may therefore be a consequence of either excitatory or inhibitory synaptic transmission.