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S G Waxman

Publications and source records attributed to S G Waxman.

At least 19 recordsLinked to original sources
PubMed1992

Different Na+ currents in P0- and P7-derived hippocampal astrocytes in vitro: evidence for a switch in Na+ channel expression in vivo.

Hippocampal astrocytes, derived from postnatal day zero (P0) rats, appear to be pluripotential with respect to sodium current expression in vitro, and display Na+ currents with h infinity midpoints close to -65 up to 5 days in vitro (DIV), and Na+ currents with midpoints close to -85 mV at 6 DIV and thereafter. These astrocytes also exhibit a biphasic pattern of Na+ current density, which is expressed at moderate levels at early times in vitro and decreases throughout the first 5 DIV, prior to expressing a second peak for the duration of time in culture. These observations have been interpreted as suggesting that astrocytes in these cultures display a 'switch' in Na+ channel biosynthesis, so that they express different types of Na+ current (with different h infinity curves) at early and later times in culture. To test the hypothesis that a similar switch in Na+ channel expression occurs in vivo, we have used patch-clamp methods to study Na+ current expression in astrocytes derived from rat hippocampus at various stages of postnatal development, P0, P4, P5 and P7. We observed a biphasic distribution of Na+ current density, which was highest in P0- and P7-derived astrocytes (18 pA/pF and 10.3 pA/pF, respectively); astrocytes derived at P4 and P5 did not express sodium currents. While P0-derived astrocytes show depolarized h infinity curves (midpoints close to -65 mV) at early times in culture, P7-derived astrocytes, studied at comparable times in vitro, display hyperpolarized h infinity curves (midpoints close to -85 mV).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Three types of sodium channels in adult rat dorsal root ganglion neurons.

Several types of Na+ currents have previously been demonstrated in dorsal root ganglion (DRG) neurons isolated from neonatal rats, but their expression in adult neurons has not been studied. Na+ current properties in adult dorsal root ganglion (DRG) neurons of defined size class were investigated in isolated neurons maintained in primary culture using a combination of microelectrode current clamp, patch voltage clamp and immunocytochemical techniques. Intracellular current clamp recordings identified differing relative contributions of TTX-sensitive and -resistant inward currents to action potential waveforms in DRG neuronal populations of defined size. Patch voltage clamp recordings identified three distinct kinetic types of Na+ current differentially distributed among these size classes of DRG neurons. 'Small' DRG neurons co-express two types of Na+ current: (i) a rapidly-inactivating, TTX-sensitive 'fast' current and (ii) a slowly-activating and -inactivating, TTX-resistant 'slow' current. The TTX-sensitive Na+ current in these cells was almost completely inactivated at typical resting potentials. 'Large' cells expressed a single TTX-sensitive Na+ current identified as 'intermediate' by its inactivation rate constants. 'Medium'-sized neurons either co-expressed 'fast' and 'slow' current or expressed only 'intermediate' current. Na+ channel expression in these size classes was also measured by immunocytochemical techniques. An antibody against brain-type Na+ channels (Ab7493)10 labeled small and large neurons with similar intensity. These results demonstrate that three types of Na+ currents can be detected which correlate with electrogenic properties of physiologically and anatomically distinct populations of adult rat DRG neurons.

Action Potentials↗
PubMed1992

Ultrastructural concomitants of anoxic injury and early post-anoxic recovery in rat optic nerve.

To study the effects of anoxia on CNS white matter, we examined the ultrastructure of axons and glial cells in a white matter tract, the rat optic nerve, that was subjected to a standardized anoxic insult in vitro. Previous electrophysiological studies showed that in this model, action potential conduction is rapidly abolished by anoxia, and conduction is restored after reoxygenation in about 30% of axons following a 60-min anoxic period. The present study examined the ultrastructural correlates of anoxic injury and early post-anoxic recovery in this model. Optic nerves examined immediately following 60 min of anoxia displayed numerous large, apparently empty zones located within myelin sheaths adjacent to the axon. The myelin remained compact and retained its periodicity. In some regions, the extracellular space was enlarged. There was mitochondrial swelling with loss of normal cristae. There was also loss of microtubules and, to a smaller degree, of neurofilaments in large-diameter axons. Some nodes of Ranvier in anoxic optic nerves displayed detachment of terminal oligodendroglial loops or retraction of the myelin from the node; the presence of tongue-like processes, extending from nearby cells under the detached myelin loops, suggested a possible role of cell-mediated damage to the paranodal myelin. Bundles of dense astrocyte processes were present, and there was vesicular degeneration of perinodal astrocyte processes. In optic nerves that had been permitted to recover for 60 min in oxygenated Ringers following the anoxic period, empty zones were only rarely observed within myelin sheaths and, when present, were smaller than in optic nerves immediately following 60 min of anoxia. The axoplasm of large fibers continued to show loss of microtubules and neurofilaments, as well as mitochondrial swelling. Myelin appeared normal, and only rare paranodal oligodendroglial processes remained unattached from the axon membrane. These results provide support for the idea that, during anoxia, myelinated axons are damaged with significant injury to cytoskeletal elements, probably due to an influx of calcium. The ultrastructural results, together with our earlier observations on the physiological correlates of anoxia and re-oxygenation, suggest that the development of intramyelinic spaces or damage to paranodes lead to conduction block in the anoxic optic nerve. These results also suggest that repair of these structural abnormalities may provide a morphological basis for the early recovery of conduction that occurs after re-oxygenation.

Animals↗
PubMed1992

Sodium channel expression in optic nerve astrocytes chronically deprived of axonal contact.

Immunocytochemical and electrophysiological methods were used to examine the effect of retinal ablation on the expression of sodium channels within optic nerve astrocytes in situ and in vitro. Enucleation was performed at postnatal day 3 (P3), and electron microscopy of the enucleated optic nerves at P28-P40 revealed complete degeneration of retinal ganglion axons, resulting in optic nerves composed predominantly of astrocytes. In contrast to control (non-enucleated) optic nerve astrocytes, which exhibited distinct sodium channel immunoreactivity following immunostaining with antibody 7493, the astrocytes in enucleated optic nerves did not display sodium channel immunoreactivity in situ. Cultures obtained from enucleated optic nerves consisted principally (greater than 90%) of glial fibrillary acidic protein (GFAP)+/A2B5- ("type-1") astrocytes, as determined by indirect immunofluorescence; GFAP+/A2B5+ ("type-2") astrocytes were not present, nor were GFAP-/A2B5+ (O-2A) progenitor cells. Sodium channel immunoreactivity was not present in GFAP+/A2B5- astrocytes obtained from enucleated optic nerves; in contrast, GFAP+/A2B5- astrocytes from control optic nerves exhibited 7493 immunostaining for the first 4-6 days in culture. Sodium current expression, studied using whole-cell patch-clamp recording, was attenuated in cultured astrocytes derived from enucleated optic nerves. Whereas 39 of 50 type-1 astrocytes cultured from intact optic nerves showed measurable sodium currents at 1-7 days in vitro, sodium currents were present in only 6 of 38 astrocytes cultured from enucleated optic nerves. Mean sodium current densities in astrocytes from the enucleated optic nerves (0.66 +/- 0.3 pA/pF) were significantly smaller than in astrocytes from control optic nerves (7.15 +/- 1.1 pA/pF). The h infinity-curves of sodium currents were similar in A2B5- astrocytes from enucleated and control rat optic nerves. These results suggest that there is neuronal modulation of sodium channel expression in type-1 optic nerve astrocytes, and that, following chronic loss of axonal association in vivo, sodium channel expression is down-regulated in this population of optic nerve astrocytes.

Animals↗
PubMed1992

Effects of temperature on evoked electrical activity and anoxic injury in CNS white matter.

Temperature is known to influence the extent of anoxic/ischemic injury in gray matter of the brain. We tested the hypothesis that small changes in temperature during anoxic exposure could affect the degree of functional injury seen in white matter, using the isolated rat optic nerve, a typical CNS white matter tract (Foster et al., 1982). Functional recovery after anoxia was monitored by quantitative assessment of the compound action potential (CAP) area. Small changes in ambient temperature, within a range of 32 to 42 degrees C, mildly affected the CAP of the optic nerve under normoxic conditions. Reducing the temperature to < 37 degrees C caused a reversible increase in the CAP area and in the latencies of all three CAP peaks; increasing the temperature to > 37 degrees C had opposite effects. Functional recovery of white matter following 60 min of anoxia was strongly influenced by temperature during the period of anoxia. The average recovery of the CAP, relative to control, after 60 min of anoxia administered at 37 degrees C was 35.4 +/- 7%; when the temperature was lowered by 2.5 degrees C (i.e., to 34.5 degrees C) for the period of anoxic exposure, the extent of functional recovery improved to 64.6 +/- 15% (p < 0.00001). Lowering the temperature to 32 degrees C during anoxic exposure for 60 min resulted in even greater functional recovery (100.5 +/- 14% of the control CAP area). Conversely, if temperature was increased to > 37 degrees C during anoxia, the functional outcome worsened, e.g., CAP recovery at 42 degrees C was 8.5 +/- 7% (p < 0.00001). Hypothermia (i.e., 32 degrees C) for 30 min immediately following anoxia at 37 degrees C did not improve the functional outcome. Many processes within the brain are temperature sensitive, including O2 consumption, and it is not clear which of these is most relevant to the observed effects of temperature on recovery of white matter from anoxic injury. Unlike the situation in gray matter, the temperature dependency of anoxic injury cannot be related to reduced release of excitotoxins like glutamate, because neurotransmitters play no role in the pathophysiology of anoxic damage in white matter (Ransom et al., 1990a). It is more likely that temperature affects the rate of ion transport by the Na(+)-Ca2+ exchanger, the transporter responsible for intracellular Ca2+ loading during anoxia in white matter, and/or the rate of some destructive intracellular enzymatic mechanism(s) activated by pathological increases in intracellular Ca2+.

Animals↗
PubMed1992

Tertiary and quaternary local anesthetics protect CNS white matter from anoxic injury at concentrations that do not block excitability.

1. Anoxic injury in CNS white matter was studied using the in vitro rat optic nerve preparation. Optic nerves were subjected to 60 min of anoxia, and functional recovery was assessed using the area under the compound action potential (CAP). In normal cerebrospinal fluid, CAP area recovered to 33.5 +/- 9.3% (SD) of control. 2. Lidocaine and procaine (0.1 or 1.0 mM), applied beginning 1 h before anoxia, significantly improved postanoxic recovery of CAP area. However, both agents also depressed the preanoxic CAP. Procaine generally allowed greater recovery with less depression compared with lidocaine. 3. The quaternary derivatives QX-314 (0.1-1.0 mM) and QX-222 (0.3-3.0 mM) resulted in more complete recovery of the CAP area from anoxia, with less depression of preanoxic excitability, compared with the tertiary compounds. At 0.3 mM, QX-314 reduced the preanoxic CAP very little (to 94.4 +/- 14% of control CAP area), yet allowed the postanoxic CAP area to recover to 99.6 +/- 19%. 4. We conclude that quaternary local anesthetics are more effective at protecting CNS white matter tracts from anoxia than tertiary compounds and that these agents can result in markedly improved recovery even at concentrations that do not block conduction. Moreover, given the relative specificity of QX-314 for noninactivating Na+ channels, we hypothesize that this channel subtype plays an important role in mediating anoxic injury in central myelinated axons.

Action Potentials↗
PubMed1992

Ion channels in spinal cord astrocytes in vitro. II. Biophysical and pharmacological analysis of two Na+ current types.

1. Na+ currents expressed in astrocytes cultured from spinal cord were studied by whole cell patch-clamp recording. Two subtypes of astrocytes, pancake and stellate cells, were morphologically differentiated and showed expression of Na+ channels at densities that are unusually high for glial cells (2-8 channels/microns2) and comparable to cultured neurons. 2. Na+ currents in stellate and pancake astrocytes were comparable to neuronal Na+ currents with regard to Na(+)-current activation (tau m) and inactivation (tau h) time constants, which were equally fast in both astrocyte types. However, they differed with respect to voltage dependence of activation, and current-voltage (I-V) curves were approximately 10 mV more positive in stellate cells (-11.1 +/- 5.6 mV, mean +/- SD) than in pancake cells (19.7 +/- 4.5 mV). Steady-state activation (m infinity curves) was 16 mV more negative in pancake (mean V1/2 = -48.8 mV) than in stellate cells (mean V1/2 = -32.7 mV). 3. Steady-state inactivation (h infinity curves) of Na+ currents was distinctly different in the two astrocyte types. In stellate astrocytes h infinity curves had midpoints close to -65 mV (-64.6 +/- 6.5 mV), similar to most cultured neurons. In pancake astrocytes h infinity-curves were approximately 25 mV more negative, with midpoints close to -85 mV (84.5 +/- 9.5 mV). 4. The two forms of Na+ currents were additionally distinguishable by their sensitivity to tetrodotoxin (TTX). Na+ currents in stellate astrocytes were highly TTX sensitive [half-maximal inhibition (Kd) = 5.7 nM] whereas Na+ currents in pancake astrocytes were relatively TTX resistant, requiring 100- to 1,000-fold higher concentrations for blockage (Kd = 1,007 nM). 5. Na+ currents were fit by the Hodgkin-Huxley (HH) model. In pancake astrocytes, as in squid gigant axons, Na(+)-current kinetics could be well described with an m3h model, whereas in stellate astrocytes Na+ currents were better described with higher-order power terms for activation (m). On average, best fits were obtained using an m4h model. 6. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp whereas stellate astrocytes were not. The h infinity curve for APs shows that membrane potentials more negative than -70 mV are required to allow these responses to occur.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Aminopyridine↗
PubMed1992

Ion channels in spinal cord astrocytes in vitro. I. Transient expression of high levels of Na+ and K+ channels.

1. Na+ and K+ channel expression was studied in cultured astrocytes derived from P--0 rat spinal cord using whole cell patch-clamp recording techniques. Two subtypes of astrocytes, pancake and stellate, were differentiated morphologically. Both astrocyte types showed Na+ channels and up to three forms of K+ channels at certain stages of in vitro development. 2. Both astrocyte types showed pronounced K+ currents immediately after plating. Stellate but not pancake astrocytes additionally showed tetrodotoxin (TTX)-sensitive inward Na+ currents, which displayed properties similar to neuronal Na+ currents. 3. Within 4-5 days in vitro (DIV), pancake astrocytes lost K(+)-current expression almost completely, but acquired Na+ currents in high densities (estimated channel density approximately 2-8 channels/microns2). Na+ channel expression in these astrocytes is approximately 10- to 100-fold higher than previously reported for glial cells. Concomitant with the loss of K+ channels, pancake astrocytes showed significantly depolarized membrane potentials (-28.1 +/- 15.4 mV, mean +/- SD), compared with stellate astrocytes (-62.5 +/- 11.9 mV, mean +/- SD). 4. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp, when clamp potential was more negative than resting potential. Both depolarizing and hyperpolarizing current injections elicited overshooting responses, provided that cells were current clamped to membrane potentials more negative than -70 mV. Anode-break spikes were evoked by large hyperpolarizations (less than -150 mV). AP-like responses in these hyperpolarized astrocytes showed a time course similar to neuronal APs under conditions of low K+ conductance. 5. In stellate astrocytes, AP-like responses were not observed, because the K+ conductance always exceeded Na+ conductance by at least a factor of 3. Thus stellate spinal cord astrocyte membranes are stabilized close to EK as previously reported for hippocampal astrocytes. 6. It is concluded that spinal cord pancake astrocytes are capable of synthesizing Na+ channels at densities that can, under some conditions, support electrogenesis. In vivo, however, AP-like responses are unlikely to occur because the cells' resting potential is too depolarized to allow current activation. Thus the absence of electrogenesis in astrocytes may be explained by two mechanisms: 1) a low Na-to-K conductance ratio, as in stellate spinal cord astrocytes and in other previously studied astrocyte preparations; or, 2) as described in detail in the companion paper, a mismatch between the h infinity curve and resting potential, which results in Na+ current inactivation in spinal cord pancake astrocytes.

4-Aminopyridine↗
PubMed1992

Ionic mechanisms of anoxic injury in mammalian CNS white matter: role of Na+ channels and Na(+)-Ca2+ exchanger.

White matter of the mammalian CNS suffers irreversible injury when subjected to anoxia/ischemia. However, the mechanisms of anoxic injury in central myelinated tracts are not well understood. Although white matter injury depends on the presence of extracellular Ca2+, the mode of entry of Ca2+ into cells has not been fully characterized. We studied the mechanisms of anoxic injury using the in vitro rat optic nerve, a representative central white matter tract. Functional integrity of the nerves was monitored electrophysiologically by quantitatively measuring the area under the compound action potential, which recovered to 33.5 +/- 9.3% of control after a standard 60 min anoxic insult. Reducing Na+ influx through voltage-gated Na+ channels during anoxia by applying Na+ channel blockers (TTX, saxitoxin) substantially improved recovery; TTX was protective even at concentrations that had little effect on the control compound action potential. Conversely, increasing Na+ channel permeability during anoxia with veratridine resulted in greater injury. Manipulating the transmembrane Na+ gradient at various times before or during anoxia greatly affected the degree of resulting injury; applying zero-Na+ solution (choline or Li+ substituted) before anoxia significantly improved recovery; paradoxically, the same solution applied after the start of anoxia resulted in more injury than control. Thus, ionic conditions that favored reversal of the normal transmembrane Na+ gradient during anoxia promoted injury, suggesting that Ca2+ loading might occur via reverse operation of the Na+)-Ca2+ exchanger. Na(+)-Ca2+ exchanger blockers (bepridil, benzamil, dichlorobenzamil) significantly protected the optic nerve from anoxic injury. Together, these results suggest the following sequence of events leading to anoxic injury in the rat optic nerve: anoxia causes rapid depletion of ATP and membrane depolarization leading to Na+ influx through incompletely inactivated Na+ channels. The resulting rise in the intracellular [Na+], coupled with membrane depolarization, causes damaging levels of Ca2+ to be admitted into the intracellular compartment through reverse operation of the Na(+)-Ca2+ exchanger. These observations emphasize that differences in the pathophysiology of gray and white matter anoxic injury are likely to necessitate multiple strategies for optimal CNS protection.

Amiloride↗
PubMed1992

Demyelination in spinal cord injury and multiple sclerosis: what can we do to enhance functional recovery?

Demyelination in white matter tracts has been observed in experimental and human spinal cord injury. The pathophysiology of demyelinated axons depends, in part, on their ion channel organization. Myelinated axons display a complementary distribution of sodium channels (clustered in the nodal axon membrane) and fast potassium channels (in the internodal axon membrane). The low density of sodium channels in the internodal axon membrane will impede conduction after demyelination. Moreover, "unmasked" potassium channels will tend to clamp the axon membrane close to EK, interfering with conduction in demyelinated axons. Pharmacologic blockade of these potassium channels can increase the safety factor for conduction in demyelinated axons. Restoration of conduction in demyelinated axons, so that action potentials can traverse the zone without myelin, appears to underly clinical remissions in patients with multiple sclerosis and may occur in some patients with spinal cord injury. At a cellular level, conduction through demyelinated axon regions can be facilitated by several mechanisms, including remyelination, development of excitability in demyelinated regions (which requires an adequate density of sodium channels), and impedance matching. Astrocytes have been shown to establish a specific relationship with sodium channel-rich regions of the axon membrane, and may play a role in the deployment and/or maintenance of sodium channels within the demyelinated axon membrane. Calcium influx appears to play a critical role in the cascade of events leading to secondary injury after spinal cord trauma. Recent observations suggest the hypothesis that myelin damage in spinal cord trauma may be mediated, at least in part, by influx of calcium into an intracellular compartment. As the route of calcium entry is identified and characterized, it may be possible to design strategies that will limit secondary injury after CNS trauma. The deleterious effects of calcium in injured white matter accumulate gradually, which suggests the potential reversibility of dysfunction in spinal cord tracts if treated early after trauma.

Animals↗
PubMed1991

Differential sensitivity to hypoxia of the peripheral versus central trajectory of primary afferent axons.

Myelinated primary afferent fibers have both peripheral and central nervous system components. As the fibers course through peripheral nerve and dorsal roots they are myelinated by Schwann cells, but after they invade the spinal cord they become myelinated by oligodendrocytes and have associations with astrocytes. This presents the opportunity to compare the pathophysiology of PNS (Schwann cell-associated) vs. CNS (oligodendrocyte/astrocyte-associated) portions of the same axonal trunk located in the dorsal roots and dorsal columns, respectively. Dorsal spinal roots and slices of dorsal columns isolated from adult rats were studied in a sucrose gap chamber from which compound action potential and membrane potential changes could be recorded. The results indicate that the peripheral component of the afferent fibers is resistant to hypoxia as evidenced by stable action and membrane potential when O2 in the bathing medium was completely replaced with N2 for periods up to 2 h. In contrast, the axons become sensitive to hypoxia as they project through the dorsal columns as evidenced by rapid reduction in action potential amplitude accompanied by membrane depolarization when O2 is replaced by N2. This differential response to hypoxia, observed on the same axon branches but over CNS vs. PNS trajectories, suggests that differences related to the extracellular environment or in axo-glial organization in dorsal root vs. dorsal column may confer different degrees of susceptibility to anoxia.

Action Potentials↗
PubMed1991

Radial glia give rise to perinodal processes.

Nodes of Ranvier in the central nervous system in mammals are characterized by the presence of perinodal astrocytic processes. This study examines the association between processes of radial glia and the axolemma at nodes of Ranvier in the spinal cord of the mature axolotl, an animal in which radial glia represent a large portion of the total glial population. The radial glial cells have their cell bodies located close to the central canal. Those situated dorsal to the canal send long processes to the dorsal surface of the spinal cord. Along this trajectory these processes coalesce into large fascicles in the midline and form the dorsal median septum. Slender branches rise from the processes in these fascicles and extend into the adjacent white matter to terminate in close apposition to the axolemma at nodes of Ranvier. This arrangement provides an intracellular pathway extending from the perinodal region to the surface of the spinal cord. Radial glia ventral to the central canal give rise to processes that project to the glia limitans adjacent to the ventral spinal artery. These ventrally projecting processes appear to be more irregular in their branching pattern than their dorsal counterparts. Multiple slender processes are seen in close apposition to the nodal axolemma of myelinated axons in the ventral white commissure, again providing an intracellular pathway that runs from the perinodal region to the cord surface. In one instance a radial glial process was observed to occupy a pocket formed by the invagination of the nodal axolemma. The axonal cytoplasm adjacent to the invagination contained a variety of organelles, e.g. multivesicular bodies, vesicles and endoplasmic reticulum, suggesting that this relationship between the radial glial process and the axon is more than a passive interaction. These observations are consistent with the view that processes of radial glial cells may regulate the extracellular environment adjacent to the nodal axolemma, and/or play an active role in the maintenance of the nodal membrane. The existence of perinodally-directed processes of radial glial cells in the salamander indicates that this axo-glial specialization reflects an important functional interaction preserved across a large segment of the phylogenetic scale.

Ambystoma mexicanum↗
PubMed1991

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.

Action Potentials↗
PubMed1991

Distribution of sodium channels in chronically demyelinated spinal cord axons: immuno-ultrastructural localization and electrophysiological observations.

The immuno-ultrastructural localization of voltage-sensitive sodium channels was demonstrated within a central demyelinating lesion induced in the rat spinal cord by ethidium bromide/irradiation using polyclonal antibody 7493. Antibody 7493 has previously been shown to immunostain intensely axon membrane at nodes of Ranvier, and also perinodal astrocyte processes. At 25-35 days post injection/irradiation, the central portion of the demyelinating lesion is populated with chronically demyelinated axons and there is an absence of glial processes. Sodium channel immunoreactivity was not observed on the chronically demyelinated axolemma within this central portion of the lesion. Within the peripheral portion of the lesion demyelinated axons were occasionally abutted by astrocyte and Schwann cell processes. At these focal sites of apposition, the axon membrane displayed intense sodium channel immunoreactivity, while the abutting astrocyte and Schwann cell processes did not exhibit immunostaining. Also in the periphery of the lesion, some axons become ensheathed and myelinated by oligodendrocytes and Schwann cells. The axon membrane of circumferentially ensheathed axons displayed antibody 7493 immunostaining, and this immunoreactivity persisted on the axolemma until the ensheathing cytoplasmic processes compacted into myelin. Internodal axon membrane beneath the myelin sheath did not display sodium channel immunoreactivity, though (putative) developing nodal axon membrane adjacent to terminal paranodal loops exhibited robust sodium channel staining. Electrophysiological recordings within the ethidium bromide/irradiation lesion demonstrated that at least some axons conducted action potentials within the lesion, while others exhibited conduction block. These results indicate that there is a reorganization of sodium channels within the axon membrane of chronically demyelinated central axons.

4-Aminopyridine↗
PubMed1991

Na(+)-Ca2+ exchanger mediates Ca2+ influx during anoxia in mammalian central nervous system white matter.

White matter of the mammalian central nervous system suffers irreversible injury after prolonged anoxia, which can result in severe neurological impairment. This type of injury is critically dependent on Ca2+ influx into cells. We present evidence that the Na+,Ca2+ exchanger mediates the majority of the damaging Ca2+ influx into cells during anoxia in white matter. Anoxic injury was studied in the isolated rat optic nerve, and functional recovery was monitored using the compound action potential. Blockers of voltage-gated Na+ channels (tetrodotoxin and saxitoxin) significantly improved recovery, as did perfusion with zero-Na+ solution; both maneuvers would prevent intracellular [Na+] from rising and thus prevent Ca2+ influx by inhibiting reverse operation of the Na+,Ca2+ exchanger. Direct pharmacological blockade of the Na+,Ca2+ exchanger during anoxia with bepridil or benzamil also significantly improved recovery. These findings suggest that reverse operation of the Na+,Ca2+ exchanger during anoxia is a critical mechanism of Ca2+ influx and subsequent white matter injury.

Amiloride↗
PubMed1991

Two types of Na(+)-currents in cultured rat optic nerve astrocytes: changes with time in culture and with age of culture derivation.

Na+ channel expression was studied in cultures of rat optic nerve astrocytes using whole-cell voltage-clamp recordings. Astrocytes from postnatal day 7 rat optic nerve (RON) expressed two distinct types of Na+ currents, which had significantly different h infinity curves. Stellate, GFAP+/A2B5+ astrocytes showed currents with h infinity curve midpoints close to -65 mV, similar to Na+ currents in most neurons. In contrast, flat fibroblast-like GFAP+/A2B5- astrocytes showed Na+ currents with h infinity midpoints around -85 mV, almost 20 mV more hyperpolarized than in neurons or A2B5+ astrocytes. Interestingly, Na+ current expression was maintained in A2B5+ astrocytes but began to decrease in A2B5- astrocytes after 6 days in vitro (DIV) and fell to or below the level of detection (i.e., 1 pA/pF) at 12 DIV. Astrocytes cultured from neonatal rats (P0) are almost exclusively GFAP+/A2B5-. These cells did not display measurable Na+ currents when studied at 2 DIV; however, Na+ current was observed after 5 DIV in A2B5- astrocytes from these neonatal (P0) cultures. These findings were substantiated by immunocytochemical experiments using 7493, an antibody raised against purified rat brain Na+ channels; in P0-derived astrocyte cultures 7493 antibody staining was initially lacking (up to 3 DIV), but it was prominent in cultures after 5 DIV, suggesting that Na+ current expression in RON astrocytes occurs postnatally.

Animals↗
PubMed1991

TEA-sensitive potassium channels and inward rectification in regenerated rat sciatic nerve.

Sucrose gap and intra-axonal recording techniques were used to identify the types of ion channels and inward rectification that are present in regenerated axons of adult (greater than 8 weeks) rat sciatic nerve after crush injury. In sucrose gap recordings, 4-aminopyridine (4-AP) led to slight broadening of the compound action potential (CAP) in normal nerve, and a greater broadening in regenerated nerves. By 12 days after sciatic nerve crush, regenerated nerves manifested an afterhyperpolarization (AHP) lasting up to 250 ms that was sensitive to tetraethylammonium (TEA). A similar TEA-sensitive AHP could be elicited with repetitive stimulation. Hyperpolarizing constant current steps (0.1 to 0.5 mA; 600-900 ms duration) applied across the sucrose gap through regenerated axons evoked membrane hyperpolarizations with a depolarizing, Cs(+)-sensitive relaxation in the response to hyperpolarization, which is characteristic of inward rectification, occurring after about 70 ms. The relaxation was present as early as 21 days after nerve crush. Intra-axonal recordings showed burst firing in 4-AP that was terminated by an AHP that temporally correlated with the TEA-sensitive AHP, and a relaxation in the response to hyperpolarizing current, similar to that of whole nerve recordings. The results demonstrate that in addition to voltage-sensitive sodium channels and 4-AP-sensitive potassium channels, there are TEA-sensitive and inwardly rectifying channels on mammalian regenerated peripheral nerve axons.

4-Aminopyridine↗