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Dorsal column axons of the rat spinal cord are partially protected from anoxic injury following blockade of voltage-sensitive Na+ channels and the Na+/--Ca2+ exchanger. To examine the potential contribution of voltage-gated Ca2+ channels to anoxic injury of spinal cord axons, we studied axonal conduction in rat dorsal columns in vitro following a 60-min period of anoxia. Glass microelectrodes were used to record field potentials from the dorsal columns following distal local surface stimulation. Perfusion solutions containing blockers of voltage-gated Ca2+ channels were introduced 60 min prior to onset of anoxia and continued until 10 min after reoxygenation. Pharmacological blocking agents which are relatively selective for L- (verapamil, diltiazem, nifedipine) and N- (omega-conotoxin GVIA) type calcium channels were significantly protective against anoxia-induced loss of conduction, as was non-specific block using divalent cations. Other Ca2+ channel blockers (neomycin and omega-conotoxin MVIIC) that affect multiple Ca2+ channel types were also neuroprotective. Ni2+, which preferentially blocks R-type Ca2+ channels more than T-type channels, was also protective in a dose-dependent manner. These data suggest that the influx of Ca2+, through L-, N- and possibly R-type voltage-gated Ca2+ channels, participates in the pathophysiology of the Ca2+-mediated injury of spinal cord axons that is triggered by anoxia., (Copyright 1999 Elsevier Science B.V.)

Olfactory ensheathing cells (OECs), which have properties of both astrocytes and Schwann cells, can remyelinate axons with a Schwann cell-like pattern of myelin. In this study the pattern and extent of remyelination and the electrophysiological properties of dorsal column axons were characterized after transplantation of OECs into a demyelinated rat spinal cord lesion. Dorsal columns of adult rat spinal cords were demyelinated by x-ray irradiation and focal injections of ethidium bromide. Cell suspensions of acutely dissociated OECs from neonatal rats were injected into the lesion 6 d after x-ray irradiation. At 21-25 d after transplantation of OECs, the spinal cords were maintained in an in vitro recording chamber to study the conduction properties of the axons. The remyelinated axons displayed improved conduction velocity and frequency-response properties, and action potentials were conducted a greater distance into the lesion, suggesting that conduction block was overcome. Quantitative histological analysis revealed remyelinated axons near and remote from the cell injection site, indicating extensive migration of OECs within the lesion. These data support the conclusion that transplantation of neonatal OECs results in quantitatively extensive and functional remyelination of demyelinated dorsal column axons.

Two distinct populations of Na+ channels (kinetically fast and slow) are present on the cell bodies and axons of cutaneous afferent neurons; the fast current is increased and the slow current reduced in amplitude following nerve injury. The present study was undertaken to determine if similar changes occur on the axons of these neurons following peripheral nerve injury. The compound action potentials from rat sural nerves were recorded in a sucrose gap chamber. Following application of 4-aminopyridine, a prominent and well-characterized depolarization (the delayed depolarization) followed the action potential. This potential, only present on cutaneous afferent axons, has been correlated with activation of a slow Na+ current. The delayed depolarization was reduced after nerve transection. The refractory period of transmission of the action potential was shortened in the transected nerves, but that of the delayed depolarization was prolonged. The changes were largest when the sural nerve was cut and ligated [control: 38.1 +/- 1.7% (n = 5); injury: 24.5 +/- 2.8% (n = 5), P < 0.05], which prevented reconnection to its peripheral target. When the nerve was crushed and allowed to reestablish peripheral target connections, the delayed depolarization was minimally effected. These results indicate that the changes in Na+ channel organization following peripheral target disconnection observed on cutaneous afferent cell bodies also occur on their axons.

The expression of c-Jun, JunB, JunD, c-Fos and ATF-2 transcription factors was studied in L4/L5 dorsal root ganglion neurons of adult rats, in order to determine the extent to know to which extend the expression of transcription factors in vitro parallels the pathophysiological expression in vivo. First, dorsal root ganglia were dissociated and cultured for up to 15 days in vitro (culture). Second, the dorsal root and the peripheral nerve fibres were transected close at the dorsal root ganglia, and the completely axotomized dorsal root ganglia were kept in artificial cerebrospinal fluid for up to 24 h. This procedure (explantation) preserves the intraganglionic morphology intact. Culture evoked a persistent expression of c-Jun and JunD in the majority of small neurons independent on neurite extension, In contrast, the number of large neurons with c-Jun decreased and with JunD increased with incubation time. JunB and c-Fos, which were also visible in the majority of neurons, strongly decreased with culture time in both small and large neurons. ATF-2 was visible in the vast majority of neurons and did not change during the observation period. Incubation with brain-derived neurotrophic factor for 15 days reduced JunB expression and raised c-Fos expression, but did not affect c-Jun or JunD labelings. Explantation of dorsal root ganglia evoked a dramatic and rapid induction of c-Jun in neurons located in the periphery of the ganglia, an area that showed prominent apoptosis as visualized by transferase dUTP nick end-labelling, followed by a delayed increase in neurons of the central parts of dorsal root ganglia. Expression of JunB showed a dramatic increase within 2 h in the whole ganglion, but disappeared within the following hours. JunD dropped from its basal levels within 4 h and was almost absent after 8 h. c-Fos did not appear until 6 h, when transferase dUTP nick end-labelling also became detectable, and remained visible in a rather small number of neurons. As with culture, incubation of explanted dorsal root ganglia with brain-derived neurotrophic factor prevented the initial rise in JunB, accelerated and enhanced c-Fos expression, but did not alter c-Jun and JunD expression. Immunoreactivity of ATF-2 declined or disappeared in those dorsal root ganglia compartments that showed a rise in c-Jun and transferase dUTP nick end-labelling. These findings demonstrate that inducible transcription factors such as Jun and Fos proteins are differentially expressed in adult neurons in vitro when compared to pathophysiological conditions in vivo such as nerve fibre transection (axotomy or rhizotomy). Moreover, the comparison between the explantation and culture experiments suggests that it is the complete axotomy of neurons that provokes those expression patterns found in neuronal cultures of adult neurons. The rapid and persisting expression of c-Jun during neurite extension and apoptosis points at the activation of a pivotal program that might be determined by the presence or absence of ATF-2 and that is involved in regeneration or degeneration.

Small (18-25 microm diam) dorsal root ganglion (DRG) neurons are known to express high levels of tetrodotoxin-resistant (TTX-R) sodium current and the mRNA for the alpha-SNS sodium channel, which encodes a TTX-R channel when expressed in oocytes. These neurons also preferentially express the high affinity receptor for nerve growth factor (NGF), TrkA. Levels of TTX-R sodium current and of alpha-SNS mRNA are reduced in these cells after axotomy. To determine whether NGF participates in the regulation of TTX-R current and alpha-SNS mRNA in small DRG neurons in vivo, we axotomized small lumbar DRG neurons by sciatic nerve transection and administered NGF or Ringer solution to the proximal nerve stump using osmotic pumps. Ten to 12 days after pump implant, whole cell patch-clamp recording demonstrated that TTX-R current density was decreased in Ringer-treated axotomized neurons (154 +/- 45 pA/pF; mean +/- SE) compared with nonaxotomized control neurons (865 +/- 123 pA/pF) and was restored partially toward control levels in NGF-treated axotomized neurons (465 +/- 78 pA/pF). The V1/2 for steady-state activation and inactivation of TTX-R currents were similar in control, Ringer- and NGF-treated axotomized neurons. Reverse transcription polymerase chain reaction revealed an upregulation of alpha-SNS mRNA levels in NGF-treated compared with Ringer-treated axotomized DRG. In situ hybridization showed that alpha-SNS mRNA levels were decreased significantly in small Ringer-treated axotomized DRG neurons in vivo and also in small DRG neurons that were dissociated and maintained in vitro, so as to correspond to the patch-clamp conditions. NGF-treated axotomized neurons had a significant increase in alpha-SNS mRNA expression, compared with Ringer-treated axotomized cells. These results show that the administration of exogenous NGF in vivo, to the proximal nerve stump of the transected sciatic nerve, results in an upregulation of TTX-R sodium current and of alpha-SNS mRNA levels in small DRG neurons. Retrogradely transported NGF thus appears to participate in the control of excitability in these cells via actions that include the regulation of sodium channel gene expression in vivo.

Outward K+ currents were recorded using a whole cell patch-clamp configuration, from acutely dissociated adult rat cutaneous afferent dorsal root ganglion (DRG) neurons (L4 and L5) identified by retrograde labeling with Fluoro-gold. Recordings were obtained 16-24 h after dissociation from cells between 39 and 49 mm in diameter with minimal processes. These cells represent medium-sized DRG neurons relative to the entire population, but are large cutaneous afferent neurons giving rise to myelinated axons. Voltage-activated K+ currents were recorded routinely during 300-ms depolarizing test pulses increasing in 10-mV steps from -40 to +50 mV; the currents were preceded by a 500-ms conditioning prepulse of either -120 or -40 mV. Coexpression of at least three components of K+ current was revealed. Separation of these components was achieved on the basis of sensitivities to the K+ channel blockers, 4-aminopyridine (4-AP) and dendrotoxin (DTx), and by the current responses to variation in conditioning voltage. Changing extracellular K+ concentration from 3 to 40 mM resulted in a shift to the right of the I-V curve commensurate with K+ being the principal charge carrier. Presentation of 100 mM 4-AP revealed a rapidly activating K+ current sensitive to low concentrations of 4-AP. High concentrations of 4-AP (6 mM) extinguished all inactivating current, leaving almost pure sustained current (IK). On the basis of the relative distribution of K+ currents neurons could be separated into three distinct categories: fast inactivating current (IA), slow inactivating current (ID), and sustained current (IK); only IA and IK; and slow inactivating current and IK. However, IK was always the dominant outward current component. These results indicate that considerable variation in K+ currents is present not only in the entire population of DRG neurons, as previously reported, but even within a restricted size and functional group (large cutaneous afferent neurons).

One of the earliest documented molecular events after sciatic nerve injury in adult rats is the rapid, long-term upregulation of the immediate early gene transcription factor c-Jun mRNA and protein in lumbar dorsal root ganglion (DRG) neurons, suggesting that c-Jun may regulate genes that are important both in the early post-injury period and during later peripheral axonal regeneration. However, neither the mechanism through which c-Jun protein is increased nor the level of its post-injury transcriptional activity in axotomized DRGs has been characterized. To determine whether transcriptional activation of c-Jun occurs in response to nerve injury in vivo and is associated with axonal regeneration, we have assayed axotomized adult rat DRGs for evidence of jun kinase activation, c-Jun phosphorylation, and activator protein-1 (AP-1) binding. We report that sciatic nerve transection resulted in chronic activation of c-Jun amino-terminal kinase-1 (JNK) in L4/L5 DRGs concomitant with c-Jun amino-terminal phosphorylation in neurons, and lasting AP-1 binding activity, with both c-Jun and JunD participating in DNA binding complexes. The timing of JNK activation was dependent on the distance of the axotomy site from the DRGs, suggesting the requirement for a retrograde transport-mediated signal. AP-1 binding and c-Jun protein returned to basal levels in DRGs as peripheral regeneration was completed but remained elevated in the case of chronic sprouting, indicating that c-Jun may regulate target genes that are involved in axonal outgrowth.

To examine the mechanisms responsible for the more rapid nerve regeneration observed after a previous (conditioning) nerve injury, adult rats were subjected to a midthigh sciatic nerve transection by using one of three protocols designed to facilitate or restrict nerve regeneration: 1) ligation, in which transected axons were prevented from regenerating; 2) cut, in which transected axons were permitted to extend into peripheral target tissue but were separated from the denervated peripheral nerve stump; and 3) crush, in which axons could regenerate normally through the denervated distal nerve tract. The affected dorsal root ganglia (DRG) were subsequently removed, dissociated, and cultured for up to 3 days, and the timing of neurite initiation, rate of outgrowth, and arborization pattern of previously injured neurons were compared with control DRG. Our results indicate that conditioning lesions have at least four distinct and differentially regulated effects on neuronal morphogenesis: 1) conditioning lesions promote earlier neurite initiation, 2) prior nerve injury decreases the ability of neurons to extend long neurites following a second axotomy, 3) exposure to the environment of a denervated peripheral nerve stimulates greater initial rates of neurite outgrowth, and 4) conditioning lesions reduces initial neuritic branching frequency, resulting in straighter neurites whose growth cones extend further distances from their cell bodies. The primary effect of all conditioning lesions on cultured DRG neurons appeared to be to advance the timing of morphogenesis, resulting in conditioning-lesioned neurons that exhibited characteristics consistent with control neurons that had been cultured for an additional day or more. A secondary effect of conditioning lesions on neurite outgrowth rates was dependent on the local environment of the axons prior to culturing.

Although the cellular mechanisms of pharmacological actions of gabapentin (Neurontin) remain incompletely described, several hypotheses have been proposed. It is possible that different mechanisms account for anticonvulsant, antinociceptive, anxiolytic and neuroprotective activity in animal models. Gabapentin is an amino acid, with a mechanism that differs from those of other anticonvulsant drugs such as phenytoin, carbamazepine or valproate. Radiotracer studies with [14C]gabapentin suggest that gabapentin is rapidly accessible to brain cell cytosol. Several hypotheses of cellular mechanisms have been proposed to explain the pharmacology of gabapentin: 1. Gabapentin crosses several membrane barriers in the body via a specific amino acid transporter (system L) and competes with leucine, isoleucine, valine and phenylalanine for transport. 2. Gabapentin increases the concentration and probably the rate of synthesis of GABA in brain, which may enhance non-vesicular GABA release during seizures. 3. Gabapentin binds with high affinity to a novel binding site in brain tissues that is associated with an auxiliary subunit of voltage-sensitive Ca2+ channels. Recent electrophysiology results suggest that gabapentin may modulate certain types of Ca2+ current. 4. Gabapentin reduces the release of several monoamine neurotransmitters. 5. Electrophysiology suggests that gabapentin inhibits voltage-activated Na+ channels, but other results contradict these findings. 6. Gabapentin increases serotonin concentrations in human whole blood, which may be relevant to neurobehavioral actions. 7. Gabapentin prevents neuronal death in several models including those designed to mimic amyotrophic lateral sclerosis (ALS). This may occur by inhibition of glutamate synthesis by branched-chain amino acid aminotransferase (BCAA-t).

Vigabatrin (gamma-vinyl GABA) is an antiepileptic drug and blocks GABA transaminase activity resulting in elevations in cellular GABA levels in the brain. Nipecotic acid (NPA) promotes release of GABA from neonatal optic nerve astrocytes, resulting in a bicuculline-sensitive depolarization of the optic nerve axons. The NPA-induced depolarization of vigabatrin-treated rats (100 mg/kg, i.p.) more than doubled, suggesting an elevation in free GABA levels; the GABA transporter inhibitor, NO-711 reduced the depolarization. These results are consistent with the known ability of vigabatrin to block the GABA degradation enzyme GABA-transaminase, suggesting that vigabatrin elevates astrocytic GABA levels, thereby favoring greater release of GABA through the GABA transporter.

In order to examine the relationship between myelination and sensitivity to anoxia in adult white matter, we studied action potential conduction in the spinal cord dorsal column of adult rats in which focal demyelinating lesions had been produced using ethidium bromide/X-irradiation. Acutely isolated spinal cords from control rats and following demyelination were maintained in vitro at 36 degrees C and compound action potentials were studied following supramaximal stimulation. The compound action potential was totally abolished within 12 min of the onset of anoxia in normal dorsal columns, but was not abolished until 50 min following the onset of anoxia in demyelinated dorsal columns. Compound action potentials showed significantly greater recovery (to 58.1 +/- 12.2% of control amplitude) in demyelinated dorsal columns compared to controls (30.8 +/- 5.3%) following 120 min of reoxygenation. These results show that focal demyelination is associated with reduced sensitivity to anoxia within white matter of the adult spinal cord.

Enhanced chemiluminescent (ECL) immunoblotting was used to quantitatively assess the initial changes in jun family transcription factor protein levels in adult rat lumbar dorsal root ganglia (DRG) after peripheral axotomy and dorsal root transection, and to study the effects of neurotrophic factor administration on these changes. Transection of central (dorsal root) or peripheral (spinal nerve) branches of DRG neurons resulted in rapid elevation of c-jun protein levels, which was transient after dorsal root transection but sustained, though slightly attenuated, after spinal nerve transection. These results suggest that injury-induced c-jun elevation is biphasic, consisting of an early, transient, injury-initiated phase and a more prolonged secondary phase specific to peripheral target disconnection. c-jun protein changes were not modulated by administration of NGF or BDNF. Immunohistochemistry was used to localize c-jun protein induction to DRG neurons. Using ECL immunoblotting, we also observed temporally regulated increases in junD protein levels after both injuries. A transient up-regulation of junB was detected by immunoblotting 5 days after peripheral axotomy, coincident with a slight decrease in c-jun protein levels.

The effects of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) on injury-induced changes in the electrophysiological properties of adult rat cutaneous afferent dorsal root ganglion (DRG) neurons were examined. Whole cell patch-clamp techniques were used to study gamma-aminobutyric acid-A (GABA(A))-receptor-mediated conductance, voltage-dependent sodium currents, and action potential waveform in cutaneous afferent neurons (35-60 microm diam) cultured from control and axotomized animals. Cutaneous afferent neurons were identified by retrograde labeling with hydroxy-stilbamidine (Fluoro-gold, a fluorescent retrograde axonal tracer); the sciatic nerve was transected 1 wk after Fluoro-gold injection and L4/L5 DRG neurons were cultured 2-3 wk after axotomy. NGF, BDNF, or Ringer (vehicle) solution was delivered in vivo directly to the transected sciatic nerve stump in axotomized rats via an osmotic pump. Recordings were obtained from neurons 5-24 h after culture. Axotomized neurons from rats treated with vehicle solution displayed a twofold increase in GABA-induced conductance and a prominent reduction in the proportion of neurons expressing action potentials that had inflections on the falling phase. The expression of kinetically slow tetrodotoxin (TTX)-resistant sodium current was markedly reduced and an increased expression of kinetically fast TTX-sensitive current was observed in neurons from vehicle-treated, axotomized rats. Treatment with NGF (0.25 microg/microl at 12 microl/day for 14 days) in axotomized animals resulted in an increase in the proportion of neurons expressing TTX-resistant, slow sodium currents and inflected action potentials, but had no effect on GABA-induced conductance. Treatment with BDNF (0.5 microg/microl at 12 microl/day for 14 days) attenuated the axotomy-induced increase in GABA(A)-receptor-mediated conductance while minimally affecting action potential waveform. The observed neurotrophin effects occurred independently of cell size changes. These findings indicate a differential regulation of GABA(A) receptor and sodium channel properties in axotomized rat cutaneous afferent neurons by specific neurotrophic factors.

To examine anoxic injury in spinal cord white matter, we studied axonal conduction in the dorsal columns during and following a standard 60 min anoxic insult at 36 degrees C. Perfusion of the spinal cord in 0-Ca2+ Ringer solution resulted in significantly improved recovery of the compound action potential. Similarly, removal of Na+ from the perfusate resulted in significantly improved recovery of conduction in dorsal column axons. Exposure of the anoxic spinal cord to the Na+ channel blocker tetrodotoxin (TTX), the Na-Ca exchange blockers benzamil and bepridil, Na(+)-H+ exchange blockers amiloride and harmaline, and perfusion in Ringer solution with pH adjusted to 6.4, all resulted in improved recovery. The tertiary anesthetics procaine and lidocaine, as well as phenytoin and carbamazepine, also resulted in improved recovery of compound action potential amplitude after 60 min of anoxia. These results demonstrate that a significant component of irreversible loss of conduction, following anoxic injury of the dorsal columns, is Ca(2+)-dependent. Moreover, these results demonstrate that TTX-inhibitable Na+ channels participate in the pathophysiology of anoxic injury in spinal cord white matter, and indicate that reverse Na-Ca exchange provides a route for at least part of the damaging influx of Ca2+ into an intracellular compartment in anoxic spinal cord white matter. Our results also suggest that extracellular acidosis may have a protective effect on anoxic spinal cord white matter, and support the hypothesis that anoxic injury of spinal cord white matter may involve the Na(+)-H+ exchanger.

In situ hybridization and immunohistochemical studies have shown that sciatic nerve crush or transection induces upregulation of the immediate early gene c-jun mRNA and protein in lumbar dorsal root ganglion neurons. Here we have used enhanced chemiluminescent (ECL) immunoblotting as a sensitive and quantitative way of measuring the time of course of c-jun protein induction following sciatic nerve transection at two distances from the L4 and L5 dorsal root ganglia. c-Jun protein was first detected within 3 h of proximal sciatic nerve transection and within 6 h of distal nerve transection. These results indicate substantially earlier increases in c-jun protein after nerve injury than previously reported, which can be attributed to the sensitivity of this detection method. The earlier induction of c-jun after proximal as compared to distal nerve transection supports the hypothesis that the c-jun response to sciatic nerve injury involves a distance-dependent signalling mechanism.

The expression of sodium channel alpha-, beta 1- and beta 2-subunit mRNAs was examined in adult rat DRG neurons in dissociated culture at 1 day in vitro and within sections of intact ganglia by in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR). The results demonstrate that sodium channel alpha-subunit mRNAs are differentially expressed in small (< 25 microns diam), medium (25-45 microns diam.) and large (> 45 microns diam.) cultured DRG neurons at 1 day in vitro (div). Sodium channel mRNA I is expressed at higher levels in large neurons than small DRG neurons, while sodium channel mRNA II is variably expressed, with most cells lacking or exhibiting low levels of detectable signal of these mRNAs and limited numbers of neurons with moderate expression levels. DRG neurons generally exhibit negligible or low levels of hybridization signal for sodium channel mRNA III. Sodium channel mRNAs Na6 and NaG show similar patterns of expression, with most large and many medium DRG neurons exhibiting high levels of expression. The mRNA for the rat cognate of human sodium channel hNE-Na is detected in virtually every DRG neuron; most cells in all size classes exhibit moderate or high levels of hNE-Na expression. Sodium channel SNS mRNA is expressed in all size classes of DRG neurons, but shows greater expression in small and medium DRG neurons than in large neurons. The mRNA for the rat cognate of mouse sodium channel mNa 2.3 is not detected, or is detected at low levels, in most DRG neurons, regardless of size, although moderate expression is detected in some neurons. Sodium channel beta 1- and beta 2-subunit mRNAs exhibit similar expression patterns; they are detected in most DRG neurons, although the level of expression tends to be greater in large neurons than in small neurons. RT-PCR and in situ hybridization of intact adult DRG showed a similar pattern of expression of sodium channel mRNAs to that observed in DRG neurons in vitro. These results demonstrate that adult DRG neurons express multiple sodium channel mRNAs in vitro and in situ and suggest a molecular basis for the biophysical heterogeneity of sodium currents observed in these cells.

1. Whole cell patch-clamp recordings were obtained from identified cutaneous and muscle afferent neurons (33-60 microns diam) in dissociated L4 and L5 dorsal root ganglia (DRGs) from normal rats and from rats 2-3 wk after sciatic nerve ligation or crush injury. gamma-Aminobutyric acid (GABA)-induced conductance was compared in normal and injured neurons from both functional classes of sensory neurons. 2. Control cutaneous afferent neurons had a peak GABA-mediated conductance of 287 +/- 27 (SE) nS compared with 457 +/- 42 nS for control muscle afferent neurons. 3. An inflection on the downslope of the action potential was observed in 47% of cutaneous afferent neurons compared with 20% of muscle afferent neurons. 4. After ligation and transection of the sciatic nerve there was no change in the GABA-mediated conductance of muscle afferent neurons or in the action potential waveform (23% inflected). However, the cutaneous afferent neurons displayed a greater than two-fold increase in their GABA-mediated conductance and displayed a prominent reduction in the number of neurons with inflected action potentials (13% inflected). Input resistance was similar in cutaneous and muscle afferent neurons and decreased after ligation in cutaneous but not muscle afferents. Resting potential averaged from -50 to -56 mV in normal and ligated groups for both cutaneous and muscle afferent neurons. 5. After crush injury in cutaneous afferent neurons where the transected axons were allowed to regenerate into the distal nerve stump, GABAA-receptor-mediated conductance was elevated compared with controls. However, action potential waveform was not altered by crush injury, suggesting a differential regulation of these two properties in cutaneous afferent neurons. 6. These data indicate that injury-induced plasticity of GABAA-receptor-mediated conductance and action potential waveform occurs in cutaneous but not muscle afferent DRG neurons. It appears that peripherally derived influences are critical in maintaining the electrophysiological phenotype of cutaneous afferent neurons but not muscle afferent neurons.

Although remyelination of demyelinated CNS axons is known to occur after transplantation of exogenous glial cells, previous studies have not determined whether cell transplantation can restore the conduction properties of demyelinated axons in the adult CNS. To examine this issue, the dorsal columns of the adult rat spinal cord were demyelinated by x-irradiation and intraspinal injections of ethidium bromide. Cell suspensions of cultured astrocytes and Schwann cells derived from neonatal rats transfected with the (beta-galactosidase) reporter gene were injected into the glial-free lesion site. After 3-4 weeks nearly all of the demyelinated axons were remyelinated by the transplanted Schwann cells. The dorsal columns were removed and maintained in an in vitro recording chamber; conduction properties were studied using field potential and intra-axonal recording techniques. The demyelinated axons exhibited conduction slowing and block, and a reduction in their ability to follow high-frequency stimulation. Axons remyelinated by transplantation of cultured Schwann cells exhibited restoration of conduction through the lesion, with reestablishment of normal conduction velocity. The axons remyelinated after transplantation showed enhanced impulse recovery to paired-pulse stimulation and greater frequency-following capability as compared with both demyelinated and control axons. These results demonstrate the functional repair of demyelinated axons in the adult CNS by transplantation of cultured myelin-forming cells from the peripheral nervous system in combination with astrocytes.

The alpha (catalytic) subunit of the Na+ pump (Na+, K(+)-ATPase) has three isoforms; alpha1 is ubiquitous, skeletal muscle expresses predominantly alpha2, and alpha3 has been localized to specific types of neurons and, possibly, to axonal processes. The alpha3 isoform mRNA is also expressed in the rat cardiac conduction system. Thus, we studied rat heart and quadriceps muscles by immunohistochemistry using isoform-specific antibodies to the Na+ pump alpha subunit and labeled alpha-bungarotoxin as a probe for the neuromuscular junction (NMJ). We found that alpha3 pump protein is localized to three sites important for impulse transmission: the junctional complex between cardiac myocytes, the heart conduction system, and the NMJ. Specifically, all levels of the conduction system expressed alpha3 immunoreactive protein, as assessed by two isoform-specific antibodies and histological conduction system markers. Specific expression at the junctional complex was confirmed by immuno-EM. Double-labeling and denervation analysis indicated that alpha3-positive areas in skeletal muscle were presynaptic and adjacent to postsynaptic bungarotoxin-positive regions, which had the classic morphology of NMJs. Thus, specific Na+,K(+)-ATPase pump isoforms may be adapted to maintenance of membrane potential and/or intracellular ion concentrations required for impulse transmission in both heart and presynaptic motor terminals contacting skeletal muscle.

Paresthesiae, dysesthesiae, and hyperesthesiae ('positive symptoms') result from ectopic nerve impulses secondary to inappropriate membrane excitability which develops in the setting of chronic sensory axonal injury. The molecular changes in the membranes of dorsal root ganglion neurons which underlie ectopic impulse generation as a result of chronic axonal injury are unknown. Preliminary evidence has suggested that voltage-dependent Na+ channels are one of the participants in the production of ectopic impulses, but the precise form of their participation remains to be determined. The present paper reviews normal sensory anatomy and Na+ channel physiology, as well as clinical syndromes heralded by positive sensations and what is so far known about the cellular and molecular mechanisms underlying them. Properties of two distinct populations of Na+ channels native to the DRG neurons which give rise to cutaneous afferents are described. The biophysical properties of each population of Na+ channels must be tuned with respect to the other in order to cooperate in the generation of action potential activity underlying normal sensory function. A novel hypothesis is put forth suggesting that chronic axonal injury leads to intraneuronal heterogeneity of the populations of Na+ channels in cutaneous afferents, as revealed by their characteristic properties. This may result in one population of Na+ channels activating the other, leading to membrane instability, and possibly to ectopic impulse generation.

The mechanism of action of the recently developed anticonvulsant gabapentin (GBP) used for treatment of partial seizures [12] is largely unknown. Rat hippocampal slices were maintained in vitro and the effects of microapplication of nipecotic acid (NPA), which promotes the release and blocks uptake of GABA, on the synaptically-evoked population excitatory postsynaptic potential (EPSP) were assessed before and after 1 h bath application of GBP. GBP treatment did not alter the population EPSP amplitude to paired or multiple stimuli, but nearly doubled the shunting effects of NPA on the EPSP with no effect on the presynaptic volley. The NPA-induced shunting of the EPSP was bicuculline-sensitive, indicating its mediation by GABAA receptor activation. These results suggest that GBP may increase free GABA levels in hippocampal cells, the release of which may be enhanced under conditions of promoted GABA release. Moreover, the study presents a methodology to electrophysiologically assess relative free GABA levels using field potential analysis in the adult rat brain.

1. The effects of axotomy on the electrophysiologic properties of adult rat dorsal root ganglion (DRG) neurons were studied to understand the changes in excitability induced by traumatic nerve injury. Nerve injury was induced in vivo by sciatic nerve ligation with distal nerve transection. Two to four weeks after nerve ligation, a time when a neuroma forms, lumbar (L4 and L5) DRG neurons were removed and placed in short-term tissue culture. Whole cell patch-clamp recordings were made 5-24 h after plating. 2. DRG neurons were grouped into large (43-65 microns)-, medium (34-42 microns)-, and small (20-32 microns)- sized classes. Large neurons had short duration action potentials with approximately 60% having inflections on the falling phase of their action potentials. In contrast, action potentials of medium and small neurons were longer in duration and approximately 68% had inflections. 3. Pressure microejection of gamma-aminobutyric acid (GABA, 100 microM) or muscimol (100 microM) onto voltage-clamped DRG neurons elicited a rapidly desensitizing inward current that was blocked by 200 microM bicuculline. To measure the peak conductance induced by GABA or muscimol, neurons were voltage-clamped at a holding potential of -60 mV, and pulses to -80 mV and -100 mV were applied at a rate of 2.5 or 5 Hz during drug application. Slope conductances were calculated from plots of whole cell current measured at each of these potentials. 4. GABA-induced currents and conductances of control DRG neurons increased progressively with cell diameter. The mean GABA conductance was 36 +/- 10 nS (mean +/- SE) in small neurons, 124 +/- 21 nS in medium neurons, and 527 +/- 65 nS in large neurons. 5. After axotomy, medium neurons had significantly larger GABA-induced conductances compared with medium control neurons (390 +/- 50 vs. 124 +/- 21; P < 0.001). The increase in GABA conductance of medium neurons was associated with a decrease in duration of action potentials. In contrast, small neurons had no change in GABA conductance or action potential duration after ligation. The GABA conductance of large control neurons was highly variable, and ligation resulted in an increase that was significant only for neurons > 50 microns. The mean action potential duration in large neurons was not significantly changed, but neurons with inflections on the falling phase of the action potential were less common after ligation. There was no difference in resting potential or input resistance between control and ligated groups, except that the resting potential was less negative in small cells after axotomy.(ABSTRACT TRUNCATED AT 400 WORDS)

Voltage-dependent Na+ currents were recorded via patch-clamp from identified adult rat lumbar cutaneous dorsal root ganglion (DRG) neurones using whole-cell and bleb-patch-clamp configurations. Na+ currents in DRG neurones studied 18 days after sciatic nerve ligation were compared with those in control neurones. Control neurones tended to have a singular kinetically slow Na+ current or net Na+ current suggestive of two kinetic varieties of channel in a single neurone. Three changes occurred following axotomy: (1) the peak Na+ current increased significantly, (2) kinetically slow TTX-resistant Na+ current, which predominated in controls, was significantly attenuated or lacking altogether, and (3) a singular, TTX-sensitive kinetically fast form of Na+ current predominated. These findings suggest that as part of the response to axonal injury, DRG neurones increase Na+ channel biosynthesis and/or modify preexisting channels such that their kinetics are accelerated. The results are consistent with the idea that axotomized DRG neurones become hyperexcitable due to the emergence of a high density of kinetically fast, pharmacologically distinguishable, Na+ channels in the membrane., (Copyright 1995 Academic Press, Inc.)

The anticonvulsant gabapentin (1-(aminomethyl)cyclohexane acetic acid) has been found to be effective for treatment of partial seizures, but the mechanism of action is unknown. Recent evidence from the rat optic nerve suggests that gabapentin may enhance promoted release of GABA, which is thought to be due to reverse operation of the GABA transporter. We have used whole-cell patch clamp recordings from CA1 pyramidal neurons in hippocampal slices to directly measure currents induced by nipecotic acid (NPA) during exposure to gabapentin. Under control conditions, pressure microejection of NPA increased whole-cell conductance with a reversal potential equal to the chloride equilibrium potential. This response was mimicked by GABA application, and blocked by bicuculline. The response to NPA was also present after blockade of synaptic transmission in the presence of calcium-free solution. These results are consistent with NPA promoting nonvesicular release of GABA from neighboring neurons or glia via reverse operation of the GABA uptake system, which then activated GABAA receptors on the recorded neurons. In control solution, the response to NPA slowly decreased over 45 min to approximately 50% of the initial response, consistent with GABAA receptor 'rundown'. However, in the presence of gabapentin there was a slow increase in the response, reaching approximately 170% of the control level after 45 min of gabapentin exposure. These results demonstrate that gabapentin enhances the promoted release of GABA by more than three-fold. The potentiation of the NPA response may be due to gabapentin increasing cytosolic GABA in neighboring cells via a delayed metabolic effect, and would have the functional effect of increasing neuronal inhibition during periods of hyperexcitability.

Adult rat DRG neurons rapidly extend extensive neuritic arbors after a 1-2-day delay in culture and generate large depolarization-induced calcium signals during this time period that are derived primarily from intracellular calcium release. To assess whether intracellular calcium mobilization is required for neurite initiation, calcium stores were depleted by brief exposure to the irreversible endoplasmic reticulum calcium ATPase inhibitor thapsigargin; cultures were then maintained for 3 days, immunostained for neurofilament and scored for percentage of neurons with neurites at least twice as long as the cell body. Brief thapsigargin treatment (20 min) during the first 24 h in culture resulted in a substantial decrease in neurite initiation frequency without affecting neuronal or nonneuronal cell survival, suggesting that intracellular calcium mobilization is necessary for triggering neurite initiation in these neurons.

1. Voltage-dependent Na+ conductances were studied in small (18-25 microns diam) adult rat dorsal root ganglion (DRG) neurons with the use of the whole cell patch-clamp technique. Na+ currents were also recorded from larger (44-50 microns diam) neurons and compared with those of the small neurons. 2. The predominant Na+ conductance in the small neurons was selective over tetramethylammonium by at least 10-fold and was resistant to 1 microM external tetrodotoxin (TTX). Na+ conductances in many larger DRG neurons were kinetically faster and, in contrast, were blocked by 1 microM TTX. 3. The Na+ conductance in the small neurons was kinetically slow. Activation half-times were voltage dependent and ranged from 2 ms at -20 mV to 0.7 ms at +50 mV. Approximately 50% of the activation half-time was comprised of an initial delay. Inactivation half-times were voltage dependent and ranged from 11 ms at -20 mV to 2 ms at +50 mV. 4. Peak slow Na+ conductances were near maximal with conditioning potentials negative to -120 mV and were significantly reduced or eliminated with conditioning potentials positive to -40 mV. The slow Na+ conductance increased gradually with test potentials extending from -40 to +40 mV. In some cells the conductance could be saturated at +10 mV. Peak conductance/voltage relationships, although stable in a given neuron, revealed marked variability among neurons, spanning > 20- and 50-mV domains for steady-state activation and inactivation (current availability), respectively. 5. Kinetics remained stable within a given neuron over the course of an experiment. However, considerable kinetic variation was exhibited from neuron to neuron, such that the half-times of activation and of inactivation spanned an order of magnitude. In all small neurons studied there appeared to be a singular kinetic component of the current, based on sensitivity to the conditioning potential, voltage dependence of activation, and inactivation half-time. 6. Unique closing properties were exhibited by Na+ channels of the small neurons. Hyperpolarization following a depolarization-induced fully inactivated state resulted in tail currents that appeared to be the consequence of reactivation of the slow Na+ conductance. Tail currents recorded at various times during a fixed level of depolarization revealed that the underlying channels accumulated into a volatile inactivated state over the course of the preceding depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The permeability of non-N-methyl-D-aspartate (non-NMDA) glutamate channels to divalent cations and specifically the entry of Ca2+ and subsequent elevations in cytoplasmic and nuclear Ca2+ signals were investigated in cultured neonatal rat retinal ganglion cells using the whole cell patch-clamp technique and Ca2+ imaging with confocal microscopy. In addition, divalent-permeable non-NMDA receptor channels were studied in retinal slices using a Co2+ staining technique. 2. Using Ca2+ (2.5 mM) as the only permeable cation in the external solution, stimulation with 100 microM kainate produced nondesensitizing, nonselective cation currents with either low or high Ca2+ permeability. Both currents were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Neurons with the low divalent-permeable currents (type 1) had reversal potentials of -41.5 +/- 4.4 mV (mean +/- SD), and neurons with the high divalent-permeable currents (type 2) had reversal potentials of -22.6 +/- 5.5 mV. The permeability ratio PCa/PCs was 3.3 for the type 1 currents and 8.5 for the type 2 currents, indicating a 2.5-fold greater permeability to Ca2+ for the type 2 non-NMDA glutamate channels. 3. Both types of non-NMDA glutamate channels showed relatively little selectivity between Ca2+ and Co2+. The type 1 neurons had a slightly higher permeability to Co2+ than to Ca2+, whereas the type 2 neurons were equally permeable to both divalent cations. The type 2 neurons had a much higher permeability for both divalent cations compared with the type 1 neurons. 4. Staining for Co2+ uptake through kainate-stimulated non-NMDA glutamate channels in retinal slices provided additional evidence for the presence of the two ganglion cell populations. Activation of the neurons by kainate in conditions isolating the non-NMDA glutamate channel caused differential uptake of Co2+. In contrast, depolarization in the presence of the non-NMDA antagonist CNQX failed to cause Co2+ influx. 5. Imaging experiments using confocal microscopy showed that kainate stimulation induced an increase in intracellular Ca2+ in both types of retinal ganglion cells, but only the type 2 neurons showed a substantial increase in cytoplasmic and nuclear Ca2+ signals. Kainate-induced Ca2+ signals in the type 2 neurons were almost nine times greater than those of the type 1 neurons. 6. When intracellular Ca2+ stores were depleted by brief treatment with thapsigargin, kainate-induced Ca2+ signals in the type 1 neurons were unchanged. However, in the type 2 neurons kainate no longer induced large Ca2+ signals in the cytoplasm and nucleus, despite normal influx of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

1. In situ hybridization with subtype-specific probes was used to ask whether there is a change in the types of sodium channels that are expressed in dorsal root ganglion (DRG) neurons after axotomy. 2. Types I and II sodium channel mRNA are expressed at moderate-to-high levels in control DRG neurons of adult rat, but type III sodium channel mRNA is not detectable. 3. When adult rat DRG neurons are examined by in situ hybridization 7-9 days following axotomy, type III sodium channel mRNA is expressed at moderate-to-high levels, in addition to types I and II mRNA that are present at relatively high levels. 4. To determine whether the expression of type III sodium channel mRNA following axotomy represents up-regulation of a gene that had been expressed at earlier developmental stages, we also studied DRG neurons from embryonic (E17) rats. In these embryonic DRG neurons, type I sodium channel mRNA is expressed at low levels, type II mRNA at high levels, and type III at high levels. 5. These results demonstrate altered expression of sodium channel mRNA in DRG neurons following axotomy, and suggest that in at least some DRG neurons, there is a de-differentiation after axotomy that includes a reversion to an embryonic mode of sodium channel expression. Different channel characteristics, as well as an altered spatial distribution of sodium channels, may contribute to the electrophysiological changes that are observed in axotomized neurons.

1. Intraaxonal recordings were obtained in vitro from the sural nerve (SN), the muscle branch of the anterior tibial nerve (ATN), or the deafferented ATN (dATN) in 5- to 7-wk-old rats. Whole-nerve sucrose gap recordings were obtained from the SN and the ATN. This allowed study of cutaneous (SN), mixed motor and muscle afferent (ATN), and isolated muscle afferent (dATN) axons. 2. Application of the potassium channel blocking agent 4-aminopyridine (4-AP) to ATN or dATN resulted in a slight prolongation of the action potential. In contrast, a distinct delayed depolarization followed the axonal action potential in cutaneous afferents (SN) exposed to 4-AP. The delayed depolarization could be induced by a single whole-nerve stimulus or by injection of constant-current depolarizing pulses into individual axons. The delayed depolarization often gave rise to bursts of action potentials and was followed by a prominent afterhyperpolarization (AHP). 3. In paired-pulse experiments on single SN axons, the recovery time (half-amplitude of the action potential) was 3.06 +/- 1.82 (SE) ms (n = 12). After exposure to 4-AP the recovery time of the delayed depolarization was considerably longer (half-recovery time: 99.0 +/- 28.3 ms; n = 15) than that of the action potential (18.8 +/- 9.1 ms; n = 16). 4. Application of tetraethylammonium (TEA) to cutaneous or muscle afferents alone had little effect on single action potential waveform. However, TEA reduced the amplitude of the AHP elicited by a single stimulus in cutaneous afferent axons after exposure to 4-AP and resulted in repetitive spike discharge. 5. The delayed depolarization and spike burst activity induced by 4-AP in SN was present in Ca(2+)-free solutions containing 1 mM ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and was not blocked by Cd2+ (1.0 mM). 6. We obtained whole-cell patch-clamp recordings to study Na+ currents from either randomly selected dorsal root ganglion neurons or cutaneous afferent neurons identified by retrograde labeling with Fluoro-Gold. The majority of the randomly selected neurons had a singular kinetically fast Na+ current. In contrast, no identified cutaneous afferent neurons had a singular fast Na+ current. Rather, they had a combination of kinetically separable fast and slow currents or a singular relatively slow Na+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane potential changes of rat neonatal optic nerves were studied in a sucrose-gap chamber. Nipecotic acid (NPA), which blocks uptake and promotes release of GABA, resulted in a bicuculline-sensitive depolarization (3.08 +/- 0.3 mV, n = 5). Pretreatment of the nerves with the anticonvulsant gabapentin (100 microM for 1 h) resulted in a near doubling of the NPA-induced depolarization (6.64 +/- 0.54 mV, n = 5). Gabapentin itself did not alter membrane potential, nor did brief applications of gabapentin enhance the NPA- or GABA-induced depolarization. Thus, gabapentin appears to enhance the releasable pool of GABA in this CNS neural system. These results have implications for the mechanism of the anticonvulsant properties of gabapentin.

Cultured adult rat dorsal root ganglion (DRG) neurons were used to study depolarization-induced Ca2+ mobilization and the effects of intracellular Ca2+ depletion on neurite outgrowth. Cytoplasmic and nuclear Ca2+ signals were visualized in dissociated DRG neurons using confocal scanning laser microscopy and the Ca2+ indicator dye fluo-3. The depolarization-induced Ca2+ signals were highest in neurons during the first few days in culture, prior to neurite extension; during this time nuclear signals exceeded those of the cytoplasm severalfold. After several days in culture, neurons began to arborize, depolarization-induced Ca2+ signals became attenuated, and nuclear signals no longer exceeded those of the cytoplasm. Elevated Ca2+ signals were dependent upon both Ca2+ influx and intact intracellular Ca2+ stores, indicating that the signals are generated by calcium-induced calcium release (CICR). Thapsigargin, an endoplasmic reticulum Ca2+ ATPase inhibitor, depleted intracellular Ca2+ stores and blocked the induction of the large nuclear Ca2+ signals. Treating DRG neurons briefly with thapsigargin (200 nM for 20 min) shortly after plating reduced subsequent neuritogenesis, implying that intact Ca2+ stores are necessary for initiating neurite outgrowth. Immunostaining of DRG neurons with antibodies to Ca2+/calmodulin-dependent kinase II (CaM kinase II) demonstrated that this enzyme is present in the nucleus at early times in culture. These observations are consistent with the idea that CICR triggered by Ca2+ entry subsequent to depolarization may elicit neurite outgrowth by activating nuclear enzymes appropriate for such outgrowth.

Confocal microscopy and the Ca(2+)-sensitive fluorescent dye fluo-3 were used to study subcellular Ca2+ signals in embryonic, neonatal, and adult dorsal root ganglion (DRG) neurons in excised dorsal root ganglia. Optical images obtained from isolated whole embryonic and neonatal ganglia revealed a marked variability in the resting Ca2+ signals of different neurons as compared to signals in adult neurons which were uniformly faint. Many of the embryonic and neonatal neurons displayed nuclear Ca2+ signals at rest which were larger than those in the cytoplasm. Embryonic DRG neurons showed a significant increase in nuclear and cytoplasmic fluorescence in response to depolarization with elevated extracellular potassium or electrical stimulation. A single brief electrical stimulus was sufficient to elicit nuclear Ca2+ signals in a subset of the embryonic neurons. The depolarization-induced Ca2+ signals were blocked by removal of extracellular Ca2+, but not by treatment with 2,5-di (tert-butyl)-1,4 benzohydroquinone (DTBHQ), a compound which depletes intracellular Ca2+ stores. The intensity of the depolarization-induced Ca2+ signals declined significantly between the late embryonic (E18-E20) and early postnatal time periods (P0-P1). The nuclear and cytoplasmic Ca2+ signals of the embryonic DRG neurons in the excised tissue preparation occur at a time of intense target innervation, suggesting a role for Ca2+ signals in the development and maturation of rat DRG neurons.

A central issue in transplantation research is to determine how and when transplantation of neural tissue can influence the development and function of the mammalian central nervous system. Of particular interest is whether electrophysiological function in the traumatized or diseased mammalian central nervous system can be improved by the replacement of cellular elements that are missing or damaged. Although it is known that transplantation of neural tissue can lead to functional improvement in models of neurological disease characterized by neuronal loss, less is known about results of transplantation in disorders of myelin. We report here that transplantation of glial cells into the dorsal columns of neonatal myelin-deficient rat spinal cords leads to myelination and a 3-fold increase in conduction velocity. We also show that impulses can propagate into and out of the transplant region and that axons myelinated by transplanted cells do not have impaired frequency-response properties. These results demonstrate that myelination following central nervous system glial cell transplantation enhances action potential conduction in myelin-deficient axons, with conduction velocity approaching normal values.

Two major glial cell types, the oligodendrocyte (which produces myelin) and the perinodal astrocyte (which contacts the sodium channel-rich axon membrane at the node of Ranvier), collaborate with the axon in forming the central myelinated fiber. Astrocytes have been demonstrated to synthesize voltage-sensitive sodium channels, and in some astrocytes these channels have neuronal properties. A variety of lines of evidence suggest that astrocytes play an important role in the reorganization of the axon membrane following demyelination. Possible functions of astrocytes include the targeting, or anchoring, of ion channels at specific sites within the axon membrane, or the specification of channel characteristics as a result of production of extracellular matrix molecules that interact with the glycosylated dome of sodium channels, or the synthesis of channels that are subsequently transferred to axons. It is also possible that astrocytes participate in ionic homeostasis at the node of Ranvier. A second major glial cell type, the oligodendrocyte, produces myelin during normal development. Morphologic studies have demonstrated that transplantation of myelin-forming cells, or their precursors, can result in the production of myelin with normal structure in the myelin-deprived CNS. In recent physiological studies, the function of axons following myelination by exogenous, transplanted glial cells has been examined. In regions of glial cell transplantation, there are significant increases in conduction velocities that approach normal values. Both of these lines of investigation, on astrocytes and myelin-forming oligodendrocytes/oligodendrocyte precursors, suggest that manipulation of glial cell properties may emerge as a viable strategy for promoting the recovery of conduction in CNS white matter axons following demyelination.

We have identified a putative cGMP-gated cation conductance in rat retinal ganglion cells. Both in situ hybridization and polymerase chain reaction amplification detected transcripts in ganglion cells that were highly homologous to the cGMP-gated cation channel expressed in rod photoreceptors. Whole-cell patch-clamp recordings detected a current stimulated by cGMP due to activation of nonselective cation channels. This current had a reversal potential near 0 mV, showed some outward rectification, and could be blocked by Cd2+. The current could also be activated by a phosphodiesterase inhibitor and the nitric oxide donors sodium nitroprusside and S-nitrosocysteine. We propose that nitric oxide released from an identified subpopulation of amacrine cells may activate this channel to modulate ganglion cell activity.

Compound action potential (CAP) conduction and Na+ channel content were studied in optic nerves from control and myelin-deficient (md) rats. Action potential propagation was approximately five times slower in the md rat, but the action potentials propagated securely and had frequency-following and refractory properties equivalent to control myelinated axons. Tritium-labelled saxitoxin ([3H]-STX) binding in md optic nerve was approximately 30% greater, per wet mass of tissue, than in the control optic nerve. However, calculations of channel density per axon based on previously published anatomical data from md and control optic nerves (Dentinger et al. 1985) show an equivalent number of sodium channels per axon, with an average density of 10 channels micron-2 in md and 11 channels micron-2 in control optic nerve axons. The amplitude of the CAP in both control and md optic nerves was significantly attenuated by 50 nM TTX, precluding the possibility that TTX-insensitive channels are responsible for the action potential in myelinated or amyelinated axons. In addition, the amplitudes of voltage-activated Na+ currents in type I and type II astrocytes cultured from control and md optic nerves were similar, suggesting that the glial component of Na+ channels is not abnormal in the optic nerve of the md rat. These results suggest that myelination (or its absence) may not directly regulate the number of axonal Na+ channels.

Immunostaining and high-pressure liquid chromatography (HPLC) were used to study the developmental time course of astrocytic gamma-aminobutyric acid (GABA) expression in rat optic nerve. GABA immunostaining was carried out on cultured astrocytes, and on whole optic nerve. Confocal scanning laser microscopy was used to obtain optical sections in excised whole tissue in order to localize the cellular origins of GABA within the relatively intact optic nerve. GABA immunoreactivity was localized in astrocytes identified by GFAP staining; GABA staining was most intense in early neonatal optic nerve and attenuated over 3 weeks of postnatal development. The staining was pronounced in the astrocyte cell bodies and processes but not in the nucleus. There was a paucity of GABA immunoreactivity by postnatal day 20, both in culture and in whole optic nerve. A biochemical assay for optic nerve GABA using HPLC indicated a relatively high concentration of GABA in the neonate, which rapidly attenuated over the first 3 postnatal weeks. Immunoreactivity for the GABA synthesis enzyme glutamic acid decarboxylase (GAD) was pronounced in neonates but also attenuated with development. These results indicate that GABA and the GABA synthesis enzyme GAD are localized in astrocytes of optic nerve, and that their expression is transient during postnatal development.

Excitability of rat dorsal root axons were studied 3 weeks after injury to the sciatic nerve. Whole nerve recordings were obtained from injured and control nerves in a sucrose gap chamber. Constant current depolarization pulses (30-200 ms) applied approximately 50% above the stimulus strength required for maximal amplitude compound action potentials (CAPs) evoked a burst of action potentials in the dorsal root which displayed spike adaptation. The depolarization-induced burst response of the dorsal roots was greatly reduced after crush or transection of the sciatic nerve. However, application of the potassium channel blocker, tetraethylammonium (TEA), restored the burst discharge in injured dorsal root axons. Brief tetanic stimulation of the dorsal root also induced an afterhyperpolarization (AHP) that was twice as large in the transection group as compared to the control group, and which was blocked by TEA. There were no changes seen in the amplitude of the compound action potential, frequency-following characteristics, refractory properties, or 4-AP sensitivity in the dorsal roots after peripheral nerve injury. These results suggest that there is enhanced spike adaptation that occurs at the same time as an increase in the sensitivity to the potassium channel blocker, TEA, in axon regions proximal to the site of nerve injury and have implications for the pathophysiology of nerve injury.

Intracellular Ca2+ was imaged in cultured neonatal rat retinal neurons using the Ca(2+)-sensitive dye fluo-3 and confocal scanning laser microscopy. Depolarization via elevation of bath K+ concentration resulted in large cytoplasmic and nuclear Ca2+ signals; responses in the nucleus exceeded those of the cytoplasm. Glutamate or kainate application elicited the same intracellular pattern of elevated Ca2+ signals. Kainate stimulation was blocked by the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and greatly reduced by removing Ca2+ from the bath and adding ethylene glycol-bis (beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA). Kainate was equally effective in eliciting Ca2+ signals when bath Na+ was replaced with equimolar concentrations of choline, or in the presence of the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV). Caffeine treatment significantly reduced the kainate-induced intracellular Ca2+ response. These results suggest that Ca2+ can enter through the kainate receptor of retinal neurons and amplify the Ca2+ signals in the cytoplasm and nucleus by releasing Ca2+ from intracellular stores.

GABA (gamma-aminobutyric acid) can modulate axonal excitability by activating GABAA receptors on some central nervous system axons. The effects of GABA on optic nerve axons decrease significantly during the course of myelination, suggesting that myelination may influence changes in GABA sensitivity. To test this hypothesis, we compared the depolarizing effect of GABA and the GABAA-receptor agonist, muscimol, on optic nerves of myelin-deficient (MD) rats and their unaffected siblings using a modified sucrose-gap technique. Optic nerves from both control and MD rats displayed relatively large GABA-induced depolarizations when studied at an early postnatal period. In both the control and MD rats, the GABA uptake inhibitor nipecotic acid led to a distinct depolarization suggesting endogenous release of GABA. Although the GABA-induced depolarization in the MD rat was significantly greater than that in the control rat at the third postnatal week, the response in the MD rat attenuated with maturation. These results demonstrate that the attenuation of the depolarization induced by GABA and nipecotic acid seen in the normal optic nerve also occurs in the MD rat optic nerve. This suggests that the attenuation of optic nerve sensitivity to GABA is not the result of myelination or interaction with myelin-associated proteins.

1. The role of synaptic activation of NMDA (N-methyl-D-aspartate) receptor-mediated conductances on CA1 hippocampal pyramidal cells in short-term excitability changes was studied with the use of a computational model. Model parameters were based on experimental recordings from dendrites and somata and previous hippocampal simulations. Representation of CA1 neurons included NMDA and non-NMDA excitatory dendritic synapses, dendritic and somatic inhibition, five intrinsic membrane conductances, and provision for activity-dependent intracellular and extracellular ion concentration changes. 2. The model simulated somatic and dendritic potentials recorded experimentally. The characteristic CA1 spike afterdepolarization was a consequence of the longitudinal spread of dendritic charge, reactivation of slow Ca(2+)-dependent K+ conductances, slow synaptic processes (NMDA-dependent depolarizing and gamma-aminobutyric acid-mediated hyperpolarizing currents) and was sensitive to extracellular potassium accumulation. Calcium currents were found to be less important in generating the spike afterdepolarization. 3. Repetitive activity was influenced by the cumulative activation of the NMDA-mediated synaptic conductances, the frequency-dependent depression of inhibitory synaptic responses, and a shift in the potassium reversal potential. NMDA receptor activation produced a transient potentiation of the excitatory postsynaptic potential (EPSP). The frequency dependence of EPSP potentiation was similar to the experimental data, reaching a maximal value near 10 Hz. 4. Although the present model did not have compartments for dendritic spines, Ca2+ accumulation was simulated in a restricted space near the intracellular surface of the dendritic membrane. The simulations demonstrated that the Ca2+ component of the NMDA-operated synaptic current can be a significant factor in increasing the Ca2+ concentration at submembrane regions, even in the absence of Ca2+ spikes. 5. Elevation of the extracellular K+ concentration enhanced the dendritic synaptic response during repetitive activity and led to an increase in intracellular Ca2+ levels. This increase in dendritic excitability was partly mediated by NMDA receptor-mediated conductances. 6. Blockade of Ca(2+)-sensitive K+ conductances in the dendrites increased the size of EPSPs leading to a facilitation of dendritic and somatic spike activity and increased [Ca2+]i. NMDA receptor-mediated conductances appeared as an amplifying component in this mechanism, activated by the relatively depolarized membrane potential. 7. The results suggest that dendritic NMDA receptors, by virtue of their voltage-dependency, can interact with a number of voltage-sensitive conductances to increase the dendritic excitatory response during periods of repetitive synaptic activation. These findings support experimental results that implicate NMDA receptor-mediated conductances in the short-term response plasticity of the CA1 hippocampal pyramidal neuron.

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.

Depolarization-induced increases in cytoplasmic and intranuclear Ca2+ were visualized in adult mammalian dorsal root ganglion (DRG) neurons during different stages of neurite extension by using confocal laser scanning microscopy and the long-wavelength Ca2+ indicator dye fluo 3-AM (acetoxymethyl ester of fluo 3). In neurons beginning to extend neurites, depolarization led to pronounced increases in nuclear and nucleolar Ca2+ levels severalfold greater than corresponding increases in the cytoplasm. The nucleolar Ca2+ signal often exceeded that of the nucleus, indicating regional heterogeneity of the nucleus. The subcellular calcium transients were dependent on extracellular Ca2+ and the level of depolarization, indicating the importance of transmembrane Ca2+ fluxes in triggering the nuclear events. After neurite extension, the nuclear Ca2+ signals were attenuated and never exceeded cytoplasmic levels. These results indicate that activity-dependent modulation of intranuclear Ca2+ levels is greater in DRG neurons during early neurite extension. Given the importance of Ca2+ in gene expression, the results may be relevant to Ca(2+)-dependent nuclear events responsible for axonal regeneration.

Spinal cords of myelin-deficient and normal age-matched (control) rats were removed and their conduction and pharmacological properties studied in an in vitro brain slice chamber. The conduction velocity of the myelin-deficient dorsal column axons was reduced to about 25% of control axons; however, the amyelinated myelin-deficient axons displayed refractory periods and the ability to sustain high-frequency action potential discharge similar to that of dorsal column axons in control rats. Pharmacological results suggest that the myelin-deficient dorsal column axons qualitatively express a normal complement of ion channels and receptors. The demonstration of a normal representation of channels and receptors on these axons supports the proposal that the oligodendrocyte, and not the axon, is the site of the primary defect in the myelin-deficient rat mutant. It is concluded that, unlike acutely demyelinated axons which display marked frequency-dependent conduction block, amyelinated axons of the myelin-deficient rat spinal cord develop compensatory mechanisms to stabilize action potential conduction.