Amyloid β-peptides inhibit Na/K-ATPase: tissue slices versus primary cultures

Aβ 1-40 (20 μM) has been reported to selectively inhibit Na⁺/K⁺-ATPase activity in rat primary hippocampal cultures after 2–6 days of exposure [10]. We expanded these studies to include Aβ’s effects on Na⁺/K⁺-ATPase activity in rat primary cortical cultures and hippocampal slices, and we correlated these effects with estimates of cell survival in rat brain primary cultures. Using optimized assay conditions, a 5-day exposure to 50 μM Aβ 25-35, 20 μM Aβ 1-40, and 20 μM Aβ 1-42 decreased Na⁺/K⁺-ATPase activity in rat primary cortical cultures 66%, 60%, and 22%, respectively. Aβ 25-35 (50 μM) at 24 h was the only condition that caused inhibition of Na⁺/K⁺-ATPase activity in the absence of cell death, defined as an extracellular shift in the localization of the cytoplasmic enzyme lactate dehydrogenase (LDH). We also found that hippocampal slices were sensitive to Aβ, exhibiting a 40–60% reduction in membrane Na⁺/K⁺-ATPase activity when exposed to 1–30 nM of Aβ 1-40 for 60 min. This inhibition was not readily reversible, as it withstood homogenization and repeated dilution and centrifugation. Additionally, this inhibition occurred only after amyloid incubation with intact hippocampal slices, not with disrupted membranes. The inhibition of Na⁺/K⁺-ATPase in brain slices by physiological, low nM concentrations of Aβ 1-40 is consistent with effects on neurotransmitter release and intrasynaptosomal calcium responses [4 and 7].     

Adenosine A2A receptor contributes to ischemic brain damage in newborn piglet

Pharmacologic inactivation or genetic deletion of adenosine A_2A receptors protects ischemic neurons in adult animals, but studies in neonatal hypoxia-ischemia (H-I) are inconclusive. The present study in neonatal piglets examined the hypothesis that A_2A receptor signaling after reoxygenation from global H-I contributes to injury in highly vulnerable striatal neurons where A_2A receptors are enriched. A_2A receptor immunoreactivity was detected in striatopallidal neurons. In nonischemic piglets, direct infusion of the selective A_2A receptor agonist CGS 21680 through microdialysis probes into putamen increased phosphorylation of N-methyl-D-aspartic acid (NMDA) receptor NR1 subunit and Na⁺,K⁺-ATPase selectively at protein kinase A (PKA)-sensitive sites. In ischemic piglets, posttreatment with SCH 58261, a selective A_2A receptor antagonist, improved early neurologic recovery and preferentially protected striatopallidal neurons. SCH 58261 selectively inhibited the ischemia-induced phosphorylation of NR1, Na⁺,K⁺-ATPase, and cAMP-regulated phosphoprotein 32 KDa (DARPP32) at PKA-sensitive sites at 3 hours of recovery and improved Na⁺,K⁺-ATPase activity. SCH 58261 also suppressed ischemia-induced protein nitration and oxidation. Thus, A_2A receptor activation during reoxygenation contributes to the loss of a subpopulation of neonatal putamen neurons after H-I. Its toxic signaling may be related to DARPP32-dependent phosphorylation of PKA-sensitive sites on NR1 and Na⁺,K⁺-ATPase, thereby augmenting excitotoxicity-induced oxidative stress after reoxygenation.     

Blood–brain barrier mechanisms involved in brain calcium and potassium homeostasis

This study examined the potential roles of the plasma membrane Ca²⁺-ATPase (PMCA) at the blood–CSF and blood–brain barriers in brain Ca²⁺ homeostasis and blood–brain barrier Na⁺/K⁺-ATPase subunits in brain K⁺ homeostasis. During dietary-induced hypo- and hypercalcemia (0.59±0.06 and 1.58±0.12 mM [Ca²⁺]) there was no significant change in choroid plexus PMCA (Western Blots) compared to normocalcemic rats (plasma [Ca²⁺]: 1.06±0.11 mM). In contrast, PMCA in cerebral microvessels isolated from hypocalcemic rats was 150% greater than that in controls (p<0.001). Comparison of the α3 subunit of Na⁺/K⁺-ATPase from cerebral microvessels isolated from hypo-, normo- and hyperkalemic rats (2.3±0.1, 3.9±0.1 and 7.2±0.6 mM [K⁺]) showed a 75% reduction in the amount of this isoform during hyperkalemia. None of the other Na⁺/K⁺-ATPase isoforms varied with plasma [K⁺]. These results suggest that both PMCA and the α3 subunit of Na⁺/K⁺-ATPase at the blood–brain barrier play a role in maintaining a constant brain microenvironment during fluctuations in plasma composition.     

Alterations of cerebromicrovascular Na, K-ATPase activity due to fatty acids and acute hypertension

Acute hypertension, induced in rats by intravenous injection of angiotensin II, previously has been shown to increase cerebrovascular permeability to macromolecules. The purpose of this study was to examine the effect of acute hypertension on Na⁺, K⁺-ATPase, the enzyme responsible for controlling ionic permeability of the cerebromicrovascular endothelium. The K⁺-dependent p-nitrophenylphosphatase activity of the cerebromicrovascular Na⁺, K⁺-ATPase was determined using microvessels prepared from hypertensive and normotensive rats. When compared to controls, a 70% decrease (P < 0.02) in the maximum rate (V_max) of the Na⁺, K⁺-ATPase from hypertensive rats was evident with no change in the Michaelis constant (K_M). In contrast, γ-glutamyltranspeptidase, a marker enzyme for cerebral endothelic cells, was not significantly affected. Sodium arachidonate (1–100 μM) inhibited the phosphatase activity of the Na⁺, K⁺-ATPase in microvessels isolated from both normotensive and hypertensive rats in a dose-dependent manner. Furthermore, poly-unsaturated fatty acids (sodium linoleate and arachidonate) evoked the greatest inhibition of the enzyme, while sodium oleate and sodium palmitate inhibited the Na⁺, K⁺-ATPase to lesser extents. This regulation of enzyme activity by fatty acids was comparable in control and hypertensive groups. In summary, the data indicate that the cerebromicrovascular Na⁺, K⁺-ATPase was altered as a consequence of acute hypertension and that poly-unsaturated fatty acids can modulate this enzyme in microvessels derived from hypertensive or control rats     

Non-uniform expression of α subunit isoforms of the Na/K pump in rat dorsal root ganglia neurons

Tissue sections and antibodies selectively recognizing isoforms of the α subunit of the Na⁺/K⁺ pump were used to determine the expression of α₁, α₂ and α₃ pump isoforms in the plasma membrane of adult rat dorsal root ganglia (DRG) neurons. There was no detectable membrane signal from DRG neurons that were probed with antibodies to the α₂ isoform of the Na⁺/K⁺ pump. The α₁ isoform of the Na⁺/K⁺ pump was found in most (77±4%) studied DRG neurons, regardless of cell size. Only 16±7% of the neurons expressed a detectable level of the α₃ Na⁺/K⁺ pump and all were apparently from a subpopulation of large DRG neurons. Comparison of cell size distributions and a study of neurons identified in serial sections suggested that of the α₃ positive DRG neurons about 75% coexpressed the α₁ isoform of the Na⁺/K⁺ pump. These data show that the expression of the protein of the α subunit isoforms of the Na⁺/K⁺ pump is not uniform throughout the population of DRG neurons and that α₁ is the predominant isoform in the plasma membrane of these neurons.     

Immunohistochemical localization of Na, K-ATPase in the choroid plexus

To determine if canine and rat choroid plexus Na⁺, K⁺-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified canine kidney medulla Na⁺, K⁺-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified canine kidney Na⁺, K⁺-ATPase and canine kidney microsomes were treated with antiserum followed by [¹²⁵I]protein A and autoradiography, the canine microsomes and purified Na⁺, K⁺-ATPase showed a prominent radioactive band coincident with the α-, β- and γ-subunits of the purified canine kidney enzyme.When the rabbit immunoglobulin that was purified from the Na⁺, K⁺-ATPase antiserum through DEAE-cellulose ion exchange chromatography was used for immunoperoxidase staining of the choroid plexus fixed with Bouin's fixative, intense immunoreactive staining was present on the epithelial cells of both choroid plexuses but was not found in the tissue around the vessel. The staining was especially confined to apical surfaces of the epithelial cells. The same procedure was performed in the canine kidney, and immunostaining was obtained in the tubules where Baskin and Stahl described the enzyme localization. No staining was seen with pre-immune serum of the normal rabbit. We concluded that both the canine and rat choroid plexus are rich in Na⁺, K⁺-ATPase, which plays an important role in cerebrospinal fluid (CSF) secretion.     

Effects of mammalian brain extracts and chlormadinone acetate on neuronal Na, K-ATPase and electrogenic Na, K-pump activity in vitro

Acid-acetone extracts of brain (from beef and guinea pig) and chlormadinone acetate (CMA) were compared with ouabain for their ability to inhibit the electrogenic Na⁺,K⁺-pump and the Na⁺,K⁺-ATPase of neuronal tissues. The membrane potential of neurones in the paravertebral sympathetic ganglion of the bullfrog was recorded in K⁺-free Ringer's solution by means of the sucrose gap technique. The potassium activated hyperpolarization (K_H⁺), induced by the re-introduction of potassium, was used as an index of electrogenic Na⁺,K⁺-pumping. The K_H⁺ was blocked by 1 μM ouabain. Na⁺,K⁺-ATPase activity was measured in microsomal membrane preparations of frog and beef brain using a continuous spectrophotometric assay. Although ouabain consistently inhibited beef brain Na⁺,K⁺-ATPase (IC₅₀ = 2.2 μM), acid-acetone extracts prepared from guinea pig and beef brain produced only partial inhibition. Neither of the extracts significantly reduced the K_H⁺ of the frog ganglion. CMA inhibited Na⁺,K⁺-ATPase prepared from bullfrog brain and spinal cord with slightly greater potency (IC₅₀ = 4.5 μM) than did ouabain (IC₅₀ = 10 μM). In contrast, electrogenic Na⁺,K⁺-pumping (i.e. the K_H⁺) in the frog ganglion was not affected by this steroid. It is concluded that although both the extracts and CMA inhibited Na⁺,K⁺-ATPase, neither can be considered ouabain-like due to their failure to affect the electrogenic Na⁺,K⁺-pump in situ.     

Glial contribution to seizure: K activation of (Na, K)-ATPase in bulk isolated glial cells and synaptosomes of epileptogenic cortex

Glial and neuronal (Na⁺, K⁺)-ATPase have dissimilar apparent affinities for K⁺ ions. Glial (Na⁺, K⁺)-ATPase is maximally activated by 20 mM K⁺ while neuronal (Na⁺, K⁺)-ATPase is maximally stimulated by 3–5 mM K⁺. Because this glial property may play an important role in the clearance of [K⁺]₀ during seizures, we investigated the K⁺ activation of (Na⁺, K⁺)-ATPase within bulk isolated glial cells and synaptosomes isolated from epileptogenic brains. The primary focus (F), the homolateral brain area around the focus (PF) and the mirror (M) or secondary focus induced by freezing lesions were studied.Results show that both glial and synaptosomal enzyme activities in the epileptogenic state change in comparison with controls, i.e. sham-operated cats. In F and M., glial enzyme decreased reaction velocities between 3 and 18 mM K⁺. In PF, maximum velocities increased in glial (Na⁺, K⁺)-ATPase. These results indicate that in actively firing epileptogenic tissue (F, M), glial (Na⁺, K⁺)-ATPase decreased rate reactions while the catalytic activity was increased in cortical tissues surrounding the focus. These phenomena appeared early, i.e. 1–3 days after production of the freezing lesion, and was associated with a sharp decrease in absolute levels of enzyme activity.Synaptosomal (Na⁺, K⁺)-ATPase from controls always exhibited a saturation curve at 3–6 mM K⁺. Synaptosomal enzyme activities in the primary (F) lesion increased slightly 24 h after lesion production, then progressively decreased 3 days after lesion production. No significant changes were seen in M and PF.     

Vulnerability of small GABAergic neurons to human β-amyloid pentapeptide

β-Amyloid peptide (Aβ), the principal component of senile plaques in Alzheimer's disease, has been found to be neurotoxic. The role of Aβ in the deficits of the GABAergic system in patients with Alzheimer's disease is unclear. It has been suggested that the cytotoxic activity of Aβ is localized to amino acid residues 25–35 of this peptide, which contains a total of 42 amino acid residues. We now report that the short amyloid peptide fragments corresponding to amino acids 31–35 (Aβ31–35) and 34–39 (Aβ34–39) are also toxic in vitro to the small GABAergic neuron population of basal forebrain cultures. Morphological changes were accompanied by an increased number of varicosities localized on the processes of the GABA-immunoreactive neurons and by the appearance of round cells without processes. The neurodegeneration was confirmed by means of scanning electron microscopy. Quantification of the morphological findings by image analysis demonstrated a size-related dependence of the degeneration of GABAergic neurons. The results suggest that fragments of Aβ shorter than Aβ25–35 may exert cytotoxic action and demonstrate the toxicity of these Aβ fragments in decreasing the number of small GABAergic neurons.     

Studies of cerebral β-hydroxybutyrate transport by carotid injection; effects of age, diet and injectant composition

Transport of β-hydroxybutyrate at the blood-brain barrier was studied by the carotid-injection technique of Oldendorf. β-Hydroxybutyrate permeability declined sharply with age (80–300 g rats) and, in adult rats, increased 5-fold during one week on a high fat diet. Acetoacetate and lactate permeabilities showed age and diet dependences which were similar in direction whereas DMO, urea and mannitol did not show age and diet dependent permeabilities. There was no apparent increase of β-hydroxybutyrateK_m with age, so the decline of permeability was attributed to a decline of V_max. β-Hydroxybutyrate permeability was inversely related to pH of the injectant in the alkaline range but not in the acid range, suggesting that the pH dependence reflected titration of a carrier rather than titration of the permeant. Permeability was independent of [Na⁺], [K⁺], [Cl⁻] and [SO²⁻₄. Replacing a portion of the Na⁺ with ammonium enhanced β-hydroxybutyrate uptake. This effect appeared to be due to trans alkalization, and was as expected of an A⁻/H⁺-symport or A⁻/H⁺-antipoport mechanism. Pyruvate, 4-hydroxy-α-cyanocinnamate and tetracaine inhibited, but SITS, DIDS, phloretin and methyl-isobutylxanthine did not. The data are consistent with transport by an A⁻/H⁺-symporter or A⁻/OH⁻-antiporter with properties similar to those found in erythrocytes and other cells. Induction of its activity during ketosis would spare carbohydrate both by favoring ketoacid uptake and by favoring lactate output.     

Blockade of bepridil on IA and IK in acutely isolated hippocampal CA1 neurons

The effects of bepridil, an antianginal agent with antiarrhythmic action, on voltage-dependent K⁺ currents in the CA1 pyramidal neurons acutely isolated from rat hippocampus were studied by means of whole-cell patch clamp techniques. Current recordings were made in the presence of TTX to block Na⁺ current. Depolarizing test pulses activated two components of outward K⁺ currents: a rapidly activating and inactivating component, I_A; and a delayed component, I_K. Results showed that bepridil reduced the amplitude of I_A and I_K, and exerted its inhibitory action in time- and dose-dependent manner. Half-blocking concentrations (IC₅₀) of bepridil on I_A and I_K were 17.8 μM and 1.7 μM, respectively. 10 μM bepridil suppressed I_A and I_K by 46.7% and 77.1% at +30 mV of depolarization, respectively. When I_K was activated nearly uncontaminated with I_A by holding at −50 mV, 10 μM bepridil inhibited I_K by 71.6% at +30 mV of depolarization; 10 μM bepridil positively shifted the voltage-dependent of activation curves of I_A and I_K 12.1 mV and 28.7 mV, respectively. These results suggested that blockade on K⁺ currents by bepridil is preferential for I_K, and contributes to the protection brain against ischemic damage.     

Effects of Dibutyryl Cyclic AMP on Na+, K+-ATPase Activity and Intracellular Na+ and K+ in Primary Cultures of Astrocytes from DBA and C57 Mice

Summary: Effects of chronic treatment of dibutyryl cyclic AMP (db-cyclic AMP) on Na⁺, K⁺-ATPase activity in cell homogenates and intracellular N a f and K+ contents [(Na⁺)_i and (K⁺)_i] were studied in primary cultures of astrocytes derived from cerebral cortex of neonatal audiogenic seizure-susceptible DBA and audiogenic seizure-resistant C57 mice. Na⁺, K⁺-ATPase activity in cell homogenates was greater and (Na⁺)_i was less in DBA astrocytes than in C57 astrocytes. There was no difference in (K⁺)_i between astrocytes from DBA and C57 mice. Addition of db-cyclic AMP to the medium from day 14 to day 21 in culture (final concentration 0.25 mM) increased Na⁺, K⁺-ATPase activity in cell homogenates and decreased (Na⁺)_i, but had no significant effect on (K⁺)_i in astrocytes from either DBA or C57 mice. Chronic treatment with db-cyclic AMP altered cell growth. Protein and DNA content of cultured astrocytes from both DBA and C57 mice was decreased. DNA was more affected than protein. Modifying K⁺ and Na⁺ concentration in medium altered Na⁺, K⁺-ATPase activity in cell homogenates as well as (Na⁺)_i and (K⁺)_i in cultured astrocytes of both DBA and C57 mice. Changes in (Na⁺)_i and (K⁺)_i at different K⁺ concentrations in medium paralleled those in Na⁺, K⁺-ATPase activity in cell homogenates. Results indicate that the ability to transport Na⁺ across the cell membrane and the response of Na⁺, K⁺-ATPase to db-cyclic AMP and to the changes in K + in medium of cultured astrocytes from audiogenic seizure-susceptible DBA mice are sufficient.     

Temperature and amiloride alter taste nerve responses to Na, K, and NH4 salts in rats

The effects of adaptation/stimulus temperature (25°C vs. 35°C) on taste nerve responses to salt stimulation and amiloride suppression were assessed in rats. We measured the integrated responses of the chorda tympani nerve to 500 mM concentrations of NaCl, Na₂SO₄, sodium acetate (NaAc), KCl, K₂SO₄, potassium acetate (KAc), NH₄Cl, (NH₄)₂SO₄, and ammonium acetate (NH₄Ac) mixed with or without 100 μM amiloride hydrochloride at 25°C and 35°C. Taste nerve responses to all Na⁺ and NH₄⁺ salts, but not K⁺ salts, were significantly smaller at 25°C than at 35°C. Amiloride significantly suppressed taste nerve responses to all salts (Na⁺ salts>K⁺ salts>NH₄⁺ salts); amiloride suppression of Na⁺ and NH₄⁺ salts was significantly greater at 25°C than at 35°C. Benzamil-HCl, a more potent Na⁺ channel blocker compared to amiloride, strongly suppressed taste nerve responses to NaCl and KCl, but not to NH₄Cl. Amiloride and benzamil suppression of NaCl responses were similar; however, amiloride suppressed KCl responses more than did benzamil. The results suggest that: (1) amiloride-sensitive Na⁺ channels are involved to varying degrees in the transduction of sodium and potassium salt taste, and (2) amiloride may inhibit membrane proteins other than passive Na⁺ channels during stimulation with potassium and ammonium salts.     

Endogenous modulators of brain Na, K-atpase at early postnatal stages of rat development

The presence of endogenous modulators (peaks I and II) of synaptosomal Na⁺, K⁺-ATPase activity from adult rat cerebral cortex was previously suggested. In this study, the presence of such modulators at different postnatal stages of rat development was examined and their effect was tested on Na⁺, K⁺-ATPase activity. Synaptosomal membrane Na⁺, K⁺-ATPase activity was enhanced 20–30% by peak I and inhibited 70–75% by peak II obtained from 4-, 10-, 20- and 35–40-day-old rats. A fraction purified from peak II by anionic exchange HPLC (termed II-E) highly inhibits enzyme activity and behaves as a ouabain-like factor. Inhibitory activity of a 4-day-old II-E fraction proved higher than the corresponding fraction obtained from adult rats. Since expression of cerebral Na⁺, K⁺-ATPase has been shown to increase 10-fold during development whereas peak II concentration was observed to remain constant, and given the higher potency of purified neonatal II-E fraction, the effect of the latter may be greater at early postnatal stages of development than during adult life. It is suggested that the II-E fraction, which contains an ouabain-like factor, may play a role in neuronal development.     

Electrophysiological properties of rat septal region neurons during development in culture

We report on the development of membrane properties of septal region neurons from embryonic rats in serum-free culture during 1–25 days in vitro (DIV). Na⁺-dependent action potentials could be evoked within 1 day after plating and 3 different types of outward current were observed by means of the patch-clamp technique: I_K, I_A and I_C. In some neurons the neurotransmitter GABA evoked a chloride current after 2 DIV. In addition a cationic current elicited by glutamate appeared after 4 DIV. Within 8–12 DIV virtually all neurons were sensitive to both GABA and glutamate. Spontaneous action potentials and postsynaptic potentials occurred after 7–10 DIV but cultured septal neurons did not generate any pacemaker-like activity.     

Deleterious brain cell membrane effects after NMDA receptor antagonist administration to newborn piglets

Previous studies have shown that administration of the N-methyl- -aspartate (NMDA) receptor antagonist 3-(2-carboxypiperazin-4-yl)-1-phosphonic acid (CPP) reduces NMDA-mediated neurotoxicity in animal models of hypoxia/ischemia but also may induce brain tissue vacuolization and alter glucose metabolism. The present study tests the hypothesis that CPP administration alters brain cell membrane structure and function in the cerebral cortex of normoxic newborn piglets through the generation of oxygen free radicals and induction of lipid peroxidation. Twenty six anesthetized, ventilated newborn piglets—13 treated with 2 mg/kg i.v. CPP and 13 untreated controls—were studied. ATP and phosphocreatine (PCr) levels were measured as an index of cellular energy metabolism and tissue glucose levels determined. Na⁺,K⁺-ATPase activity was measured as an index of brain cell membrane function and the lipid peroxidation products conjugated dienes (CD) and fluorescent compounds (FC) measured. Free radical generation was detected on cortical biopsies homogenized with α-phenyl-N-tert-butyl-nitrone (PBN) through electron spin resonance spectroscopy. Signal height of spectrum was divided by dry tissue weight and expressed as mm/g tissue. In the two groups brain tissue ATP and PCr levels were not different. Tissue glucose levels were higher in the CPP group (24±5 mg/dl) than in controls (14±3 mg/dl), p<0.05, whereas Na⁺,K⁺-ATPase activity was lower in the CPP group than in controls (34±4 vs. 43±6 μmol Pi/mg protein/h), p<0.05. Lipid peroxidation products were higher in the CPP group (CD: 57±19 nmol/g brain, FC: 1.5±0.3 μg/g brain) than in controls (CD: 0±0 nmol/g brain, FC: 0.9±0.2 μg/g brain), p<0.05. Free radical intensity was higher in the CPP group (493±397 mm/g tissue) than in controls (51±83 mm/g tissue), p<0.05. In vitro administration of CPP to brain cell membranes did not change Na⁺,K⁺-ATPase activity or the generation of lipid peroxidation products. The data demonstrate that administration of CPP induces lipid peroxidation, results in free radical generation, decreases brain cell membrane Na⁺,K⁺-ATPase activity and alters glucose metabolism in the cerebral cortex of newborn piglets. Since CPP is a potent antagonist of the NMDA receptor, we speculate that CPP generates free radicals through a pathway independent of the NMDA receptor by altering cellular metabolism and possibly glucose utilization during normoxia in newborn piglets.     

Differentiation of Ionic Currents in CNS Progenitor Cells: Dependence upon Substrate Attachment and Epidermal Growth Factor

Multipotential progenitor cells grown from central nervous system (CNS) tissues in defined media supplemented with epidermal growth factor (EGF), when attached to a suitable substratum, differentiate to express neural and glial histochemical markers and morphologies. To assess the functional characteristics of such cells, expression of voltage-gated Na⁺and K⁺currents (I_Na, I_K) was studied by whole-cell patch clamp methods in progenitors raised from postnatal rat forebrain. Undifferentiated cells were acutely dissociated from proliferative “spheres,” and differentiated cells were studied 1–25 days after plating spheres onto polylysine/laminin-treated coverslips.I_NaandI_Kwere detected together in 58%,I_Naalone in 11%, andI_Kalone in 19% of differentiated cells recorded with K⁺-containing pipettes. With internal Cs⁺(to isolateI_Na),I_Naup to 45 pA/pF was observed in some cells within 1 day after plating.I_Naranged up to 150 pA/pF subsequently. Overall, 84% of cells expressedI_Na, with an average of 38 pA/pF.I_Nahad fast kinetics, as in neurons, but steady-state inactivation curves were strongly negative, resembling those of glialI_Na. Inward tail currents sensitive to [K⁺]_outwere observed upon repolarization after the 10-ms test pulse with internal Cs⁺, indicating the expression of K⁺channels in 82% of cells. In contrast to the substantial currents observed in differentiating cells, little or noI_NaorI_K-tail currents were detected in recordings from cells acutely dissociated from spheres. Thus, in the presence of EGF, ionic currents develop early during differentiation induced by attachment to an appropriate substratum. Cells switched from EGF to basic fibroblast growth factor (bFGF) when plated onto coverslips showed greatly reduced proliferation and developed less neuron-like morphologies than cells plated in the presence of EGF.I_Nawas observed in only 53% of bFGF-treated cells, with an average of 9 pA/pF. Thus, in contrast to reports that bFGF promotes neuronal differentiation in some CNS progenitor populations, our EGF-generated postnatal rat CNS progenitors do not develop neuronal characteristics when switched to medium containing bFGF. Thus, differentiated CNS progenitors can express a mix of neuronal and glial molecular, morphological, and electrophysiological properties that can be modified by culture conditions.     

Familial hemiplegic migraine

Familial hemiplegic migraine (FHM) is a rare and genetically heterogeneous autosomal dominant subtype of migraine with aura. Mutations in the genes CACNA1A and SCNA1A, encoding the pore-forming α₁ subunits of the neuronal voltage-gated Ca²⁺ channels Ca_v2.1 and Na⁺ channels Na_v1.1, are responsible for FHM1 and FHM3, respectively, whereas mutations in ATP1A2, encoding the α₂ subunit of the Na⁺, K⁺ adenosinetriphosphatase (ATPase), are responsible for FHM2. This review discusses the functional studies of two FHM1 knockin mice and of several FHM mutants in heterologous expression systems (12 FHM1, 8 FHM2, and 1 FHM3). These studies show the following: (1) FHM1 mutations produce gain-of-function of the Ca_v2.1 channel and, as a consequence, increased Ca_v2.1-dependent neurotransmitter release from cortical neurons and facilitation of in vivo induction and propagation of cortical spreading depression (CSD: the phenomenon underlying migraine aura); (2) FHM2 mutations produce loss-of-function of the α₂ Na⁺,K⁺-ATPase; and (3) the FHM3 mutation accelerates recovery from fast inactivation of Na_v1.5 (and presumably Na_v1.1) channels. These findings are consistent with the hypothesis that FHM mutations share the ability of rendering the brain more susceptible to CSD by causing either excessive synaptic glutamate release (FHM1) or decreased removal of K⁺ and glutamate from the synaptic cleft (FHM2) or excessive extracellular K⁺ (FHM3). The FHM data support a key role of CSD in migraine pathogenesis and point to cortical hyperexcitability as the basis for vulnerability to CSD and to migraine attacks. Hence, they support novel therapeutic strategics that consider CSD and cortical hyperexcitability as key targets for preventive migraine treatment.     

Control of the Na, K -pump by nerve growth factor is essential to neuronal survival

Recently we have shown that nerve growth factor (NGF) controls the performance of the Na⁺, K⁺ -pump in its target ganglionic neurons in suspension cultures. In the present study, enriched neuronal preparations of embryonic day 8 (E8) chick dorsal root ganglia (DRG) were obtained by means of a differential attachment procedure using tissue culture plastic dishes. Neurons were routinely seeded into polyornithine-coated 16 mm culture wells in the presence of NGF. After 18 h, cultures were switched to media with or without NGF, and containing either⁸⁶Rb⁺ (as a tracer for K⁺) or²²Na⁺ (as a tracer for Na ions). Over the next 12–15 h the cultures were assessed for numbers of surviving neurons and accumulated radioactivity. Cultured E8 chick DRG neurons fail to maintain their intracellular K⁺ concentration when deprived of NGF over 4–6 h. The NGF-deprived and K⁺- depleted neurons reaccumulate K⁺ within minutes of delayed NGF administration. The occurrence of this K⁺ response in culture to added NGF parallels the response occurring in E8 neuronal suspensions, including the time of onset of irreversibility. Similar experiments performed with²²Na⁺ indicate corresponding ionic behaviors for cultured E8 DRG neurons. These NGF-controlled ionic responses in monolayer cultures occur for E7 and E10 neurons, but not E14 neurons and parallel the survival response to NGF of the same neurons. Blocking the pump performance by NGF deprivation leads to neuronal death. Identical results are obtained by addition of oubain or omission of external K⁺ in the presence of NGF. Partial reduction of pump performance by any one of these treatments leads to partial survival of the neuronal population in a precisely predictable manner. Therefore, control of the pump by NGF is an essential component of the NGF action on neuronal survival.