Edward M. Lieberman

Glutamate regulation of non-quantal release of acetylcholine in the rat neuromuscular junction

Glutamate, previously demonstrated to participate in regulation of the resting membrane potential in skeletal muscles, also regulates non‐quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non‐quantal ACh secretion was estimated by the amplitude of endplate hyperpolarization (H‐effect) following blockade of skeletal muscle post‐synaptic nicotinic receptors by (+)‐tubocurarine and cholinesterase by armin (diethoxy‐p‐nitrophenyl phosphate). Glutamate was shown to inhibit non‐quantal release but not spontaneous and evoked quantal secretion of ACh. Glutamate‐induced decrease of the H‐effect was enhanced by glycine. Glycine alone also lowered the H‐effect, probably due to potentiation of the effect of endogenous glutamate present in the synaptic cleft. Inhibition of N‐methyl‐d‐aspartate (NMDA) receptors with (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzocyclohepten‐5,10‐imine (MK801), dl‐2‐amino‐5‐phosphopentanoic acid (AP5) and 7‐chlorokynurenic acid or the elimination of Ca2+ from the bathing solution prevented the glutamate‐induced decrease of the H‐effect with or without glycine. Inhibition of muscle nitric oxide synthase by NG‐nitro‐l‐arginine methyl ester (l‐NAME), soluble guanylyl cyclase by 1H[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) and binding and inactivation of extracellular nitric oxide (NO) by haemoglobin removed the action of glutamate and glycine on the H‐effect. The results suggest that glutamate, acting on post‐synaptic NMDA receptors to induce sarcoplasmic synthesis and release of NO, selectively inhibits non‐quantal secretion of ACh from motor nerve terminals. Non‐quantal ACh is known to modulate the resting membrane potential of muscle membrane via control of activity of chloride transport and a decrease in secretion of non‐quantal transmitter following muscle denervation triggers the early post‐denervation depolarization of muscle fibres.

Synthesis and release of N-acetylaspartylglutamate (NAAG) by crayfish nerve fibers: implications for axon–glia signaling

Early physiological and pharmacological studies of crayfish and squid giant nerve fibers suggested that glutamate released from the axon during action potential generation initiates metabolic and electrical responses of periaxonal glia. However, more recent investigations in our laboratories suggest that N-acetylaspartylglutamate (NAAG) may be the released agent active at the glial cell membrane. The investigation described in this paper focused on NAAG metabolism and release, and its contribution to the appearance of glutamate extracellularly. Axoplasm and periaxonal glial cell cytoplasm collected from medial giant nerve fibers (MGNFs) incubated with radiolabeled L-glutamate contained radiolabeled glutamate, glutamine, NAAG, aspartate, and GABA. Total radiolabel release was not altered by electrical stimulation of nerve cord loaded with [(14)C]glutamate by bath application or loaded with [(14)C]glutamate, [(3)H]-D-aspartate or [(3)H]NAAG by axonal injection. However, when radiolabeled glutamate was used for bath loading, radiolabel distribution among glutamate and its metabolic products in the superfusate was changed by stimulation. NAAG was the largest fraction, accounting for approximately 50% of the total recovered radiolabel in control conditions. The stimulated increase in radioactive NAAG in the superfusate coincided with its virtual clearance from the medial giant axon (MGA). A small, stimulation-induced increase in radiolabeled glutamate in the superfusate was detected only when a glutamate uptake inhibitor was present. The increase in [(3)H]glutamate in the superfusion solution of nerve incubated with [(3)H]NAAG was reduced when beta-NAAG, a competitive glutamate carboxypeptidase II (GCP II) inhibitor, was present.Overall, these results suggest that glutamate is metabolized to NAAG in the giant axon and its periaxonal glia and that, upon stimulation, NAAG is released from the axon and converted in part to glutamate by GCP II. A quisqualate- and beta-NAAG-sensitive GCP II activity was detected in nerve cord homogenates. These results, together with those in the accompanying paper demonstrating that NAAG can activate a glial electrophysiological response comparable to that initiated by glutamate, implicate NAAG as a probable mediator of interactions between the MGA and its periaxonal glia.

The nature of the membrane potential of glial cells associated with the medial giant axon of the crayfish

Abstract Electrophysiological, pharmacological and electron microscopic methods were used to characterize the satellite glial (Schwann-like) cells associated with the medial giant axon of the crayfish, Procambarus clarkii. The satellite glial cell layer surrounding the axon is formed by 15–20 cells deeply interdigitated into each other, forming a vast system of intercellular channels which communicate the axo-glial space with the external medium. The satellite cell layer varies from 0.2–3 μm in thickness. Membrane potentials of the giant axon and the satellite glial cells were monitored before, during and after treatment with ouabain and a variety of cholinergic agonists and antagonists. The membrane potentials of 63 control satellite cells averaged−42.6 ± 0.6 mV. The intracellular localization of the glial cell potential difference was corroborated by lithium carminate marking of the microelectrode tip recording site. Superfusion of satellite cells with 10−7 m carbachol, nicotine or acetylcholine caused a 15 to 20 mV hyperpolarization from resting level. Muscarine (10−6 m ) had no effect on the glial cell potential. The effect of nicotine was prevented or reversed by-tubocurarine (10−9 m ). The effects of cholinomimetics were reversed by washing the cells in drug-free solution. None of these agents had an obvious effect on axon membrane potential or action potential generation at the concentration used in this study. Ouabain (10−3 m ) also caused a rapid hyperpolarization of the glial cells. The effect lasted 15–18 min after which the membrane rapidly depolarized. The results suggest that (1) the satellite glial cell of the crayfish giant axon system may be studied simultaneously with the axon using electrophysiological techniques; (2) the satellite glial cell membrane appears to have typical acetylcholine receptors of the nicotinic type; (3) the membrane potential of the glial cell is sensitive to ouabain and (4) properties of the glial cell associated with the giant axon of the crayfish are similar to those of the Schwann cell of the tropical squid Sepioteuthis sepioidea.

Mechanisms of glutamate activation of axon-to-Schwann cell signaling in the squid

Membrane potentials from Schwann cells associated with giant axons of the small squid (Alloteuthis and Loliguncula) and the large squid (Loligo) were monitored with glass microelectrodes following 100 Hz/15 s axonal stimulation, or the application of 10(-7) M glutamate and ion substitutions, in the presence or absence of 10(-7) M d-tubocurarine. Glutamate or stimulation caused the membrane of the Schwann cell to depolarize to approximately -32 mV. This was rapidly replaced by a transient hyperpolarization to approximately -55 mV; the potential returning to the resting level (-40 mV) in approximately 7 min. In the presence of d-tubocurarine only the initial depolarization was evident. Nominally zero [Na+]o or treatment with 10(-7) M tetrodotoxin (in normal [Na+]o) blocked the stimulation- and glutamate-induced depolarization while low Clo- hyperpolarized the Schwann cell without effect on the glutamate- or stimulation-induced depolarization. Nao+ depletion or pretreatment with tetrodotoxin in normal Nao+ did not affect the development of the Schwann cell hyperpolarization. These results do not support the hypothesis that the glutamate-induced depolarization is the trigger leading to the Schwann cell hyperpolarization. Preliminary experiments to test the possibility that inositol phosphate second messenger and an increase in [Ca2+]i are triggered by glutamate receptor activation showed that nominally 0 Cao2+/75 mM Mgo2+ only slightly reduced the hyperpolarizing response to stimulation or glutamate while intracellular Bapta (20-30 microM) blocked the hyperpolarization but not the depolarization. [3H]Myoinositol incorporation into axon-Schwann cell plasma membranes was high.(ABSTRACT TRUNCATED AT 250 WORDS)

N-acetylaspartylglutamate (NAAG) is the probable mediator of axon-to-glia signaling in the crayfish medial giant nerve fiber.

Glial cell hyperpolarization previously has been reported to be induced by high frequency stimulation or glutamate. We now report that it also is produced by the glutamate-containing dipeptide N-acetylaspartylglutamate (NAAG), by its non-hydrolyzable analog beta-NAAG, and by NAAG in the presence of 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), a potent inhibitor of the NAAG degradative enzyme glutamate carboxypeptidase II. The results indicate that NAAG mimics the effect of nerve fiber stimulation on the glia. Although glutamate has a similar effect, the other presumed product of NAAG hydrolysis, N-acetylaspartate, is without effect on glial cell membrane potential, as is aspartylglutamate (in the presence of 2-PMPA). The hyperpolarization induced by stimulation, glutamate, NAAG, beta-NAAG, or NAAG plus 2-PMPA is completely blocked by the Group II metabotropic glutamate receptor antagonist (S)-alpha-ethylglutamate but is not altered by antagonists of Group I or III metabotropic glutamate receptors. The N-methyl-D-aspartate receptor antagonist MK801 reduces but does not eliminate the hyperpolarization generated by glutamate, NAAG or stimulation. These results, in combination with those of the preceding paper, are consistent with the premise that NAAG could be the primary axon-to-glia signaling agent. When the unstimulated nerve fiber is treated with cysteate, a glutamate reuptake blocker, there is a small hyperpolarization of the glial cell that can be substantially reduced by pretreatment with 2-PMPA before addition of cysteate. A similar effect of cysteate is seen during a 50 Hz/5 s stimulation. From these results we suggest that glutamate derived from NAAG hydrolysis appears in the periaxonal space under the conditions of these experiments and may contribute to the glial hyperpolarization.

Studies of axon-glial cell interactions and periaxonal K+ homeostasis—III. The effect of anisosmotic media and potassium on the relationship between the resistance in series with the axon membrane and glial cell volume

The effect of anisosmotic physiological solutions and [K+]o on the resistance in series with the axon membrane were studied in medial giant axons of the crayfish, Procambarus clarkii, to determine if changes in series resistance are correlated with changes in glial cell volume and volume regulatory responses. Series resistance was estimated from computer analysed voltage waveforms generated by constant current and space clamp techniques using piggy-back axial wire current passing and glass pipette recording electrodes. Axons subjected to anisosmotic physiological solution in the range of 23 to 175% of isosmolar solution demonstrated that the series resistance of axons changes in a manner similar to that expected for a volume change in isolated cells. In hyperosmotic solution the series resistance changes biphasically, initially decreasing followed by a recovery of the series resistance, similar to the regulatory volume increase described for glial cells in culture. The increase in series resistance following the initial decrease is inhibited by bumetanide (0.1 mM). Ouabain (1 mM), an inhibitor of the volume decreasing Na-K pump, causes the series resistance to increase significantly above that seen for the no-drug control. Bumetanide, an inhibitor of the volume increasing Na-K-Cl cotransporter, inhibits the volume regulatory response to anisosmotic media. Treating the axon with three times normal external [K+] causes the series resistance to decrease approximately 15% while five times normal [K+] leads to a 15% increase in series resistance. Both ouabain and d-tubocurare (10(-p8) M) prevent the three-fold [K+]-induced decrease in series resistance while carbachol (10(-7) M) and bumetanide have little effect. On the other hand, ouabain enhances the five-fold [K+]-induced increase in series resistance while carbachol and bumetanide cause the five-fold [K+] response to be in a decreasing direction. d-Tubocurare has little effect on the five-fold [K+]-induced increase in series resistance. The study demonstrates that under the conditions of these experiments changes in series resistance are a reflection of changes in cell volume modulated by ouabain- and bumetanide-sensitive K+ uptake mechanisms. The effects of carbachol and d-tubocurare on the series resistance suggest that their effects are modulated through their actions on the glial cell membrane potential and the electrochemical gradient for K+, which in turn controls the amount of K+ that appears in the periaxonal space.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies of axon-glial cell interactions and periaxonal K+ homeostasis—I. The influence of Na+, K+, Cl− and cholinergic agents on the membrane potential of the adaxonal glia of the crayfish medial giant axon

The ionic basis for the low (-40 mV) resting membrane potential of glial cells surrounding the giant axons of the crayfish and their hyperpolarization by cholinergic agents (to -55 mV) was studied using standard electrophysiological techniques, ionic substitutions and pharmacological agents. The resting membrane potential of the glial cell was depolarized by increasing [K+]o, but the response was not Nernstian. Na+ depletion caused a small depolarization of the glial resting membrane potential, whereas Cl- depletion resulted in a hyperpolarization comparable to that seen with carbachol at various [K+]o. Both furosemide (1 mM) and bumetanide (0.1 mM) produced an 8-10 mV hyperpolarization as compared to 15-17 mV seen with Cl- depletion or carbachol. Carbachol has no further effect on the potential following furosemide treatment or Cl- depletion. After carbachol administration or Cl- depletion the resting membrane potential of the glial cell responded to [K+]o in a more Nernstian manner. The data indicate that the low resting membrane potential of glial cells is due to a combination of a low [K+]i and an outwardly-directed (depolarizing) Cl- electrochemical gradient. Carbachol acts to decrease Cl- conductance, resulting in the hyperpolarization of the glial cell membrane and a decrease in the outwardly-directed K+ electrochemical gradient by approximately two-thirds. We hypothesize that this mechanism for modulation of the glial cell membrane potential and the K+ electrochemical gradient serves to enhance the uptake of K+ by the glial cell transport system.

Effect of N‐acetylaspartylglutamate (NAAG) on non‐quantal and spontaneous quantal release of acetylcholine at the neuromuscular synapse of rat

N‐Acetylaspartylglutamate (NAAG), known to be present in rat motor neurons, may participate in neuronal modulation of non‐quantal secretion of acetylcholine (ACh) from motor nerve terminals. Non‐quantal release of ACh was estimated by the amplitude of the endplate membrane hyperpolarization (H‐effect) caused by inhibition of nicotinic receptors by (+)‐tubocurarine and acetylcholinesterase by armin (diethoxy‐p‐nitrophenyl phosphate). Application of exogenous NAAG decreased the H‐effect in a dose‐dependent manner. The reduction of the H‐effect by NAAG was completely removed when N‐acetyl‐β‐aspartylglutamate (βNAAG) or 2‐(phosphonomethyl)‐pentanedioic acid (2‐PMPA) was used to inhibit glutamate carboxypeptidase II (GCP II), a presynaptic Schwann cell membrane‐associated ectoenzyme that hydrolyzes NAAG to glutamate and N‐acetylaspartate. Bath application of glutamate decreased the H‐effect similarly to the action of NAAG but N‐acetylaspartate was without effect. Inhibition of NMDA receptors by dl‐2‐amino‐5‐phosphopentanoic acid, (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzocyclohepten‐5,10‐imine (MK801), and 7‐chlorokynurenic acid or inhibition of muscle nitric oxide synthase (NO synthase) by NG‐nitro‐l‐arginine methyl ester and 3‐bromo‐7‐nitroindazole completely prevented the decrease of the H‐effect by NAAG. These results suggest that glutamate, produced by enzymatic hydrolysis of bath‐applied NAAG, can modulate non‐quantal secretion of ACh from the presynaptic terminal of the neuromuscular synapse via activation of postsynaptic NMDA receptors and synthesis of nitric oxide (NO) in muscle fibers. NAAG also increased the frequency of miniature endplate potentials (mEPPs) generated by spontaneous quantal secretion of ACh, whereas the mean amplitude and time constants for rise time and for decay of mEPPs did not change.

Studies of axon-glial cell interactions and periaxonal K+ homeostasis--II. The effect of axonal stimulation, cholinergic agents and transport inhibitors on the resistance in series with the axon membrane.

The small electrical resistance in series with the axon membrane is generally modeled as the intercellular pathway for current flow through the periaxonal glial (Schwann cell) sheath. The series resistance of the medial giant axon of the crayfish, Procambarus clarkii, was found to vary with conditions known to affect the electrical properties of the periaxonal glia. Series resistance was estimated from computer analysed voltage waveforms generated by axial wire-constant current and space clamp techniques. The average series resistance for all axons was 6.2 +/- 0.5 omega cm2 (n = 128). Values ranged between 1 and 30 omega cm2. The series resistance of axons with low resting membrane resistance (less than 1500 omega cm2) increased an average of 30% when stimulated for 45 s to 7 min (50 Hz) whereas the series resistance of high membrane resistance (greater than 1500 omega cm2) axons decreased an average of 10%. Carbachol (10(-7) M) caused the series resistance of low membrane resistance axons to decrease during stimulation but had no effect on high membrane resistance axons. d-Tubocurare (10(-8) M) caused the series resistance of high membrane resistance axons to increase during stimulation but had no effect on low membrane resistance axons. Bumetanide, a Na-K-Cl cotransport inhibitor and low [K+]o, prevented the stimulation-induced increase in series resistance of low membrane resistance axons but had no effect on the high membrane resistance axons. The results suggest that the series resistance of axons varies in response to the activity of the glial K+ uptake mechanisms stimulated by the appearance of K+ in the periaxonal space during action potential generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms for clearance of released N-acetylaspartylglutamate in crayfish nerve fibers: Implications for axon-glia signaling

Crayfish nerve fibers incubated with radiolabeled glutamate or glutamine accumulate these substrates and synthesize radioactive N-acetylaspartylglutamate (NAAG). Upon stimulation of the medial giant nerve fiber, NAAG is the primary radioactive metabolite released. Since NAAG activates a glial hyperpolarization comparable to that initiated by glutamate or axonal stimulation through the same receptor, we have proposed that it is the likely mediator of interactions between the medial giant axon and its periaxonal glia. This manuscript reports investigations of possible mechanisms for termination of NAAG-signaling activity. N-acetylaspartyl-[(3)H]glutamate was not accumulated from the bath saline by unstimulated crayfish giant axons or their associated glia during a 30-min incubation. Stimulation of the central nerve cord at 50 Hz during the last minute of the incubation dramatically increased the levels of radiolabeled glutamate, NAAG, and glutamine in the medial giant axon and its associated glia. These results indicate that stimulation-sensitive peptide hydrolysis and metabolic recycling of the radiolabeled glutamate occurred. There was a beta-NAAG-, quisqualate- and 2-(phosphonomethyl)-pentanedioic acid-inhibitable glutamate carboxypeptidase II activity in the membrane fraction of central nerve fibers, but not in axonal or glial cytoplasmic fractions. Inactivation of this enzyme by 2-(phosphonomethyl)-pentanedioic acid or inhibition of N-methyl-D-aspartate (NMDA) receptors by MK801 reduced the glial hyperpolarization activated by high-frequency stimulation. These results indicate that axon-to-glia signaling is terminated by NAAG hydrolysis and that the glutamate formed contributes to the glial electrical response in part via activation of NMDA receptors. Both NAAG release and an increase in glutamate carboxypeptidase II activity appear to be induced by nerve stimulation.