Nerve impulse-enhanced release of amino acids from non-synaptic regions of peripheral and central nerve trunks of bullfrog

Brain Research, 84 (1975) 137-142 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

137

Short Communications

Nerve impulse-enhanced release of amino acids from non-synaptic regions of peripheral and central nerve trunks of bullfrog

DANIEL WEINREICH* AND RICHARD HAMMERSCHLAG Division of Neurosciences, City of Hope National Medical Center, Duarte, Calif. 91010 (U.S.A.)

(Accepted October 24th, 1974)

The release of glutamate from vertebrate central nervous tissue following electrical stimulation has been demonstrated in a variety of preparations: spinal cord slices 11,13, spinal hemicord 19, brain slices 17, exposed cerebral cortex 14,~8, exposed hippocampus 4, retina 2~, and cerebral cortical synaptosomes 3. These findings are frequently discussed in the context of a possible synaptic transmitter role for glutamate in the vertebrate central nervous systemS,10, ~5. A cautionary note to such discussions is that an efflux of this amino acid from non-synaptic regions of neurons could contribute to the release observed in many of the above studies. Such a nonsynaptic release of glutamate has been described from desheathed sciatic nerves 7,~3, and more importantly, this release appears enhanced during neuronal activity. In the present study, isolated dorsal and ventral spinal roots of bullfrog were chosen to further characterize amino acid efflux from nerve trunks during impulse conduction, and to compare parameters of this release with those established for the release of substances from synaptic regions. Since spinal roots lack the thick perineurium--epineurium that surrounds peripheral nerve trunks, diffusion rates of small molecules are similar to those found in desheathed sciatic nerves6,2°; in addition, problems of tissue swelling and metabolic alterations subsequent to desheathing 2° are avoided. Both types of roots were examined to determine if the glutamate release previously observed from sciatic nervesT,23 was solely from afferent fibers where it may serve a transmitter role 10, or was equally released from the cholinergic efferent fibers. Impulse-enhanced release of amino acids was also examined in isolated optic nerves of bullfrog to study the phenomenon in the central nervous system. A preliminary report of these findings has appeared 22. Dorsal and ventral spinal roots and optic nerves were excised from 4-6 in. bullfrogs, Rana catesbeiana, during the months of March through early July. The * Present address: Department of Cell Biology and Pharmacology, University of Maryland School of Medicine, Baltimore, Md. 21201, U.S.A.

138 isolated nerve trunks, manipulated by ligatures placed at both ends, were checked for their ability to conduct action potentials and then placed in capped plastic vials, 2 nerves/0.5 ml oxygenated Ringer solution. The incubation medium contained [t4C]glutamate and [3H]mannitol (at final concentrations given in the legend to Fig. 1). Incubations were carried out at 7 °C for periods up to 24 h. While initial uptake of glutamate appears to be predominantly into glial cellsa, ~2, relatively long incubation periods were employed in the present study to allow equilibration of glutamate and its metabolites between satellite cells and axons2, 9. Following incubation, each nerve was briefly dipped in non-radioactive Ringer solution and then placed for 10 min periods into 35 mm × 10 mm disposable Petri dishes each containing 3 ml of Ringer solution maintained at 18 °C. Sample aliquots of 1.5 ml were mixed with 12 ml of Aquasol (New England Nuclear Corp., Boston, Mass.) in counting vials for determination of total radioactivity during each etttux period. The remaining 1.5 ml of each sample were frozen at --20 °C for subsequent 50/sec 20000- A.

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Fig. 1. Nerve impulse-enhanced efttux of 14C- and ~H-materials from isolated 8th and 9th, dorsal and ventral spinal roots, or optic nerves of bullfrog, following incubation (24 h, 7 °C) in [14C]glutamate (30 # M final concentration) and [aH]mannitol (7.5 ffM). To normalize differences between nerve trunks, in B - D the effiux of isotope in each 10 min collection period was expressed as a percentage of the effiux during the third collection period (eft Wheeler et al.2Z). Periods of nerve stimulation, in B - D , are indicated by hatched bars. A: efflux of 14C (O O) and aH ( 0 - - - - ( 3 ) materials from a single dorsal root. Ten min period of nerve stimulation indicated by symbol (4.). B: effects of 20 m M K + and electrical stimulation on efltux of 14C () and 3H ( . . . . . ) materials from a single ventral root. C: effiux of 14C-materials from dorsal ( ) and ventral ( . . . . . ) roots in Ca~+-free medium supplemented with 1 m M EGTA and 10 m M MgC12. D : efflux of 14C-materials (-) from optic nerve. The counts/rain ratios of [~4C]glutamate : [~4C]glutamine (right ordinate) released during each 10 min collection period are indicated on the bars by ( x × × x ) .

139 identification of radioactive materials. The frog Ringer solution used for the efflux studies and for the initial incubations was essentially that of Wheeler et aL 23 (mM): NaC1, 1I0.0, KC1, 2.0; CaC12, 1.0; NaHCOs, 2.0. After the plateau phase of the washout curve was established, usually 60 min after initiation of the experiment (see Fig. 1A), the nerve was placed on a pair of chloridized silver electrodes and stimulated for 10 rain with a train of rectangular current pulses 80 #sec in duration at 50 Hz. The presence of compound action potentials was monitored at the beginning and at the termination of each stimulation period. Release of laC-material from spinal roots under resting conditions occurred in two phases: an initial rapid component, and a slower plateau-like phase (Fig. 1A). During the plateau phase, in both dorsal and ventral roots, a single 10 min period of electrical s t i m u l a t i o n increased the rate of 14C-etttux, b u t had n o p r o n o u n c e d effects o n the release o f 3H (Fig. 1A). I n 7 frogs, s t i m u l a t i o n increased the efflux f r o m 7 dorsal roots to 3 3 6 ~ :/_ 64 (S.E.M.), range 161-615, a n d to 3 5 7 ~ & 53, range 266-566 from 5 ventral roots. While the percentage increase was similar for both root types, the total a m o u n t o f 14C-material released f r o m dorsal roots, b o t h at rest a n d d u r i n g stimulation, tended to be greater t h a n c o r r e s p o n d i n g material released from ventral roots (Table TABLE I DISTRIBUTIONOF [14C]AMINOACIDSIN EFFLUENTSFROMFROGSPINALROOTS Samples of effluent medium from periods of rest and stimulation were passed through the cationexchange resin AG50WX8 (Na+), 0.3 ml settled resin/1.0 ml medium. Amino acids were eluted from the resin with 3 M NH4OH, dried in a vacuum centrifuge, and dissolved in H20. Samples and standards were subjected to paper electrophoresis at pH 4.1 (0.2 M pyridine-acetate, 2.5 h/800 V) or thin-layer chromatography on cellulose (BuOH-HOAC-H20, 3:1 :l). Each value for spontaneous or stimulated release represents mean for 10 rain collection period. S.E.M. shown within parentheses. Glutamate counts/mini 10 rain

Glutamine %

counts/min/ 10 rain

Aspartate %

counts/mini 10 rain

%

(A) Spontaneous release Dorsal (6) 1252 (241) Ventral (5) 757 (194)

47.6 (3.1) 36.4 (6.7)

1076 (192) 1016 (192)

38.1 (2.6) 47.2 (3.9)

428 (147) 312 (84)

13.5 (2.6) 15.4 (4.6)

(B) Stimulated release Dorsal (6) 6371 * (2261) Ventral (5) 2715" (697)

60.2 (3.5) 51.2 (3.0)

3354* * (1520) 1940"** (692)

28.5 (3.8) 33.8 (5.2)

954** (257) 504*** (143)

11.4 (2.6) 14.7 (4.9)

(C) Ratio of stimulated : spontaneous release Dorsal 5.09 3.12 Ventral 3.59 1.91

2.23 1.62

* P < 0.05 compared to spontaneous release. ** 0.05 < P < 0.10. *** Stimulated release not significantly different from spontaneous release.

140 I,A and B). Analysis of 14C-materials (see legend of Table I for methods) consistently revealed the presence of significant amounts of labeled glutamine and aspartate, in addition to glutamate. Stimulation increased the efflux rates of all 3 amino acids as indicated by ratios greater than unity (Table IC). The dorsal/ventral differences do not appear to reflect radioactivity levels within the roots since both types of roots contained comparable amounts of total t4C-material as well as [14C]glutamate per unit protein, after the 24 h incubation period. If the increased release of amino acids observed during the generation of nerve action potentials is related to membrane depolarization, similar increases in efflux might be initiated by raising the extracellular concentration of K + ions. Exposure of roots to K + concentrations of 20 (Fig. 1B), 50, or 100 m M had no marked effect on efflux of 14C-substances relative to [3H]mannitol release. The mean percentage increase during exposure to high K + was 12 (range - - 2 to +20, n = 5). As an internal control, each of these same roots was subsequently electrically stimulated to produce action potentials, conditions that, as expected, resulted in a clear increase in efflux of radioactivity (Fig. 1B). Since glutamate and aspartate are being proposed as neurotransmitters at both central and peripheral synapses, it was important to test whether the release of these amino acids from non-synaptic regions might be Ca2+-dependent. Ca2+-free, Mg 2+and EGTA-supplemented medium had no apparent effect on the spontaneous or stimulated efflux rates of 14C-substances (Fig. 1C). In 5 dorsal and 7 ventral roots excised from 3 frogs, a single 10 rain period of stimulation in Ca2+-free conditions increased the etttux to 3 3 6 ~ ~ 64 (S.E.M.), compared to 3 4 5 ~ ~z 59 for 12 roots in normal Ringer solution. Even in roots incubated with [14C]glutamate for 24 h in CaZ+-free solutions, subsequent nerve stimulation resulted in an increased effiux similar in magnitude to the value obtained in normal Ca2+-Ringer. To examine selectivity of amino acid release, spinal roots were incubated with [aH]leucine, [3H]lysine, or [aH]a-aminoisobutyrate together with [14C]glutamate. While the subsequent washout curves were similar for both isotopes, the etttux rates of 3H were not significantly increased during periods of electrical stimulation (2 experiments with each [3H]amino acid), under conditions where release of 14Cmaterials was markedly increased. A 10 min period of electrical stimulation of optic nerves, treated in a manner similar to spinal roots, increased the effluent radioactivity to 3 3 0 ~ ± 60 (S.E.M., n = 6). Nerve stimulation also produced a large increase in the etttux ratio of [14C]glutamate to [14C]glutamine (Fig. 1D), similar to that observed during electrical stimulation of spinal roots (Table I,A and B), and of retina preloaded with [14C]glutamate2L The impulse-enhanced release of [14C]glutamate from dorsal and ventral spinal roots, and from optic nerve, confirms and extends previous observations of glutamate release from desheathed sciatic nerve72 a. A major difference from these studies z3 is that effluents from spinal roots (and from optic nerves) contained significant amounts of labeled glutamine and aspartate, as well as glutamate, suggesting that the release phenomenon may be more complex than previously described. The observation that

141 nerve stimulation enhances the efflux rates of glutamate, glutamine, and aspartate to different extents, further suggests that nerve impulses induce a more specific effect on efflux of these amino acids than merely accelerating the rate of an ongoing process. This specificity is also reflected in the observations that not all amino acids are released by nerve impulses (see text and ref. 23). It appears that depolarization p e r se is not a stimulus for enhanced amino acid release since exposing roots to increased concentrations of extracellular K + over a 50-fold range had no major effect on the rate of efflux. A similar lack of effect of high K + on [14C]glutamate etttux from squid axon was observed by Baker and Potashner 1. Impulse-enhanced efflux of amino acids may be linked to a particular event(s) associated with the generation of action potentials that must occur repetitively to lead to a detectable level of 14C-material in the effluent. Such a process would not be simulated by K + which acts to maintain the membrane in a depolarized state. Amino acid release from conducting axons thus differs from neurotransmitter release at nerve terminals which can be initiated either by nerve impulses or by increased extracellular K +. The apparent lack of Ca 2+ dependence of the mechanism(s) for amino acid release from spinal roots implies a second fundamental difference from the mechanism for neurotransmitter release from nerve terminals, where a reduction in extracellular Ca 2+ ions markedly reduces the rate of release of transmitter 16. The observations that these [14C]amino acids are released from such anatomically distinct nerve trunks as dorsal and ventral roots, and optic nerves, suggest that stimulus-enhanced release from nerve trunks may be a general phenomenon in frog, unrelated to any possible transmitter role o f glutamate (or aspartate). These findings pose potential difficulties for studies attempting to demonstrate a stimulus-dependent release of this amino acid from primary afferent nerve endings. A high Ca2+-independent background release of glutamate during stimulation might well mask a smaller Ca2+-dependent release from afferent terminals. When assessing glutamate and aspartate as potential transmitter compounds in preparations containing polysynaptic pathways, the present findings suggest that results could be obtained from which misleading conclusions might be drawn. A diminution in glutamate or aspartate release observed subsequent to decreased extracellular Ca 2+ concentration might only reflect a diminished amino acid release from nerve fibers (and not necessarily from synapses). Such ~ de.crease would be expected since reduced levels of Ca 2+ result in a blockade of synaptic transmission that in turn reduces the number of axons conducting action potentials. We wish to express our appreciation to Ms. Arlene Chiu for her creative assistance, and to Dr. H. K. Borys for many discussions and critical reading of the manuscript. This work was supported by a PHS Research Grant NS09885 to R . H . The facilities and support of Dr. R. E. McCaman (PHS-NS9339) are greatly appreciated by D. W. (Postdoctoral Fellowship M H 53357).

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