glutamine cycle enzymes in the crayfish giant nerve fiber: implications for axon-to-glia signaling

GLIA 14:19%208 (1995)

Glutamine Cycle Enzymes in the Crayfish Giant Nerve Fiber: Implications for

Axon-to-Glia Signaling ELIZABETH McKINNON,' PAL T. HARGITTAI,' ROBERT M. GROSSFELD?

AND EDWARD M. LIEBERMAN' 'Department of Physiology, East Carolina University School of Medicine, Greenuille, North Carolina 27858 and

'Department of Zoology and Physiology Program, North Carolina State University, Raleigh, NC 27695-761 7

KEY WORDS Glutamate metabolism, Glutaminase, Glutamine synthetase

ABSTRACT Two of the key enzymes involved in glutamate metabolism, glutaminase and glutamine synthetase, were quantitatively localized to axons and glia of the crayfish giant nerve fiber by immunocytochemistry and electron microscopy of antibody-linked gold microspheres. In Western blots, rabbit antisera for glutamine synthetase and glutaminase specifically recognized crayfish polypeptides corresponding approximately in size to subunits of purified mammalian brain enzymes. Glutamine synthetase immunoreactivity was found to be 11 times greater in the adaxonal glial cells than in the axon. Glutaminase immunoreactivity was found in somewhat greater concentration (2.5:l) in glia as compared to axoplasm. Glutamate immunoreactivity also was evaluated and found to be present in high concentration in both glia and axons, as might be expected for an important substrate of cellular metabolism. Using radiolabeled substrates it was demonstrated that glutamine and glutamate were interconverted by the native enzymes in the intact crayfish giant nerve fiber and that the formation of glutamine from glutamate occurred in the axoplasm-free nerve fiber, the cellular component of which is primarily periaxonal glia.

The results of this investigation provide immunocytochemical and metabolic evidence consistent with an intercellular glutamine cycle that modulates the concentration of periaxonal glutamate and glutamine in a manner similar to that described for perisynap- tic regions of the vertebrate central nervous system. These findings further corroborate previous electrophysiological evidence that glutamate serves as the axon-to-glial cell neurochemical signal that activates glial cell mechanisms responsible for periaxonal ion homeostasis. o 1995 Wiley-Liss, Inc.

INTRODUCTION

Previous investigations from this laboratory (Lieber- man and Hassan, 1988; Lieberman et al., 1989; Lieber- man, 1991; Lieberman and Sanzenbacher, 1992; Lie- berman et al., 1994) and others (Evans et al., 1991, 1992a,b; Villegas, 1984) have demonstrated that glutamate is the likely mediator of axon to glial cell signaling in the crayfish and squid. Pharmacological studies have characterized glutamate receptors on the Schwann cell plasma membrane as quisqualatekainate-sensitive and blocked by 2-amino-4-phosphonobutyrate (2APB) and L-glutamic acid diethylester (GDEE) (Lieberman et al., 0 1995 Wiley-Liss, Inc.

1989). These agents block both glutamate-induced and nerve stimulation-induced membrane potential changes in the adaxonal Schwann cell. NMDA-type receptors also appear to be present on the squid Schwann cell membrane, although they do not appear to be activated during action potential generation in the associated axon (Evans et al., 1991,1992a).

To definitively prove that a substance acts as a neu- rotransmitter or neurohumor, a number of stringent

Received September 29,1994; accepted March 1, 1995 Address reprint requests to Edward M. Lieberman, Department of Physiology,

East Carolina University School of Medicine, Greenville, NC 27858.

GLUTAMINE CYCLE ENZYMES 199

criteria must be satisfied (Kravitz et al., 1970; Wer- man, 1966). They include the following: a) the cell that releases the neuroactive agent has enzymes for its synthesis and facilities for its storage or is in close associa- tion with a cell that does; b) the agent is released from the cell by physiologically appropriate stimuli; c) the target cell has receptors specific for the agent which generates a consistent and physiologically relevant re- sponse; and d) mechanisms for removal of the agent from the environment of the target cell exist to terminate its action (e.g., by diffusion; by uptake into the releasing target cell, or nearby cells; and/or by metabolic degradation to inactivate products). Glutamate as a signaling agent at non-synaptic regions between axons and glia of squid and crayfish giant nerve fibers has been shown to meet criterion (c) and there is evidence consistent with criterion (b) (Lieberman et al., 1989; Lieberman and Sanzenbacher, 1992). The existence of cellular mechanisms to satisfy criteria (a) and (d) have yet to be demonstrated.

This investigation was designed to determine if glutamine-glutamate cycle enzymes such as those known to be present in mammalian central nervous systems for the regulation of transmitter glutamate metabolism (Berl and Clarke, 1978) also are present in the giant axon and associated glial cells with sufficient activity to regulate the interconversion of glutamate and glutamine. Our results demonstrate that the relevant enzymes are present in the crayfish giant nerve fiber and effectively interconvert glutamate and glutamine accumulated from the bath solution. These findings provide further evidence for the role of glutamate as the axon-to-glial cell signal.

Portions of this work have appeared in abstract form (McKinnon et al., 1993; Grossfeld et al., 1994).

MATERIALS AND METHODS Animals and Tissue Preparation

Crayfish, Procambarus clarkii, ranging in length from 4-6 inches, were obtained commercially from Waubun Laboratories in Shriver, LA. All experiments were performed on medial giant nerve fibers using a method of isolation modified from Wallin (1967). The ventral nerve cord was removed from the animal and placed in a lucite chamber continuously superfused with a normal physiological crayfish solution (NCS) containing, in mmoVL, 190 NaC1, 5.4 KC1, 13.5 CaCl,, 2.7 MgCl,, 20 Tris-HC1, at pH 7.4 (van Harreveld, 1936). The intact nerve cord was cleaned of excess tissue in preparation for fixation for immunocytochemistry and electron microscopy. The perineural sheath was removed by dis- section prior to incubation of the tissue with radiolabeled amino acid substrates.

Histological Processing

Tissue to be fixed for immunocytochemistry was superfused for 5 min with 2.5% glutaraldehyde: 3.0%

paraformaldehyde in a veronal acetate-buffered crayfish saline (NCSva). Fixation was continued in the same solution overnight at 4°C by immersion. NCSva contains veronal acetate (40 mmol/L) substituted for Tris.HC1, normal concentrations of KCl, CaCl,, and MgCl, with sufficient NaCl to maintain osmolarity. After fixation the tissue was dehydrated in an increasing series of ethanols, infiltrated with LR White resin (Polysciences, Inc., Warrington, PA), and polymerized at 50°C for 48 h.

Tissue to be embedded for morphometric analysis of structural detail was fixed overnight in 2% glutaraldehyde at 4"C, post-fixed using 1% osmium tetroxide in NCSva at 4°C for 1 h, and stained en bloc with 0.5% uranyl acetate a t room temperature. After dehydration, the tissue was embedded in Embed 812 resin (Poly- sciences, Inc.) a t 65°C in flat molds.

Antibodies and Western Blotting

The three primary antibodies used in this study were raised against glutamate (Arne1 Laboratories, New York, NY 17661, glutaminase (gift from Dr. N. Curthoys of Colorado State University), and glutamine synthetase. The latter antiserum was produced in house and, like the others, raised in rabbits using a procedure described by Martinez-Hernandez et al. (1977). Since all antisera were raised in rabbits, only one secondary antibody, a goat anti-rabbit IgG conjugated with a 30 nm gold label (Amersham, Arlington Heights, IL; Auroprobe, EM GAR G3-0, RPN423), was needed for immunocytochemical protocols.

Western Blot analysis was performed to insure that antibodies raised in rabbits were effective and specific in crayfish. Crayfish nerve cord was homogenized in 200 p1 denaturing solution consisting of P-mercaptoeth- anol, urea, and sodium dodecylsulphate and aliquots were fractionated by SDS-PAGE in 10% gels containing AcrylAide (Xue and Grossfeld, 1993). Proteins were transferred electrophoretically to nitrocellulose, non-specific binding sites were blocked with 10% non-fat powdered milk, and the blot was probed with glutamine synthetase or glutaminase antisera at 1:10,000, as described previously (Xue and Grossfield, 1993). Non-immune rabbit serum (Sigma, St. Louis, MO R4505) was substituted for primary antibody as a negative control. Bound antibody was detected by enhanced chemiluminescence (Am- ersham, RPN2109) after incubation of the blot with peroxidase-linked goat anti-rabbit IgG (Amersham, NIF 824 or Bio-Rad, Hercules, CA, 170-6515) at 1:25,000. PurifiedE. coli glutamine synthetase (EC 6.3.1.2; Sigma (3-3144) or glutaminase (EC 3.5.1.2; Sigma, G-5382) were included as positive controls. Prestained molecular weight standards (Sigma, SDS-7B) were run concurrently to estimate molecular masses.

Immunocy tochemistry

Following fixation and embedding in LR White resin, thick sections were cut and checked by light microscopy

200 McKINNON ET AL.

Fig. 1. Electron micrographs of four cross-sections of the medial giant nerve fiber of the crayfish, illustrating the various cellular structures characteristic of the anatomic relationship between the axon and the associated Schwann cells. Nerve cords fixed in glutaraldehyde and embedded in Embed 812 resin for high quality electron microscopy were used for morphometric analysis of the cytoplasmic and extracellular fractions of the nerve fiber. These data were used for analysis of immunocytochemical localization of glutamate, glutamine synthetase, and glutaminase. Regions of the cross-section related to the axon (Axon) and glial layer (Glia) are shown in A and can be used as a frame of

reference for all electron micrographs presented. The glial layer consists of alternating layers of glial cell cytoplasm (G1) as indicated in B and collagen matrix ( C ) in A and C with the “adaxonal” layer closest to the axon membrane always being glial cytoplasm. Other significant structures are tubular lattice (TL) and mitochondria (MI in A; Schwann cell nuclei (NS) in B; endoplasmic reticulum (ER) and trophospongium (Tr) in C . The relationship between the axon and two adjacent glial cells is shown in D (arrow). The point ofjunction between the glial cells nearest the axolemma is the mesaxon (Mx).

GLUTAMINE CYCLE ENZYMES 201

Fig. 2. Electron micrographs depicting glutamate, glutamine synthetase and glutaminase immunoreactivities in tissue embedded in LR White resin. Visualization of the antibody bound to the substrate and enzymes was accomplished using a secondary antibody conjugated with an electron dense 30 nm gold probe (Au) which is seen as small dark spheres in the micrographs (A). A. Glutamate immunoreactivity is evident in both the glial layer and axoplasm. It is evenly distributed throughout the glial cytoplasm but appears to be in higher concentration in the cortical region of the axoplasm than elsewhere in axon. Labeling also is seen in the trophospongia (Tr). The axon-glial

cell interface is marked by the arrow labeled “MGl.” B: Electron micrograph depicting glutaminase immunoreactivity. The labeled antibody appears to associate with membranous or tubular structures and appears to be uniformly distributed throughout both the glial and axoplasmic regions of the nerve fiber. C: Electron micrograph of glutamine synthetase immunoreactivity. Glutamine synthetase immunoreactivity is differentially distributed in the nerve fiber with a highly concentrated representation in the glial layer as compared to axoplasm. Quantitation of the distribution of immunoreactivity is tabu- lated in Table 1 and in the text.

202 McKINNON ET AL.

ASE SYN HOM 116 - 85 - 60 - 48 - 25 - A

ASE SYN HOM 116 - 85 - 60 - 48 - 25 - B

Fig. 3. Western blot of enzyme immunoreactivities of crayfish nerve homogenates. Blots were probed with rabbit antisera to E. coli glutamine synthetase (A, upper panel) or glutaminase (B, lower panel). The purified E. coli enzymes, glutamine synthetase (SYN) and glutaminase (ASE) were fractionated concurrently for comparison with crayfish nervous tissue extract (HOM). In both cases the antibodies used marked specific bands of the homogenate similar to those of the purified enzyme.

for orientation and location of a suitable area for ul- trathin sectioning. Dark gold sections were obtained, ranging in thickness from 80-90 nm, and mounted on cleaned 400 mesh nickel grids. Then, they were blocked with 1% chicken egg albumin for 30 min and incubated overnight with primary antiserum. Antisera for glutamate, glutaminase, and glutamine synthetase were used at 1:10,000, 1:4,000, and 1:10,000, respectively. Con- trols included a) pre-absorbing the antibody with the pure antigen before applying it to the section and b) substituting non-immune serum or buffer for the primary antiserum. After washing with PBS containing 0.5% Tween 20 for 5 min, the sections were incubated with 30 nm gold-labeled goat anti-rabbit IgG at 1:15 for 30 min, washed, and counterstained with uranyl mag- nesium acetate for 1 h and lead citrate for 10 min at room temperature. The methods used were comparable

to those used by other investigators to immunocytochem- ically localize glutamate and glutamine synthetase in sections of mammalian brain (Storm-Mathisen et al., 1981; Martinez-Hernandez et al., 1977). Sections were examined with a JEOL transmission electron micro- scope and micrographs were recorded on Kodak 4489 electron microscopy film.

Morphometrics

During electron microscopic examination, the giant axon and its surrounding glial cell layer were located and photographed at an initial magnification of X5,OOO. The giant nerve fibers from a total of 12 animals were processed for immunocytochemistry/electron microscopy, groups of 4 treated with one of the three antibodies. Five sections from three of the four animals in the group were fully analyzed providing data from at least 23 tissue areas for each antibody tested. Prints made at a final magnification of x 13,500 were used to determine the number of gold particles located in the axoplasm and glial layer of the tissue cross-section with the aid of a transparent 1.5 cm2 grid overlay. Approximately ten micrographs for each antibody also were analyzed in areas of resin only, to estimate the extent of non-specific binding of the antibody to the plastic. The average non-specific value was subtracted from the tissue-associated values to determine specific tissue binding, the values quoted in the text.

The giant axon is surrounded by a periaxonal sheath composed of alternating layers of glial cytoplasm and extracellular space. The glial volume density is the frac- tional representation of glial cell volume within this sheath. To measure this parameter, mounted sections were photographed at a magnification of x10,OOO and x 20,000, and morphological analysis was performed with the same grid overlay on prints at a final magnification of x27,OOO and ~54,000, respectively. Grid in- tersections were counted for areas of glial cytoplasm and total sheath volume. The results indicated are based on 15 electron micrographs, from two animals. These data were used to calculate the numerical density values quoted in the text, which represent the number of gold particles per unit cell volume (Weibel, 1979).

Glutamate and Glutamine Metabolism

Desheathed nerve cords (perineurial sheath removed) were incubated in NCS for 2-7 h with 1 pCi [14C] glutamate (New England Nuclear, Boston, MA, NEC 290E; 261.6 mCi/mmol) or I3H1 glutamine (New England Nu- clear; NET 551; 43.98 Ci/mmol) confined to the bath solution surrounding the thoracic or circumesophageal connectives. Upon termination of the incubation, the labeled portion of the giant nerve fibers was transferred to 70% ethanol in water and stored at 4°C until assayed within a few days to minimize decomposition of glutamine to glutamate. In several experiments, the giant

203 GLUTAMINE CYCLE ENZYMES

axon was cannulated with a micropipet and flushed with oil and NCS before or after radiolabeling, to evaluate the metabolic capacity of the axoplasm-free nerve fiber. This provides a measure of the inherent metabolism of glial cells of the periaxonal sheath. Radiolabeled metabolites were separated by low voltage paper elec- trophoresis (100 V for 7 h) with a potassium hydrogen phthalate buffer, pH 4.0 (Smith, 1968, 1969) and detected with a Bioscan 600 Radiochromatogram scanner. Non-radioactive amino acid standards added to the samples served to confirm the identities of the radioactive peaks when the paper was stained afterwards with 2% ninhydrin in acetone.

Statistical Methods

Results of immunocytochemical localization studies were reported as the mean 2 S.E.M. Significance of the results was determined using the Student t-test, taking P s 0.05 as significant.

RESULTS

Cellular Morphology

The fixation and embedding procedure employed in this study generally resulted in well-preserved tissues samples for morphometric analysis (Fig. 1A-D). Plasma membranes were intact and euchromatin and hetero- chromatin were apparent. There were several layers of membrane-enclosed glial cell cytoplasm surrounding the giant axon, interspersed with layers of extracellular collagen matrix. The collagen matrix accounts for a significant fraction of the tissue surrounding the axon. If the adaxonal layer is considered to include the cellular and collagen layers 3 to 5 pm lateral to the axon membrane (see review by Lieberman et al., 19941, then the cytoplasmic volume of the adaxonal glial cells represents 48 2 2% of the total non-axonal tissue volume and the remainder is extracellular space. At sites where the membranes of adjacent glia make contact, the processes were highly interdigitated (Fig. 1D). Schwann cell nuclei were electron dense and showed clusters of chromatin at the periphery (Fig. 1B). Well-preserved mitochondria were interspersed throughout the glial cytoplasm whereas axonal mitochondria are found primarily in the cytoplasmic region adjacent to the axolemma. Trophospongia (Nordlander et al., 1975) of the adaxonal glia projected finger-like extensions into the axoplasm (Fig. 1C). Tubular lattice structures were evident within the cytoplasm of the glia (Figs. lA,C). These structures opened into the periaxonal space, the glia- glia interface, and the glia-collagen interface. Struc- tures described here have been described previously in crayfish by Hama (19611, Shivers (19761, Lieberman et al. (19811, and Hargittai and Lieberman (1991).

Immunocy tochemistry

Although cellular structure was not as well defined for tissue embedded in LR White as for tissue embedded in Embed 812, minimal shrinkage of the tissue and only slight morphological change in mitochondria were detected. The advantage of LR White is that it pre- serves the antigenic sites of the tissue and allows access to them without etching, as is required with Embed 812 (Newman et al., 1982). In spite of the reduced structural resolution, axonal material was easily distinguished from periaxonal structures, allowing accurate morphometric data collection and analysis (see Fig. 2A-C).

General Observations

Non-specific staining of the resin in areas lacking tissue was negligible, accounting for less than 5% of the total staining present in the tissue. Furthermore, there was no specific labeling when non-immune serum or buffer was substituted for primary antisera or the antibody was pre-absorbed with the pure antigen before its application to the tissue section.

As seen in Figure 3 (upper panel), staining of the Western blot was specific for bands of about 48 kDa and 100 kDa for the purified E. coli glutamine synthetase and for the crayfish homogenate. These results are consistent with the molecular mass of 600 kDa for the bacterial enzyme, which consists of 12 identical subunits of 50 kDa that could form a 100 kDa dimer if the samples were not completely depolymerized (Rhee et al., 1985). Also in Figure 3 (lower panel), it can be seen that specific staining for the purified E. coli glutaminase was observed at about 52 kDa and for the crayfish nerve extract at 50 and 25 kDa. The difference may reflect species diversity of this enzyme. These results also are consistent with the reported size of the subunits (64 kDa) for pig brain enzyme (Kvamme et al., 1988).

Glutaminase and glutamine synthetase appeared to be co-localized with intracellular microtubules and fila- ments (Fig. 2B,C), suggesting that they may not be free cytoplasmic enzymes. Glutamate was ubiquitously distributed, showing the highest labeling density at the glial-axonal interface (Fig. 2A). Glutamate immunoreactivity was associated with electron dense material in the axoplasm presumably because of cross-linking to available cytoplasmic protein by the aldehyde fixative. Density of gold-labeled antibody, representing its concentration in the axon, was greatest near the axolemma for both glutamate and the enzymes under study. The numerical density of all three antigens decreases pro- gressively in the first 4 pm of cortical axoplasm, by 70% for glutamate, 50% for glutamine synthetase, and 40% for glutaminase. There was no labeling trend or gradi- ent observed in the periaxonal glia for any of the three antigens used in this study in comparing the proportion of glial cytoplasm in the adaxonal layer with that on more lateral portions of the glial layer. The collated

204 McKINNON ET AL.

TABLE 1 . Numerical density values of glutamate, glutaminase, and glutamine synthetase in the crayfish glia-axonal preparation”

~- Axoplasm Glia Ratios ____ Axoplasmic Axoplasmic Glial layer Glial cell

space cortex volume volume D B

Glutamate 5.5 t- 0.5 9.5 f 1.1 11.7 2 0.8 24.1 2.1 4.4 2.5 Glutaminase 1.1 2 0.2 1.4 2 0.4 1.3 t- 0.2 2.7 1.1 2.5 1.9 Glutamine synthetase 0.5 * 0.05 0.7 t- 0.1 2.8 t 0.3 5.8 5.6 11.6 8.3

Values given as means -c 1 S.E.M. and are based on analyses of 15 to 37 electron micrographs of 2-4 animals. (A) Axoplasmic space represents the volume in a ten micrometer ring of axoplasm adjacent to the axolemma. (B) Axoplasmic cortex represents the volume in a one micrometer ring of axoplasm adjacent to the axolemma. (C) Glial layer volume is the volume of the glial layer including extracellular space, collagen and glial cells, between the axolemma and the intermediate glial layer (Butt et al., 1990). (D) GIial cell volume represents the cytoplasmic fraction ofglial layer valume.

- A B C D C:A D:A

morphological data are based on the analysis of 15 to 37 micrographs from two to four animals and presented in Table 1.

Quantitative Distribution of Glutamate, Glutaminase, and Glutamine Synthetase

Glutamate

The overall numerical density of glutamate in the axon averaged 5.5 5 0.5 gold particles per unit volume. Labeling of glutamate in the entire periaxonal sheath averaged 11.7 2 0.8 gold particles per unit volume, approximately two times that found in the axon. When this value is corrected for glial cytoplasmic volume (about 48% of the sheath volume), the numerical density is 24.1 gold particles per unit glial cell volume, representing a glutamate immunoreactivity 4.4 times higher than in the axon. Comparing the numerical density of glutamate immunoreactivity in the first p-m of axoplasm nearest the axolemma to its immunoreactivity in glial cytoplasm, glutamate is 2.5 times more concentrated in glia (Table 1).

Glutaminase

The overall glutaminase-associated antibody in the 4 pm of axoplasm nearest the axolemma averaged 1.1 t 0.2 particles per unit volume. Glutaminase labeling of the entire periaxonal sheath was 1.3 -t 0.2 particles per unit volume, not significantly higher than the labeling seen in the 4 pm of axoplasm nearest the axolemma. When taking into account the glial cytoplasmic volume, labeling of glutaminase is 2.7 particles per unit glial cell volume. Comparing this value to axoplasm labeling, glutaminase is 2.5 times more concentrated in the glia. Comparing only the first 1 p-m numerical density in the axon to the glial cytoplasmic numerical density for glutaminase labeling, this enzyme is 1.9 times more concentrated in the glia (Table 1).

Glutamine Synthetase

Glutamine synthetase labeling of the entire periaxonal sheath was 2.8 -t- 0.3 particles per unit volume as

compared to 0.5 +- 0.05 particles per unit volume average labeling of the 4 pm of axoplasm nearest the axolemma, or approximately 6 times higher in the glial layer than in the axoplasm. The glial labeling value, when corrected for glial cytoplasmic volume, is 5.8 particles per unit glial cell volume, or 11.6 times higher in glia than in the axon. Using just the first 1 pm interval in the axoplasm compared to the glial cytoplasm (Table 11, glutamine synthetase is 8.3 times more concentrated in the glia.

Metabolic Interconversion of Glutamate and Glutamine

If the immunoreactivities reflect the cellular distribution and concentration of active enzymes, then the axon and glia in the intact nerve fiber should differ- ently interconvert glutamate and glutamine. The giant nerve fiber accumulated radioactive glutamate from the bath and formed detectable amounts of radioactive glutamine within 2 h (Fig. 4A). Similarly, it accumulated exogenous radioactive glutamine, from which it made radioactive glutamate (Fig. 4B). To confirm that glutamine synthetase activity is associated with the periaxonal glia, axons were perfused with mineral oil and NCS before (not shown) or after (Fig. 4C) incubation of the nerve fiber with radioactive glutamate. This procedure flushes the bulk of axoplasm, including the dense cortical axoplasm, from the axon, leaving an axoplasm- free axon attached to the periaxonal sheath (Brown and Lasek, 1990). When this was done, radioactive glutamine still was present in the isolated nerve fiber. Figures 4D,E represents chromatographic runs of radiolabeled glutamine and glutamate used to load the cells to control

~~

Fig. 4. Metabolic interconversion of glutamate and glutamine by intact nerve fiber. Alcohol extracts of intact nerve fibers (A and B) or of a nerve fiber perfused after incubation to eliminate axoplasm (C) were spotted at 10 cm (arrow at bottom offigure) and electrophoresed at low voltage on different occasions. Glutamate (“GLU”) migrates towards the anode (to the right of center) and glutamine (”GLN”) towards the cathode (to the left of center). Slight variations among peak positions reflect slight variations in running conditions. In A, the tissue was incubated for 7 h with L3Hl-glutamine, and in B and C it was incubated for 6.33 h with [14Cl-glutamate. B and C were from the same nerve cord. The results with aliquots of the radioactive tracers used for the incubations are shown in panels D ([3Hl-glutamine) and E ([‘4C]-glutamate). Each plot illustrates CPM versus distance in cm.

GLUTAMINE CYCLE ENZYMES

DPP,

205

A

B

C

f 1

C

F

1 D

k 0

E

C

149,

89

Fig. 4

McKINNON ET AL. 206

for their position in chromatography of the biological samples and indicate the extent of their purity. The slight differences in position of the samples from one chromatographic run to the next likely results from slightly different chromatographic conditions (buffer, voltage, pH, etc.). Although these results demonstrate conversion of glutamate to glutamine by glia and interconversion of these substances by axons andlor glia further experiments will be required to evaluate the extent of compartmentalization of the enzymatic activities.

DISCUSSION

This study is part of a continuing effort of these laboratories to define the role of axon-to-glial cell signaling during neuronal activity. Pharmacological studies from this and other laboratories (Evans et al., 1992a,b; Lie- berman et al., 1989; Lieberman, 1991; Lieberman and Sanzenbacher, 1992) have provided strong evidence that glutamate is the likely chemical signal released from axons during action potential generation and propaga- tion. Thus, metabolic mechanisms for the uptake, synthesis, and degradation of the glutamate signal should be present in the giant nerve fiber if glutamate is the communication link between axon and glia, as it is a t many synapses between neurons or between neurons and muscle fibers of invertebrates. To determine whether these mechanisms exist and are appropriately distributed between axons and glia, we used immunocytochemical methods to quantitatively localize glutamate, glutaminase, and glutamine synthetase in axons and glia of the crayfish medial giant nerve fiber and radiotracer metabolism to demonstrate that these enzymes are func- tional in the living crayfish nerve fiber.

Immunocytochemical analysis of the localization of glutamate, glutaminase, and glutamine synthetase showed that they all are more concentrated in glia than in axoplasm. Comparing immunoreactivities per cellular volume, glutamate is 4.4x, glutaminase is 2.5x, and glutamine synthetase is 1 1 . 6 ~ greater in glial cytoplasm than in axoplasm. Roots (1981) also found glutamine synthetase to be localized to perineural glial cells of the abdominal ganglion of ApZysia californica. The specific binding of the same antibodies to polypeptides of comparable size in extracts of crayfish nervous tissue and in samples of the purified mammalian enzymes suggests that the immunoreactivities accurately reflect the distribution of the enzyme proteins in crayfish tissue.

Calculated densities of axoplasmic immunoreactivity were limited to the 4 p.m nearest the axolemma because of the large size of the axon relative to the magnification required to accurately discern gold particles in pho- tographic images. However, there is good reason to be- lieve that this is the most relevant portion of the axoplasm to study. As shown by the results, the densities of glutamate and both enzymes decreased considerably over this interval. This finding is not unexpected, since the cortical axoplasm of squid (Brown and Lasek, 1990) and

crayfish (Viancour et al., 1987) giant axons contains the bulk of subcellular organelles and cytoplasmic matrix proteins. During normal neuronal electrical activity, diffusion of ions occurs within msec and involves no more than this few pm of axoplasm and extracellular space adjacent to the axolemma. If the axoplasmic glutamate nearest the axolemma represents the excitation-releasable pool of axon-glia transmitter, then it is present in highest concentration in the region of greatest need. It would also be expected that appropriate membrane transporters and synthetic and degrada- tive enzymes for amino acids would be localized within this axolemma-axoplasmic volume, as they were found to be.

In mammalian central nervous system tissue, glutaminase activity typically is slightly greater (roughly 2 x ) in neurons than in astrocytes, whereas glutamine synthetase is found at much greater activity in the glia (Schousboe et al., 1988). In fact, it was the immunocytochemical demonstration of a differential distribution of the latter enzyme that solidified the concept of metabolic compartmentation in mammalian central nervous system. That concept was introduced by Walesch and his co-workers (Berl et al., 1961; Lajtha et al., 1959) more than 30 years ago to explain their results on metabolism of radioactive glutamate. They, and others who have followed them, have shown that the adult mammalian brain contains a t least two distinct compartments, a large pool containing most of the glutamate, and a small glutamate pool, from which glutamine is rapidly formed (Clarke et al., 1974; Garfinkel, 1972). It has been suggested that neurons comprise the large pool and that glia make up the small pool (Balazs and Cremer, 1972; Van den Berg, 1972). Based on studies of mouse brain slices exposed to drugs which act selec- tively on neurons, Benjamin and Quastel (1972, 1975) suggested a glutamate-glutamine cycle in which glutamate released from neurons is taken up by glial cells and converted to glutamine, which is then returned to the neurons for hydrolytic deamidation to form glutamate and ammonia. At nerve terminals, this would re- plenish much of the releasable pool of transmitter glutamate. The significant drop in synaptic release of glutamate during pharmacological inhibition of glutamine synthetase or glutaminase strongly supports this no- tion (Nicklas, 1983; Rothstein and Tabakoff, 1984). Be- cause glial cells have a more efficient glutamate uptake than neurons (Schousboe et al., 19881, this property, together with glial conversion of glutamate to glutamine, not only terminates the immediate transmitter activity, but also contributes to its perpetuation over the long term.

For the crayfish giant nerve fiber, we have reported here that glutamine synthetase is localized primarily to the glia and that, as expected, glutamine is formed in this location from glutamate in intact, living tissue. Furthermore, we have shown that there is only a rela- tively small differential distribution of glutaminase and glutamate immunoreactivity between axon and glia and that the tissue converts exogenous glutamine to gluta-

GLLJTAMINE CYCLE ENZYMES 207

mate. Considering the central role of glutamate and glutamine in energy, nitrogen and protein metabolism, and osmotic homeostasis, in addition to intercellular signal transmission, a uniform distribution of glutamate and glutaminase is not unexpected. The goal for the near future is to examine the cellular specificities of glutamate-glutamine metabolic interconversion, and associated transport processes in the functioning tissue.

The results presented here suggest that the glutamate-glutamine cycle may not be confined to synaptic regions of the central nervous system of either verte- brates or invertebrates but may also function in chemical signaling between neuronal processes and glia. In this regard, it may serve to terminate the immediate action of released glutamate while insuring the preservation of signaling during continued neuronal impulse generation. In fact, glutamate has been shown to be released from sciatic nerve of amphibians during stimulation (DeFeudis, 1971; Weinreich et al., 1975; Wheeler et al., 1966) and indirect pharmacological evidence also exists for glutamate release from optic nerve of mammals (Kriegler and Chiu, 1993). Although the evidence for axonal release of glutamate during stimulation in crayfish and squid also is primarily pharmacological (Lieberman et al., 1989; Lieberman and Sanzenbacher 1992), recently we have reported that stimulation of crayfish giant nerve fibers loaded with radiolabeled glu-

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tamate or glutamine results in the appearance of these compounds in the superfusion fluid Of the crayfish (Gross- feld et al., 1994). We speculate that the glutamate-glu- tarnine cycle between and glia may Serve an important role at both synaptic and non-synaptic sites in the nervous system. It has been established that glutamate during excitation is a signal to enhance the dial clearance of KC from the

fiber: Implications "for axon-to-glia signalkg. SOC. Neuyosci. Abs., 20:914.

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from

periaxonal space (Krieaer and Chiu, 1993; Lieberman et al., 1994). A glutamate-glutamine cycle between axons and glia would be essential for termination of the glutamate signal.

ACKNOWLEDGMENTS

The authors thank Mike Means, Carrie Armel, Drew Jones, and Tracey Warren for their help with the experiments related to nerve metabolism and Mike Means with the photography that generated the plates of the electron micrographs. This study was supported in part by a grant from the Army Research Office DAAG29-86- K-0023 to EML.

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glutamine cycle enzymes in the crayfish giant nerve fiber: implications for axon-to-glia signaling

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