REVIEW ARTICLE
published: 27 May 2013
doi: 10.3389/fendo.2013.00059
The glutamate–glutamine (GABA) cycle: importance of late
postnatal development and potential reciprocal
interactions between biosynthesis and degradation
Leif Hertz*
Clinical Pharmacology, Medical University of China, Shenyang, China
Edited by:
Tiago B. Rodrigues, University of
Cambridge, UK
Reviewed by:
Douglas L. Rothman,Yale University,
USA
Arne Schousboe, University of
Copenhagen, Denmark
*Correspondence:
Leif Hertz, RR 2, Box 245, Gilmour,
K0L 1W0, Ontario, Canada.
e-mail: leifhertz@xplornet.ca
The gold standard for studies of glutamate–glutamine (GABA) cycling and its connections
to brain biosynthesis from glucose of glutamate and GABA and their subsequent metab-
olism are the elegant in vivo studies by
13
C magnetic resonance spectroscopy (NMR),
showing the large fluxes in the cycle. However, simpler experiments in intact brain tis-
sue (e.g., immunohistochemistry), brain slices, cultured brain cells, and mitochondria have
also made important contributions to the understanding of details, mechanisms, and func-
tional consequences of glutamate/GABA biosynthesis and degradation.The purpose of this
review is to attempt to integrate evidence from different sources regarding (i) the enzyme(s)
responsible for the initial conversion of α-ketoglutarate to glutamate; (ii) the possibility that
especially glutamate oxidation is essentially confined to astrocytes; and (iii) the ontoge-
netically very late onset and maturation of glutamine–glutamate (GABA) cycle function.
Pathway models based on the functional importance of aspartate for glutamate synthesis
suggest the possibility of interacting pathways for biosynthesis and degradation of gluta-
mate and GABA and the use of transamination as the default mechanism for initiation of
glutamate oxidation. The late development and maturation are related to the late cortical
gliogenesis and convert brain cortical function from being purely neuronal to becoming
neuronal-astrocytic. This conversion is associated with huge increases in energy demand
and production, and the character of potentially incurred gains of function are discussed.
These may include alterations in learning mechanisms, in mice indicated by lack of pairing
of odor learning with aversive stimuli in newborn animals but the development of such
an association 10–12 days later. The possibility is suggested that analogous maturational
changes may contribute to differences in the way learning is accomplished in the newborn
human brain and during later development.
Keywords: aspartate aminotransferase, glutamine–glutamate cycle, postnatal metabolic enzyme development
GLUTAMATE AND GABA
The function of glutamate and γ-aminobutyric acid (GABA) asthe
key excitatory and inhibitory transmitters in mammalian brain
was not realized until the second half of the twentieth century
(Okamoto, 1951; Florey, 1956; Roberts, 1956; Curtis et al., 1960;
Watkins, 2000). Relatively soon thereafter evidence was obtained
that a cycle of neuronal-astrocytic interactions plays a majorrole in
the production from glucose and the metabolism of both amino
acid transmitters (van den Berg and Garfinkel, 1971; Benjamin
and Quastel, 1972), and intense uptake of the transmitters, espe-
cially glutamate, was demonstrated and quantitated in astrocytic
preparations (McLennan, 1976; Schousboe et al., 1977; Hertz et al.,
1978a,b). Although the importance of glutamatergic/GABAergic
activation of endocrine responses was suggested already at that
time (Ondo and Pass,1976; Ondo etal., 1976),the full consequence
of the involvement of the amino acid transmitters only became
realized during the last decade. More recently, direct evidence is
emerging that astrocytes may also account for much of gluta-
mate degradation (Bauer et al., 2012; McKenna, 2012; McKenna,
present Research Topic; Whitelaw and Robinson, present Research
Topic), and that production and degradation pathways may inter-
act (Hertz, 2011a). These recent conclusions and observations
place an increased focus on identification of the enzymes(s) car-
rying out the undisputed initial conversion of glutamate to α-
ketoglutarate (α-KG) (almost certainly mainly transamination)
and, especially vice-versa. This paper will deal with these ques-
tions and discuss a possible interaction between the pathways
mediating synthesis and degradation of the two amino acid trans-
mitters. It will also discuss an observed late maturation of the
metabolic processes involved. Many of the developmental obser-
vations were made decades ago, but their full importance can only
now be understood after the realization in the living rodent and
human brain of the huge glutamine–glutamate (GABA) cycle flux
determined in the brain in vivo and described below.
THE GLUTAMINE–GLUTAMATE (GABA) SHUTTLE AND ITS
RELATION TO GLUCOSE METABOLISM
Figure 1 is a cartoon of selected parts of glucose metabolism
in astrocytes (right) to neurons (left). They are connected by a
flow of glutamine (produced directly from glutamate, generated as
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Hertz Astrocytes, glutamine, glutamate, and GABA
FIGURE 1 | Cartoon of glucose metabolism via pyruvate in neurons
(left – N) and astrocytes (right – A) and of glutamine–glutamate
(GABA) cycling. In both cell types pyruvate metabolism via acetyl
Coenzyme A (ac.CoA) leads to formation of citrate by condensation with
pre-existing oxaloacetate (OAA) in the tricarboxylic acid (TCA), an end-result
of the previous turn of the cycle. Citrate oxidation in the TCA cycle includes
two decarboxylations, leading to re-formation of oxaloacetate, ready for
another turn of the cycle, and to production of large amounts of energy
(ATP). Pyruvate carboxylation creates a new molecule of oxaloacetate,
which after condensation with acetyl Coenzyme A, derived from a second
molecule of pyruvate, forms a new molecule of citrate.This process can be
used for replacement of worn TCA cycle intermediates. More important in
the present context is that α-ketoglutarate (α-KG), one of the intermediates
of the TCA cycle can leave the cycle to form glutamate (glu) and, catalyzed
by the cytosolic and astrocyte-specific enzyme glutamine synthetase,
glutamine (gln). After release from astrocytes glutamine is accumulated in
glutamatergic and GABAergic neurons [lower line (V
gln
in the
13
C-NMR
studies) of the glutamine–glutamate (GABA) cycle (glu–gln cycle), converted
to glutamate (and in GABAergic cells onward to GABA)] and released as
transmitter. Released glutamate is almost quantitatively re-accumulated in
astrocytes, together with part of the released GABA [upper line (V
cyc
in the
13
C-NMR studies) of the glutamine–glutamate (GABA) cycle (glu–gln cycle)]
and re-accumulated in the astrocytic cytosol. Here, about 85% is converted
to glutamine and re-enters the glutamine–glutamate (GABA) cycle. The
remaining 15% is oxidatively degraded after re-conversion via
α-ketoglutarate to malate, exit of malate to the cytosol, decarboxylation to
pyruvate by the already mentioned cytosolic malic enzyme and further
pyruvate oxidation in theTCA cycle via acetyl Coenzyme A. Combined
astrocytic formation and oxidation of glutamate creates almost as much
ATP as direct oxidation of glutamate (Hertz et al., 2007).
discussed below) from astrocytes to neurons. In the neurons gluta-
mate is converted to transmitter glutamate and GABA. However,
after their release as transmitters most glutamate and a consid-
erable amount of GABA are returned to astrocytes. This is the
glutamine–glutamate (GABA) cycle. Elegant
13
C-NMR analysis
(in vivo injection of labeled glucose, or in some cases acetate,
and determination of labeled metabolites) has shown that the
glutamine flux in the cycle, V
gln
in the
13
C-NMR studies, is
slightly greater than the flux, V
cyc
in the
13
C-NMR studies, of
released transmitter glutamate and GABA in the opposite direc-
tion (Rothman et al., 2011), and that GABA fluxes account for up
to 20% of total flux in V
cyc
(Patel et al., 2005; Chowdhury et al.,
2007a). The reason for the slightly smaller V
cyc
than V
gln
may
mainly be that some GABA is re-accumulated in GABAergic neu-
rons (Schousboe, this Research Topic), where it can be oxidized
(Yu, 1984). However, as discussed above, a considerable amount
of GABA is also transferred to astrocytes, where it is taken up
(Hertz et al., 1978a), transaminated to succinic acid semialdehyde
(SSA), oxidized to succinate and then either (i) exits the tricar-
boxylic acid (TCA) cycle as malate (Figure 1); or (ii) is converted
via α-ketoglutarate and glutamate to glutamine and returned
to neurons in the glutamine–glutamate (GABA) cycle. Cytoso-
lic malate is decarboxylated by the astrocyte-specific (Kurz et al.,
1993), remarkably active (McKenna et al., 1995; Vogel et al., 1998)
cytosolic malic enzyme to pyruvate, which can then be completely
oxidized in the TCA cycle. Using this route, released glutamate is
almost quantitatively taken up by astrocytes (Danbolt, 2001), and
either converted to glutamine and reintroduced in the glutamine–
glutamate (GABA) cycle, or metabolized to α-ketoglutarate by
glutamate-dehydrogenase (GDH) or aspartate–glutamate trans-
ferase (AAT), followed by α-ketoglutarate oxidation after malate
exit and decarboxylation. Glutamate oxidation is intense in cul-
tured astrocytes (Yu et al.,1982; Hertz et al.,1988; McKenna, 2012),
and increases with increasing glutamate concentration (McKenna
et al., 1996). In vivo 85% of the accumulated glutamate is con-
verted to glutamine and re-used,whereas the last 15% is oxidatively
degraded (Rothman et al., 2011). The close quantitative correlation
between V
cyc
and rate of glucose oxidation suggests that over 80% of
neuronal oxidative ATP production is coupled to neuronal signaling
even in the absence of specific stimulation (Rothman et al., 2011).
The operation of glutamine–glutamate (GABA) cycle in one
direction only is a result of the astrocyte-specific (probably not
glia-specific) localizations of the enzymes pyruvate carboxylase,
PC (Yu et al., 1983; Shank et al., 1985; Hutson et al., 2008) and glut-
amine synthetase, GS (Norenberg and Martinez-Hernandez, 1979;
Derouiche, 2004). Pyruvate carboxylase is the enzyme catalyzing
formation of oxaloacetate (OAA in Figure 1) from pyruvate. This
is the only enzyme catalyzing net synthesis from glucose of a new
TCA intermediate. Cytosolic malic enzyme normally only oper-
ates toward decarboxylation. The ubiquitously expressed pyruvate
dehydrogenase (PDH) carries pyruvate, via pyruvate dehydro-
genation and formation of acetyl Coenzyme A, into the TCA cycle
in both neurons and astrocytes, but no new TCA cycle intermedi-
ate is generated by the action of this enzyme alone. This is because
the citrate (citr), which is formed by condensation of acetyl Coen-
zyme A with pre-existing oxaloacetate in the TCA cycle loses two
molecules of CO
2
during the turn of the cycle, which leads to
re-generation of oxaloacetate. This mechanism allows addition of
another molecule of pyruvate in the next turn of the TCA cycle
to continue the process, but it does not provide a new molecule
of a TCA cycle intermediate that the cycle can afford to release
and convert to glutamate. In contrast joint activity of PDH and
PC activity creates a new molecule of citrate (Figure 1), which via
α-ketoglutarate can be converted to glutamate, and by the aid of
glutamine synthetase converted to glutamine. The pyruvate car-
boxylase is activated by enhanced brain function, as shown by an
increase in CO
2
fixation with brain activity in the awake rat brain
(Öz et al., 2004). After termination of increased brain activity this
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Hertz Astrocytes, glutamine, glutamate, and GABA
effect may be reversed by increased glutamate degradation (see
also below). Pyruvate can also be formed glycogenolytically from
glycogen, previously generated from glucose (not shown), but
glycogen turnover and glycogenolysis are slow processes (Watan-
abe and Passonneau, 1973; Dienel et al., 2002; Öz et al., 2012),
except perhaps for occasional rapid bouts of glycogenolysis dur-
ing very short time periods (Hertz et al., 2003). Glycogenolysis
seems thus to be incapable of contributing much to metabolic
fluxes, although blockade of glycogenolysis during sensory stimu-
lation of awake rats does increase glucose utilization (Dienel et al.,
2007). As discussed below, glycogenolysis seems mainly to serve as
a fuel for signaling pathways, which are activated by stimulation
of glycogenolysis, either by increased extracellular K
+
concentra-
tions (Hof et al., 1988) or transmitter effects (Magistretti, 1988;
Subbarao and Hertz, 1990).
As illustrated in Table 1 the rate of flux in the glutamine–
glutamate (GABA) cycle in normal rat brain cortex is only slightly
lower than that of neuronal glucose oxidation (Sibson et al., 1998;
Rothman et al., 2011, 2012; Hyder et al., 2013). Publications by
these authors also show that the slight difference between the two
fluxes is due to the persistence during deep anesthesia of a small
amount of glucose oxidation but no glutamine–glutamate (GABA)
cycling, whereas there is an approximately 1:1 ratio between the
two parameters under all other conditions. This includes brain
stimulation (Chhina et al., 2001; Patel et al., 2004).
Stimulated brain activity is accompanied by a small immedi-
ate increase in glutamate content, associated with a quantitatively
similar decrease in content of aspartate and with a slower decrease
in content of glutamine (Dienel et al., 2002; Mangia et al., 2007;
Lin et al., 2012). The matched increase in glutamate and decrease
in aspartate may suggest an activity-induced alteration in relative
distribution of these two amino acids in their association with the
malate–aspartate shuttle (MAS) (Mangia et al., 2012). However, a
larger increase in glutamate content without concomitant decrease
in aspartate observed in an epileptic patient almost certainly rep-
resents increased de novo synthesis (Mangia et al., 2012). The same
probably applies to a short-lasting increase in glutamate, together
with a similar increase in glutamine (Figure 2) and aspartate (not
shown) during learning (Hertz et al., 2003; Gibbs et al., 2007). The
rapid subsequent return to normal amino acid levels is most likely
brought about by enhanced degradation.
Oxidative metabolism in astrocytes is a sine qua non-for opera-
tion of the glutamine–glutamate (GABA) cycle. Pioneering studies
Table 1 | Approximate metabolic rates in the non-anesthetized brain
cortex from a multitude of
13
C-NMR studies cited in text.
Parameter µmol/g wet wt per min
Rate of glucose oxidation 0.7
Rate of brain glucose utilization in astrocytes 20% of 0.7 = 0.14
Rate of glutamine–glutamate (GABA) cycle 0.6
Pyruvate carboxylase-mediated flux 50% of 0.14 = 0.07
Rate of glycogenolysis (Öz et al., 2012) 0.003
Except for glycogenolysis, measured in humans, the values apply to the rat brain.
For more details, see references cited in the text.
early in this century (Gruetter et al., 2001; Lebon et al., 2002)
showed that neurons account for up to 75% of oxidative glu-
cose metabolism in the living brain and that astrocytes contribute
most of the rest. These studies have been consistently and repeat-
edly confirmed in both human and rodent brain, and many of the
rates are tabulated by Hertz (2011b). Since the volume occupied
by astrocytes is similar to, or smaller, than the relative contribu-
tion of these cells to energy metabolism, their rate of oxidative
metabolism per cell volume must be as high, if not higher, than
that of neurons (Hertz, 2011b). This conclusion is consistent with
an at least similarly high expression of most enzymes involved
in oxidative metabolism of glucose in astrocytic as in neuronal
cell fractions freshly obtained from the mouse brain (Lovatt et al.,
2007).
THE GLUCOSE-TO-GLUTAMATE PATHWAY
In cultured cerebellar astrocytes conversion of glutamate to α-
ketoglutarate at least mainly occurs via a transamination (Wester-
gaard et al., 1996). This is consistent with a recent in vivo study by
Pardo et al. (2011), which established that the contents of gluta-
mate and glutamine in cultured astrocytes increase by 50% in the
presence of aspartate at a concentration of 100 µM, but not in
the presence of alanine or leucine. On the basis of this finding the
authors suggested the pathway shown in Figure 3A, according to
which glucose-derived α-ketoglutarate leaves the astrocytic TCA
cycle in exchange with malate, generated via oxaloacetate (OAA),
which in turn had been formed from aspartate in a transamina-
tion process. Subsequently OAA is reduced to malate (MAL), with
concomitant oxidation of NADH to NAD
+
. The entire process
requires operation of the α-ketoglutarate/malate exchanger (OGC
in Figure 3A), but not of the aspartate/glutamate exchanger
FIGURE 2 | Learning-induced changes in glutamate and glutamine
content in the equivalent of the mammalian brain cortex in day-old
chicken. Pre-learning contents are indicated by open symbols and
post-learning contents with filled-in symbols. From Hertz et al. (2003).
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Hertz Astrocytes, glutamine, glutamate, and GABA
FIGURE 3 | (A) Cartoon describing metabolic pathway from pyruvate to
glutamate/glutamine in astrocytes, as suggested by Pardo et al. (2011).
Joint pyruvate carboxylase and pyruvate dehydrogenase activation
generates a “new” molecule of citrate as described above. Citrate-derived
α-ketoglutarate exiting the mitochondrial membrane leaves the astrocytic
TCA cycle and is transaminated with aspartate to form glutamate, with
concomitant oxaloacetate (OAA) formation from aspartate. The
mitochondrial exit of α-ketoglutarate occurs via the ketoglutarate/malate
exchanger, generally acknowledged to be expressed in astrocytes, and the
cytosolic malate with which is exchanged, is generated via
NADH-supported reduction of aspartate-generated oxaloacetate. No
aralar-requiring aspartate/glutamate exchanger (Slc1) activity is involved. (B)
Proposed expansion by Hertz (2011a) of the model shown in (A). The
expanded model shows astrocytic production of glutamine (pathway 1), its
transfer to glutamatergic neurons (without indication of any extracellular
space, because there is no other function for extracellular glutamine than
astrocyte-to-neuron transfer) and extracellular release as the transmitter
glutamate (pathway 2), and subsequent reuptake of glutamate and oxidative
metabolism in astrocytes (pathway 3), with connections between pathways
1 and 3 shown as pathway 4. Biosynthesis of glutamine is shown in brown
and metabolic degradation of glutamate in blue. Redox shuttling and
astrocytic release of glutamine and uptake of glutamate are shown in black,
and neuronal uptake of glutamine, hydrolysis to glutamate, and its release is
shown in red. Reactions involving or resulting from transamination between
aspartate and oxaloacetate (OAA) are shown in green. Small blue oval is
pyruvate carrier into mitochondria and small purple oval malate carrier out
from mitochondria. AGC1, aspartate/glutamate exchanger, aralar; α-KG,
α-ketoglutarate; Glc, glucose; Pyr, pyruvate; OGC, malate/α-ketoglutarate
exchanger. It should be noted that (i) aralar activity is required initially for
reversal of cytosolic NAD
+
/NADH changes occurring during the one
oxidative process occurring during pyruvate formation, but subsequently not
in astrocytes until the oxidation of glutamate, probably allowing rapid
glutamate synthesis, and (ii) all reactions are stoichiometrically accounted
for (A) From Pardo et al. (2011); (B) From Hertz (2011a).
AGC, in brain AGC1. On the basis of their own and previous
immunocytochemical observations in brain tissue by themselves
and others (Ramos et al., 2003; Berkich et al., 2007), Pardo et al.
(2011) regarded this exchanger as absent or sparsely expressed in
astrocytes because of deficient expression of aralar, a necessary
component of AGC1.
Subsequently Hertz (2011a), suggested that (i) the reduction
of oxaloacetate to malate was a necessary compensatory conse-
quence of the reduction of NAD
+
to NADH during the one
oxidative process during glycolysis (glyceraldehyde-3-phosphate
to 1-3-biphosphoglycerate), without which normally no produc-
tion of α-ketoglutarate can occur from glucose, and (ii) that
aspartate, formed from OAA in astrocytes when glutamate dur-
ing its oxidation is transaminated to α-ketoglutarate, supplied the
needed aspartate, as illustrated in Figure 3B. The latter suggestion
required exit to the cytosol of mitochondrially located aspartate
via the aralar-dependent AGC1 in the MAS. The involvement of
the MAS during glutamate oxidation, but not during its synthesis
(Figure 3A) might contribute to the development of MAS-based
alteration in glutamate/aspartate ratio during brain activation sug-
gested by Mangia et al. (2012). The suggestion of malate–aspartate
participation in Figure 3B was felt to be justified by the finding by
Lovatt et al. (2007) of equal expression of mRNA for aralar, deter-
mined by microarray analysis, in freshly isolated astrocytes and
neurons. Moreover, it was calculated (based on data by Berkich
et al., 2007) that the aralar expression found by Pardo et al. (2011)
sufficed to produce enough aralar for the proposed model to
function, although malate–aspartate cycle activity needed for syn-
thesis of α-ketoglutarate at the beginning of the process increase
demands. Equally high levels of mRNA aralar expression is astro-
cytes were later confirmed, and its protein expression (Figure 4)
shown in freshly separated astrocytes and neurons from isolated
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Hertz Astrocytes, glutamine, glutamate, and GABA
FIGURE 4 | Protein expression of aralar in neuronal and astrocytic cell
fractions are similar and develop at identical rates. Neuronal and
astrocytic cell fractions were gently isolated from two mouse strains, one
expressing a neuronal marker with a specific fluorescence and the second
expressing an astrocytic fluorescent signal (Lovatt et al., 2007). Samples
were applied to slab gels of 12% polyacrylamide, separated by
electrophoresis, and transferred to nitrocellulose membranes. After
blocking with 5% skim milk powder and washing, these membranes were
incubated for 2h at room temperature with the primary anti-aralar antibody
sc-271056 (from Santa Cruz Biotechnology, CA, USA) after dilution
(1 × 100), shown not to react with the related carrier citrin, and followed by
incubation with a goat anti-mouse HRP-conjugated secondary antibody,
also from Santa Cruz Biotechnology (dilution: 1 × 200) for 2 h at room
temperature. Anti β-actin (Sigma, St. Louis, MO, USA) was applied in the
same lanes as the aralar antibody to use β-actin as a housekeeping protein
providing an internal control for protein load. The intensities of both bands
on the blots were scanned, and the ratios between aralar and β-actin were
calculated and shown in the Figure. From Li et al. (2012b).
cell fractions (Li et al., 2012b). The separation procedure used
selects astrocytes indiscriminately, but among neurons it mainly
isolates glutamatergic projection neurons. These experiments also
demonstrated remarkably large differences in aralar expression in
young and mature animals. This finding was replicated in cul-
tured astrocytes, whereas homogeneous neuronal cultures are too
short-lived to provide meaningful results.
The model suggested in Figure 3B is consistent with the impor-
tant
13
C labeling data in the study by Pardo et al. (2011) in young
aralar
/
animals, showing incorporation of [
13
C]glucose into
glutamate but not into glutamine. This is because the absence of
aralar does not exclude mitochondrial glutamate synthesis, espe-
cially if a substantial amount of α-ketoglutarate is supplied from
non-glucose source in these young animals. However, in aralar
/
animals de-amidation of glutamine in neurons may be impaired
as will be discussed below, which may prevent glutamine synthesis
in astrocytes.
Formation of glutamate from glucose requires glycogenoly-
sis, both in the intact chicken brain (Gibbs et al., 2007) and in
cultured astrocytes (Sickmann et al., 2009). Absence of glycogen
phosphorylase in oligodendrocytes (Richter et al., 1996) therefore
is a powerful argument against functioning pyruvate carboxylase
activity in oligodendrocytes. The rate of glycogenolysis in brain
(Table 1) is not high enough that pyruvate derived from glycogen
could be used by the astrocytes as the sole source of pyruvate for
carboxylation. Most, although probably not all glucose oxidation
in astrocytes proceeds via glutamate formation, astrocytes account
for 20% of total glucose oxidation rate, or 0.14 µmol/g per min
(in the rat), and one half of glutamate formation (0.07 µmol/g per
min) occurs via pyruvate carboxylation (with the other one half
mediated by PDH). This exceeds the rate of glycogenolysis by at
least 10 times. Rather, as in the case of other astrocytic processes
requiring activation of specific signaling pathways (Xu et al., 2013),
glycogenolysis seems to be required for signaling processes needed
to activate pyruvate carboxylase activity. Glycogenolysis is stimu-
lated by even very small increases in extracellular K
+
concentra-
tions above their normal level (Hof et al., 1988), and in astrocyte
cultures pyruvate carboxylation is increased by an elevation of the
K
+
concentration in the medium (Kaufman and Driscoll, 1993).
Pyruvate carboxylation at least in other cell types (Garrison and
Borland, 1979) is also stimulated by noradrenaline, as is astrocytic
glycogenolysis (Magistretti, 1988; Subbarao and Hertz, 1990). This
does not mean that a very brief increase in glutamate content, as
shown in Figure 2 might not, at least partly, be derived from glyco-
gen,which showed a simultaneous precipitous and large fall (Hertz
et al., 2003).
Formation of glutamine from glutamate in the astrocytic
cytosol is in agreement with the astrocyte-specific expression
of glutamine synthetase (Norenberg and Martinez-Hernandez,
1979), with probable lack of expression in oligodendrocytes
confirmed by Derouiche (2004). In cultured astrocytes reduced
function of the glutamine synthetase after administration of its
inhibitor, methionine sulfoximine (MSO), causes an increase in
glutamate and aspartate formation, the latter probably reflecting
increased glutamate oxidation, when glutamine synthesis is inhib-
ited (Zwingmann et al., 1998). Increased content of aspartate in
brain slices during MSO inhibition has also been shown by Nicklas
(1983). Aspartate production by this route might under adverse
conditions supplement the aspartate needed for transamination,
when α-ketoglutarate is converted to glutamate (Figures 3A,B).
Chronic infusion of MSO into rat hippocampus increases gluta-
mate content, specifically in astrocytes, by almost 50%, but has
remarkably little effect on glutamate in synaptic endings (Perez
et al., 2012). The animals develop seizures, and the authors sug-
gested that the extracellular brain glutamate concentration had
become increased, perhaps due to excessive release of glutamate
and/or decreased extracellular clearance.
Glutamine can travel between gap-coupled astrocytes, and
the distance it reaches increases during brain activation (Cruz
et al., 2007). Different transporters have been proposed to direct
its transport from astrocytes to neurons, but it now appears
well established that glutamine release occurs via the amino
acid transporter SN1. This transporter is densely expressed in
astrocytic processes abutting glutamatergic and GABAergic neu-
rons (Boulland et al., 2002). Efflux through SN1 is increased
by acidic extracellular pH and by increased intracellular Na
+
concentrations (Bröer et al., 2002). Uptake of Na
+
in astro-
cytes during re-accumulation of excess extracellular K
+
from
the extracellular space after neuronal excitation (Xu et al., 2013)
might therefore increase glutamine release. Extracellular gluta-
mine is taken up into neurons by SAT1,2 (Kanamori and Ross,
2006; Blot et al., 2009; Jenstad et al., 2009). This topic is dis-
cussed in detail in the paper by Chaudhry et al. in this Research
Topic.
Although not shown in Figure 3B (for the sake of simplicity),
the subsequent de-amidation of glutamine to glutamate appears
to be somewhat complex, probably reflecting the subcellular local-
ization of the phosphate-activated glutaminase (PAG). In cultured
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Hertz Astrocytes, glutamine, glutamate, and GABA
FIGURE 5 | Metabolic pathway for conversion of glutamine to
glutamate in cultured cerebellar granule neurons. Glutamine enters the
intermembranaceus space from the cytosol (red arrow at bottom of Figure).
Here glutamate is formed by phosphate-activated glutaminase (PAG), but
instead of returning to the cytosol, it enters the mitochondrial lumen and is
transaminated to aspartate, coupled with transamination of oxaloacetate
(OAA) to α-ketoglutarate (α-KG). As in the malate–aspartate shuttle α-KG
exits the mitochondrial membrane in exchange with incoming malate, and
intramitochondrial malate is oxidized to oxaloacetate. The mitochondrially
generated aspartate is the source of the cytosolic malate exchanging with
α-KG after it has been transaminated to oxaloacetate and reduced to
malate. Two transmitochondrial carriers are involved, (1) the
glutamate/aspartate exchanger AGC1, shown by a red circle, which requires
aralar [undisputed presence in neurons, except in the aralar
/
mice studied
by Pardo et al. (2011), where glutamate synthesis may have been
abrogated], and (2) the α-ketoglutarate/malate carrier (OGC), shown by a
blue circle. As mentioned in the text this process is similar to that operating
in the malate–aspartate shuttle, with the exception that glutamate in the
latter originates in the cytosol, not in the mitochondrial intermembranaceus
space. The interactions between oxaloacetate and malate must be coupled
to conversion of NADH to NAD
+
in the cytosol and the reverse change in
the mitochondria (top of Figure). This contributes to the quantitative
correlation between glucose flux and glutamine/glutamate (GABA) cycle
activity at different activity ranges. Modified from Palaiologos et al. (1988).
glutamatergic neurons inhibitor studies have suggested the path-
way indicated in Figure 5 (Palaiologos et al., 1988). This Figure
shows conversion of glutamine to glutamate by PAG, followed
by a process similar to that occurring in the MAS, with the only
exception that the glutamate molecule involved does not originate
in the cytosol, but from PAG-activated de-amidation of glut-
amine in the intermembranaceus space of the mitochondrion.
This mechanism implies a concomitant mitochondrial reduc-
tion of NAD
+
to NADH, associated with malate oxidation to
OAA, cytosolic oxidation of NADH to NAD
+
, and reduction of
oxaloacetate to malate. Evidence that a similar process occurs in
freshly isolated mitochondria (Bak et al., 2008) and description
of perhaps even more complicated processes in GABAergic neu-
rons are discussed by Schousboe et al. in the present Research
Topic.
NH
+
4
released during the glutaminase reaction (and/or the
small amount of NH
3
present at physiological pH) may easily
traverse the outer, permeable mitochondrial membrane to reach
the neuronal cytosol. A large number of studies have attempted
to investigate ammonia transport from here to the astrocytic
cytosol, where it is needed for continuous glutamine produc-
tion. Many of these have focused on potential amino acid shut-
tles, capable of mediating this transport, but a recent review by
Rothman et al. (2012) has shown too little transport capacity
of these cycles in the brain in vivo to be entirely responsi-
ble for this function. This does not mean that they could not
have a back-up function. This conclusion may re-focus atten-
tion on channel- and/or transporter-mediated contributions to
efflux from neurons and influx into astrocytes (Benjamin, 1987).
A major ion extruder in neurons is the K
+
–Cl
co-transporter
KCC2 (Chamma et al., 2012; Löscher et al., 2013), and Marcaggi
and Coles (2000) has shown rapid ammonia exit from neurons in
the bee retina via a co-transporter. Fittingly, in cultured astrocytes
Nagaraja and Brookes (1998) showed that channel- and NKCC1-
mediated NH
+
4
uptake together accounted for an uptake, which
was similar in magnitude to the glutamate uptake in similar cul-
tures. At 1 mM extracellular NH
4
Cl it amounted to 30 nmol/mg
protein per min, which in vivo (100 g protein/g wet wt.) would
equal 3 µmol/g wet wt per min, or about one half of the in vivo
rate of the glutamine–glutamate (GABA) cycle (Table 1). At the
same time the cytosol became acidic. Reversal of intracellular aci-
dosis by NHE1 and NBCe1 acid extruders (Song et al., 2008, 2012)
would create extracellular acidosis, stimulating SN1-mediated
glutamine release. Na
+
,K
+
-ATPase activity is required both to
support NKCC1 function, since NKCC1 operates as a secondary
active transporter supported by Na
+
,K
+
-ATPase-generated ion
gradients, and to maintain conditions allowing inward channel-
mediated NH
+
4
transport. Inhibition of glutamine formation and
retention in rat brain slices by ouabain (Benjamin, 1987) might
therefore result from impairment of channel and transporter
activity.
Regardless of detailed mechanisms involved, ATP requirement
for glutamine synthesis and ammonia uptake in astrocytes and for
glutamine (or GABA) uptake in neurons makes neuronal trans-
mitter supply from astrocytes an approximately two–three times
more expensive process than neuronal production or uptake from
the extracellular fluid would have been.
RETURN AND RE-USE OF RELEASED GLUTAMATE
It is well established that virtually all released transmitter glu-
tamate is accumulated specifically into astrocytes by the two
transporters GLAST (EAAT1) and Glt1 (EAAT2) (Danbolt, 2001).
Claims to the contrary can generally be discounted as due to arti-
facts, e.g., homo- or hetero-exchange with intracellular amino
acids. There is also no doubt that intact brain tissue can oxi-
dize glutamate. Brain slices show higher respiratory activity during
incubation in a medium containing l-glutamate as the only sub-
strate than in the absence of any substrate (Dickens and Greville,
1935; Abadom and Scholefield, 1962). Nevertheless, according to
most authors (Lipsett and Crescitelli, 1950; Ghosh and Quastel,
1954) rodent brain slices incubated with l-glutamate show a lower
rate of oxygen consumption than during incubation with glucose
alone. These findings are consistent with glutamate being a meta-
bolic substrate that can be utilized by some, but not all cells, in the
tissue. A recently demonstrated ability of glutamate to decrease
the rate of glucose oxidation in incubated rat hippocampi (Torres
et al., 2013) is reproduced in Figure 6 and shows that oxidation of
glutamate can supply energy supporting not only the increased
demand created by its own uptake (e.g., McKenna, 2012), but
also demands normally fueled by glucose oxidation. The relative
Frontiers in Endocrinology | Cellular Endocrinology May 2013 | Volume 4 | Article 59 | 6
Hertz Astrocytes, glutamine, glutamate, and GABA
FIGURE 6 | Glucose oxidation rates of intact rat hippocampi, incubated
in tissue culture medium for 2 h with [U-
14
C] glucose. From Torres et al.
(2013).
low rates of apparent glucose oxidation in such experiments is
well known, partly due to the incubation in vitro and partly to
the late turns of the TCA cycle during which some the labeled
atoms are oxidized. Although it may be doubtful if glutamate
oxidation is necessary metabolically, it may be functionally very
important for complete disposal of glutamate in the brain in vivo,
with continuous glutamate de novo synthesis and degradation and
paucity/absence of other disposal or dilution routes.
In astrocyte cultures (Eriksson et al., 1995; Hertz and Hertz,
2003) l-glutamate is an excellent substrate for oxidative metab-
olism. The observation by Yu et al. (1982) that glutamate
oxidation is insensitive to the transamination inhibitor AOAA
and therefore initiated by GDH activity has been confirmed
by virtually all authors with the exception of Hutson et al.
(1998). However, this in vitro observation may not be valid
for the brain in vivo, and in glutamate-dehydrogenase knock-
out mice glutamate oxidation, determined in astrocyte cultures
is reduced, but absolutely not abolished (Frigerio et al., 2012).
Moreover, glutamate-dehydrogenase-mediated glutamate oxida-
tion disagrees with the observation by Balazs (1965) that in brain
mitochondria by far most of the glutamate conversion to α-
ketoglutarate is catalyzed by the aspartate aminotransferase, an
observation confirmed in both synaptic and non-synaptic mito-
chondria by Berkich et al. (2007). These observations raise the
question whether lack of some of the many mechanisms regu-
lating GDH activity (McKenna, 2011; Li et al., 2012a) may not
function in isolated mitochondria,or whether use of isolated astro-
cytes without the possibility of metabolic interactions between
glutamate synthesis ad glutamate oxidation may have caused the
GDH dependency (see also below). Observations by Wysmyk-
Cybula et al. (1991) in freshly isolated cerebral astrocytes and
by Rao and Murthy (1993) in isolated cerebellar astrocytes that
glutamate oxidation is mainly transaminase-dependent, support
the latter conclusion. Additional support may be provided by the
stimulation of glutamate production by aspartate shown by Pardo
et al. (2011), since transactivation during glutamate oxidation can
provide the needed aspartate (Figure 3B), but GDH-mediated α-
ketoglutarate cannot. In contrast to glutamate oxidation via GDH,
that by the aspartate transaminase also requiresuse of oxaloacetate,
in the proposed model generated during glutamate production
(labeled 4 close to the lower edge of Figure 3B).
Patients suffering from temporal lobe epilepsy, with sclerotic
astrocytes and neuronal loss, often display a combination of highly
elevated extracellular glutamate concentrations, also interictally,
interictal hypometabolism, reduced glutamine synthetase activity
and greatly reduced glutamine formation from glutamate (Petroff
et al., 2002a,b; Eid et al., 2012). As during chronic perfusion of
normal animals with MSO (Perez et al., 2012), the interictal ele-
vated glutamate may largely reflect higher cellular glutamate levels
within astrocytes, probably due to chronic impaired glutamine
synthetase function and perhaps also impaired oxidation, since
neuronal number and volume is reduced much more than total
tissue glutamate levels (D. L. Rothman, personal communication).
It would be interesting actually to determine extracellular gluta-
mate concentration in the animals treated by Perez et al. (2012),
since Exposito et al. (1994) previously showed that acute intrastri-
atal administration of MSO decreases the extracellular glutamate
concentration.
Glutamate synthesis and degradation in differently located
astrocytes was envisaged to present a possible problem in the
suggested interacting pathways of glutamate production and
oxidation with its exclusive use of aspartate aminotransferase
(Figure 3B) rather than GDH (Hertz, 2011a). It was discussed
that trans-astrocytic transport of co-factors and metabolites and
lactate formation may alleviate this problem, but this may not
always be sufficient, and lack of GABA return, and thus oxi-
dation, in astrocytes will aggravate the situation. Pronounced
expression in non-synaptic mitochondria of both AOAA- and
non-AOAA-sensitive glutamate oxidation pathways (Table 2) may
suggest a back-up function of the GDH. This would be con-
sistent with the findings by Balazs (1965) and Wysmyk-Cybula
et al. (1991) that transaminase-dependent glutamate oxidation
accounted for most, but not all, glutamate oxidation. However,
in GDH 1 knock-out mice (only humans express GDH 2) most
functions, except the already mentioned reduced glutamate oxida-
tion in cultured astrocytes, are remarkably intact (Frigerio et al.,
2012). This applies to synaptic activity and long-term potentia-
tion (LTP), an observation which may exclude that GDH in intact
tissue should be essential for glutamate oxidation in astrocytes.
However, glutaminase activity, glutamine content, and expression
of the two astrocytic transporters Glt1 and GLAST were mod-
erately increased in the knock-out animals. These alterations are
consistent with, but do not prove, that glutamate production is
rendered slightly more difficult, but certainly not abolished, when
GDH is silenced.
Based on the sum of cited evidence it appears reasonable to
suggest initiation of glutamate oxidation by transamination as the
default pathway, but participation of GDH may occur in situa-
tions when an insufficient match between glutamate biosynthesis
and degradation is not possible because of different rates, too
long distances between biosynthesis and degradation sites, or
prevailing conditions in vitro. The ability of the transaminase-
mediated pathway to provide aspartate for glutamate synthesis
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Hertz Astrocytes, glutamine, glutamate, and GABA
Table 2 | Oxygen consumption rates as indications of kinetics of glutamate-dehydrogenase and aspartate–glutamate transferase activities in
non-synaptic mitochondria from the rat.
High-affinity Km (mM) High-affinity V max
(µmol O
2
/min per g)
Low-affinity Km (mM) Low-affinity V max
(µmol O
2
/min per g)
Glutamate + malate 0.26 3.75 1.3 7.65
Glutamate + malate + AOAA 0.19 2.45
From Lai and Clark (1976).
is a strong argument for its function, and the increased aspar-
tate content in brain slices after administration of MSO found
by Perez et al. (2012) might partly reflect a decreased glutamate
oxidation. In addition to the pathway suggested in Figure 3A,
Hutson et al. (1998) has proposed a different pathway for aspartate
transaminase-mediated glutamate oxidation. This model relies on
many complex interactions, including branched chain amino acid
cycling, but Rothman et al. (2012) concluded that it would be able
to function in the mammalian brain in vivo, since in vivo data
showed high enough fluxes and enzyme expression levels for this
to be possible.
The total of four conversions of NAD
+
to NADH in the joint
glutamate synthesis/oxidation process suggested [two in astro-
cytes during the production of α-ketoglutarate and one during
glutamate oxidation (Figure 3B), together with one in neurons
(Figure 5)] may explain the close to 1:1 ratio between rates
of glucose oxidation and of glutamine/glutamate (GABA) cycle
flux. The repeated NAD
+
/NADH conversions also suggest much
more complicated and repeated oxidation/reduction responses
in brain cortex during neuronal and astrocytic activities than
previously considered (e.g., Kasischke et al., 2004). It was also
mentioned above that over 80% of neuronal oxidative ATP produc-
tion is coupled to neuronal signaling even in the absence of specific
stimulation (Rothman et al., 2011). What is peculiar, though, is
that three of the four NAD
+
/NADH conversions occur in astro-
cytes. The initial formations of α-ketoglutarate from glucose do
require malate–aspartate cycle activity, but there seem to be agree-
ment that only 15–20% of glutamine–glutamate (GABA) cycle
activity is connected with de novo synthesis of glutamate. Could
glutamate destined for oxidation and for glutamine synthesis be
segregated, and the pathways suggested in Figure 3A apply only to
the former? This question makes it so important to determine the
pathway(s) for glutamate degradation not only in isolated astro-
cytes but also in intact brain tissue (see papers by McKenna and
by Whitelaw and Robinson in this Research Topic). Would studies
of metabolism of labeled glutamate (with appropriate receptor
antagonists) at least in brain slices be useful? Also, could glu-
tamate conversion to α-ketoglutarate be catalyzed by aspartate
aminotransferase in one potential subfraction and by GDH in the
other? These are critical questions. Finally, even if virtually all glu-
tamate is oxidized in astrocytes, pyruvate formed from malate
could be converted to lactate and transferred to neurons, but
most evidence does not support lactate transfer from astrocytes
to neurons. Nevertheless, astrocytes do contribute energetically to
glutamine, glutamate, and GABA homeostasis (by uptake and glu-
tamine synthesis) to a similar degree as neurons (cellular uptake
and vesicular accumulation of glutamate and GABA). The same is
the case for clearance of excess extracellular K
+
(astrocytic uptake
followed by release and neuronal uptake after extracellular K
+
clearance, and they seem even to be responsible for the post-
excitatory undershoot in extracellular K
+
concentration (Hertz
et al., 2013). Since glutamate is the major excitatory transmitter, its
release will cause efflux of neuronal K
+
, followed by extracellular
K
+
clearance and, after extensive stimulation, also post-excitatory
undershoot in extracellular K
+
concentration. Thus, glutamate-
mediated, K
+
-associated excitatory activity may increase astro-
cytic energy demands as much as neuronal. One may wonder (i)
if this dual uptake (and thus double metabolic billing), apparently
of both glutamate and K
+
is the major reason for the extremely
high energy metabolism in brain, and (ii) whether signaling pos-
sibilities especially exist during transport through the astrocytic
syncytium.
RETURN AND RE-USE OF RELEASED GABA
Although GABA contributes at most 10–20% of fluxes in the
glutamine–glutamate (GABA) shuttle, GABAergic signaling is
essential in the regulation of endocrine functions and in brain
information processing. It is therefore an important question
whether oxidation of GABA in astrocytes might also be associ-
ated with the biosynthetic pathway. Pathways for such a potential
interaction are suggested in Figure 7. The synthetic pathway is
identical to that for glutamate up till GABA formation from gluta-
mate, which is discussed by Schousboe et al. in this Research Topic.
During metabolism the most uncertain point is how GABA enters
the mitochondrion. The model suggests an exchange with gluta-
mate, but no mitochondrial GABA/glutamate exchanger is known
in mammalian cells, although an exchanger has been demon-
strated in plants (Michaeli et al., 2011), and the cell membrane of
prokaryotes (which have no mitochondria) express a glutamate–
GABA exchanger, GadC (Ma et al., 2012). After its mitochondr-
ial exit cytosolic glutamate follows a similar pathway as during
glutamate oxidation. The relatively low content of glutamate in
astrocytes (Storm-Mathisen and Ottersen, 1983; Storm-Mathisen
et al., 1992), and thus also in astrocytic mitochondria, will not
be affected, because mitochondrial glutamate is re-established via
MAS-mediated uptake, initial transamination to α-ketoglutarate,
coupled to transamination of oxaloacetate, and a second transam-
ination coupled to GABA transaminase-mediated formation of
SSA. The steps of GABA transaminations are conventional, as
is its subsequent complete oxidation via succinate, malate, and
pyruvate.
An increase in aspartate but a decrease in glutamate and glu-
tamine contents and synthesis has been observed in brain cortex
from 17-day-old succinic-semialdehyde dehydrogenase-deficient
Frontiers in Endocrinology | Cellular Endocrinology May 2013 | Volume 4 | Article 59 | 8
Hertz Astrocytes, glutamine, glutamate, and GABA
FIGURE 7 | Proposed model for cytosolic–mitochondrial trafficking
associated with astrocytic production of glutamine (pathway 1), its
transfer to GABAergic neurons (without indication of any extracellular
space, because astrocyte-to-neuron transfer is the only major function
for extracellular glutamine), neuronal GABA formation via glutamate
(without details), and extracellular release as transmitter GABA
(pathway 2), with subsequent reuptake of GABA and oxidative
metabolism in astrocytes (pathway 3), and connections between
pathways 1 and 3 shown as pathway 4. Biosynthesis of glutamine is
shown in brown and metabolic degradation of GABA in blue. GABA is
suggested to enter the mitochondria in exchange with glutamate, although
no such exchanger is presently known, and subsequently be transaminated
to succinic-semialdehyde and oxidized to succinate. α-KG is initially used for
the transamination, but later re-generated during metabolism of glutamate
entering the mitochondria and metabolized as previously suggested for
transmitter glutamate, with the mitochondrial malate extruder shown as a
small purple oval. Redox shuttling and astrocytic release of glutamine and
uptake of GABA are shown in black, and neuronal uptake of glutamine,
followed by GABA formation and release in red. Reactions involving or
resulting from transamination between aspartate and oxaloacetate (OAA)
are shown in green. Note that (i) aralar activity is required not only for the
initial production of each of two molecules of pyruvate (light blue oval in
pathway 1), but also during the re-entry of glutamate into the TCA cycle and
for the final entry of pyruvate into the mitochondria (light blue oval in
pathway 3), (ii) all reactions are stoichiometrically accounted for, although
only when GABA synthesis and oxidation are integrated. AGC1,
aspartate/glutamate exchanger, aralar; α-KG, α-ketoglutarate; Glc, glucose;
Pyr, pyruvate; OGC, malate/α-ketoglutarate exchanger.
mice together with a pronounced decrease (40%) in glutamine
synthetase expression, whereas GABA production was virtu-
ally unaffected (Chowdhury et al., 2007b). From Figures 3A,B
follows that reduction of glutamate production, including ini-
tial transamination from α-ketoglutarate might explain both
the increase in aspartate content and the reduced glutamine
synthetase expression. On the other hand deficient GABA
transamination should decrease aspartate production in the return
route. Although degradation-synthesis coupling would be consis-
tent with Figures 3A,B, it is also unexplained why metabolism of
GABA should be so important for glutamate production, since
GABA in adult animals contributes so relatively little to the return
flux toward astrocytes (V
cyc
). However, by necessity 17-day-old
mice were used (the gene-deficient animals die around day 21). As
will be discussed below, the glutamine–glutamate (GABA) cycle is
not fully developed in these immature animals. Moreover, in rat
brain slices both GABA and glutamate uptake and release rates
show very rapid and pronounced quantitative fluctuations during
early development (Schousboe et al., 1976), and GABA fluxes may
have been considerable in 16-day-old animals.
Metabolism of GABA along the pathway suggested in Figure 7
would eliminate a need for uptake of exogenous aspartate and
its conversion to malate in astrocytes in an aspartate-dependent
pathway model for GABA formation suggested by LaNoue et al.
(2007). Such a pathway is not likely to operate, since aspar-
tate itself must be synthesized in an astrocyte–neuron meta-
bolic co-operation. Also, in contrast to glutamate, aspartate can-
not sustain its own uptake in cultured astrocytes by oxidative
metabolism (Peng et al., 2001), as it should have been able to
do in order for the subsequent oxaloacetate-to-malate reduc-
tion suggested by LaNoue et al. (2007) to occur. These models
were based on the assumption that astrocytes express no aralar.
The repeated findings of aralar mRNA together with the addi-
tional demonstration of its protein expression in freshly isolated
astrocytes probably mean that they can now be regarded as
outdated.
HOW DOES THE BRAIN MANAGE BEFORE THE
DEVELOPMENT OF THE GLUTAMINE–GLUTAMATE (GABA)
CYCLE?
The strikingly slow development of full aralar expression in rat
brain and isolated brain cells (Figure 4) probably mainly reflects
that gliogenesis in the rodent cerebral cortex is mainly postnatal
(Altman, 1969b). A glial cell population, predominantly contain-
ing astrocytes, expands in the rodent brain cortex during the first
3 weeks of postnatal development, largely by local division (Ge
et al., 2012). This is in contrast to a virtually completed neu-
rogenesis at birth (Altman, 1969a; Bhardwaj et al., 2006). Mori
et al. (1970) and Schousboe (1972) took advantage of the develop-
mental difference between neurogenesis and gliogenesis to regard
measured cell division in brain cortical tissue from postnatal day
6 and onward (by incorporation of label from [
14
C]thymidine)
as mainly reflecting formation of astrocytes. This appears jus-
tified, since simultaneous proliferation of oligodendrocytes and
vascular cells probably contribute less to total volume in gray mat-
ter than astrocytes and may be less condensed time-wise. Both
groups found similarly intense and virtually constant cell prolif-
eration rate for slightly more than one subsequent week, followed
by its abrupt termination around postnatal day 15 (Figure 8A).
The cell proliferation is accompanied by huge increases in brain
weight and total DNA, also stopping around day 14 (Figure 8B).
The postnatal gliogenesis is accompanied and followed by many
biochemical alterations. As could be expected, the activity of core
astrocyte-specific enzymes depend upon the formation of astro-
cytes, although some enzymes that later become astrocyte-specific
are neuronal during early development. The activity of the pyru-
vate carboxylase is very low in 8-day-old rat brain and only reaches
adult activity after >30 days (Wilbur and Patel, 1974) (Figure 9). A
fivefold higher pyruvate carboxylase activity in adult mouse brain
than in newborn mouse brain was confirmed by Yu et al. (1983),
and Yu (1984), showed a 10- to 15-fold increase in the activity
of this enzyme in astrocyte cultures between the ages of 1 and
3 weeks. This contrasts a much faster development of glutamate
www.frontiersin.org May 2013 | Volume 4 | Article 59 | 9
Hertz Astrocytes, glutamine, glutamate, and GABA
FIGURE 8 | (A) Rate of DNA synthesis, measured in unincubated brain
slices by incorporation
14
C after [
14
C]thymidine exposure 12 h earlier. From
Schousboe (1972). (B) Developmental increase in weight and DNA content
of rat brain. From Mori et al. (1970).
FIGURE 9 | Decarboxylation of [l-14C]pyruvate by pyruvate
dehydrogenase and the fixation of HCO
3
by pyruvate carboxylase in
rat brain homogenates obtained from animals of different ages
between birth and adulthood. From Wilbur and Patel (1974).
uptake in cultured astrocytes,which is quite pronounced,although
not mature, in 1-week-old cultures (Figure 10A). The activity of
glutamine synthetase increases steeply during all the first 3 weeks
of development both in cultured astrocytes and in brain in vivo
(Hertz et al., 1978c; Patel et al., 1982). Glycogen content as well as
activity of its degrading enzyme, glycogen phosphorylase, are low
in brain at birth and increase during early postnatal development
(Ferris and Himwich, 1946; Folbergrová, 1995).
The activities of many enzymes that are not specific for astro-
cytes also show drastic increases in activity during this period.
This applies to glucose-metabolizing enzymes (e.g., hexokinase,
aldolase, lactate dehydrogenase, phosphofructokinase, and PDH),
which increase considerably in activity between the ages of 15
and 30 days (Takagaki, 1974; Wilbur and Patel, 1974; Land et al.,
1977; Leong and Clark, 1984; – see also Figure 9). Synaptic mito-
chondria mature earlier (Almeida et al., 1995) than non-synaptic
mitochondria (Bates et al., 1994), and cytosolic malate dehy-
drogenase (MDHc), which operates in the MAS but not in the
TCA cycle, matures much more slowly than mitochondrial malate
FIGURE 10 | Developmental changes in glutamate uptake in primary
cultures of mouse astrocytes (A) and cerebral cortical neurons (B). (A)
open circles, open squares and filled circles: 7, 18 and 30 days in culture; (B)
open triangles 4 and closed triangles 8 days in culture. From Peng (1995).
dehydrogenase (MDHm), which functions both in the MAS
(Figure 1) and in the TCA cycle (Malik et al., 1993). Glutamate-
metabolizing enzymes also show changes during the early post-
natal period. Thus, the glutamate dehydrogenase activity falls
between the ages of 2 and 3 weeks in astrocyte cultures, whereas
that of aspartate aminotransferase increases (Hertz et al., 1978c).
These observations point toward a slow postnatal development
of the glutamine–glutamate (GABA) cycle, and perhaps decreased
importance of GDH.
A three fourfold increase in cycle flux has been shown by
13
C-
NMR in rat brain cortex by Chowdhury et al. (2007a) between
postnatal days 10 and 30, and energy metabolism in the gluta-
matergic and GABAergic neurons increased proportionately with
cycle flux, leading to an threefold increase in TCA cycle activ-
ity between postnatal days 10 and 30. Since, as was illustrated
in Figure 8B, the amount of respiring brain tissue also increases
hugely, total rate of energy metabolism in the rat brain cor-
tex must increase about 10 times within these 20 days. That a
small amount of astrocytic activity did occur, even at day 10
is indicated by the finding of detectable, although low incorpo-
ration of label from the astrocyte-specific substrate acetate into
glutamate and GABA. Thus, postnatal day 10 must be close to
the beginning of neuronal–astrocytic interactions involved in the
glutamine–glutamate (GABA) cycle. Re-analysis of the acetate
data may allow determination of the developmental pattern also
of astrocytic TCA cycle flux (D. L. Rothman and K. L. Behar,
Frontiers in Endocrinology | Cellular Endocrinology May 2013 | Volume 4 | Article 59 | 10
Hertz Astrocytes, glutamine, glutamate, and GABA
personal communication). Measuring rapid incorporation of
14
C
from glucose into amino acids in rat brain, which also is an indi-
cation of glutamine–glutamate (GABA) cycle function, at many
different developmental stages, Gaitonde and Richter (1966) and
Patel and Balázs (1970) had also found a sharp increase between
postnatal days 10 and 20, and that a maximum was not reached
until around postnatal day 25. Maximum metabolic compart-
mentation between glutamate and glutamine, another indicator
of glutamine–glutamate cycle activity was not found until a few
days later. Thus old-style biochemical studies and cutting-edge
13
C-NMR determinations have similarly identified the time period
during which the glutamine–glutamate (GABA) cycle develops in
the rat to between postnatal days 10 and 30.
Other aspects of TCA cycle function in brain are com-
pleted around postnatal day 15, indicated by maximum oxidative
response in rat brain slices to stimuli at this age (Holtzman et al.,
1982), and by sensory-evoked increases in brain glucose utiliza-
tion in vivo by day 10 in barrel cortex (Melzer et al., 1994) and
between days 13 to 18 in auditory and visual areas (Nehlig
and de Vasconcelos, 1993). A functioning brain cortex is obviously
working at that time, which is consistent with the completion of
neurogenesis except at a few specific locations (Altman, 1969a).
This is also exemplified by active GABAergic signaling at early
neonatal stages (Lauder et al., 1986), and glutamatergic activa-
tion of synchronized spike waves in 3–5-day-old rats (Seki et al.,
2012). Astrocytes are among the targets of glutamatergic signal-
ing, which plays a role during astrocytic differentiation (Oppelt
et al., 2004; Stipursky et al., 2012; Sun et al., 2013). Such an
ontogenetic development from a purely neuronal nervous sys-
tem to a neuronal–glial system also occurs during phylogenesis
(Reichenbach and Pannicke, 2008). It is generally accepted that
mammalian brain function should be studied in mammals, but
far too often tissues from rats or mice younger than 4 weeks are
studied.
In the absence of pyruvate carboxylase activity during early
postnatal development (Figure 9) glutamate synthesis within the
brain must be replaced by import of glutamate or a precursor. A
relatively fast uptake of glutamine, glutamate, and GABA from the
systemic circulation occurs across the blood-brain barrier at this
age (Pardridge and Mietus, 1982; Al-Sarraf et al., 1997; Al-Sarraf,
2002), and it might be the source of neuronal amino acid transmit-
ters.Also,during early postnatal development GLT1 is expressed in
neurons (Shibata et al., 1996), although it later becomes astrocyte-
specific. Glutamate oxidation by neurons occurs at much higher
rates during early development, when little if any glutamate can
be metabolized by astrocytes. This is illustrated in Figure 10B,
showing a 50% reduction in rate of glutamate uptake in the glu-
tamatergic cerebellar granule neurons between the ages of 4 and
8 days in culture (Peng, 1995). Similarly, other accounts of neu-
ronal ability to oxidize glutamate may reflect the young age of the
neurons (Olstad et al., 2007).
The large increase in metabolic demand during the first post-
natal month may partly reflect that instead of purely neuronal
uptake of glutamate and GABA during very early postnatal devel-
opment, virtually all glutamate and some GABA now becomes
accumulated twice. The first uptake is of glutamate and GABA
into astrocytes, where a large fraction is converted to glutamine in
FIGURE 11 | Developmental changes in synaptic density in frontal
gyrus, peaking at 12–15 months of age, and before or during
adolescence decreasing to similar levels as in the newborn. From
Huttenlocher (1979).
an energy-requiring process, and the second uptake is that of glut-
amine into neurons. However, this alone cannot explain a 10-fold
increase in energy demand, and many other processes may also
become very costly in energy. Thus, Na
+
,K
+
-ATPase-mediated
uptake of K
+
released during neuronal excitation into astrocytes
seems also to precede neuronal re-accumulation of K
+
in adult
astrocytes and brain (Xuet al.,2013). Since the astrocytic K
+
uptake
also depends on glycogenolysis-activated signaling (to facilitate
Na
+
access to the Na
+
,K
+
-ATPase’s Na
+
-sensitive site), it must
also be absent in neonatal brain. Greatly increased growth and
branching of neuronal processes also occur during the first year
(Conel, cited by Altman, 1967), and synaptic density increases, in
frontal gyrus peaking at 12–15 months of age (Figure 11) and
before or during adolescence decreasing to similar levels as in
the newborn (Huttenlocher, 1979; Huttenlocher and Dabholkar,
1997).
Since the early postnatal rat brain functions well on the much
smaller budget and without astrocytic participation in glutamate
and GABA biosynthesis, one may ask which advantages could be
associated with the developing dependence on astrocytic func-
tions. One potential answer may be enhanced precision and more
autonomy from the periphery. The changes may affect mental
function, including learning. The day-old chick is a precocious
animal. Its brain contains glycogen, and both glycogenolysis and
glutamate formation, events occurring during glutamate produc-
tion in the combined neuronal–astrocytic system, are essential
for learning of a one-trial aversive memory task (Gibbs et al.,
2007). Moreover, learning is inhibited by a glial-specific metabolic
inhibitor (Ng et al., 1992). In this task the bird learns to associate
a specific color on an artificial bead tainted with an aversively tast-
ing compound and as a result later refuses to peck at beads of this
color, even when untainted. Noradrenaline, released from locus
coeruleus, acts mainly on astrocytes during learning in the day-
old chick (Gibbs et al., 2008), but in the non-precocious newborn
rat pups odor learning occurs via a direct noradrenaline effect on
the neuronal mitral cells of the olfactory glomerulus (Wilson and
www.frontiersin.org May 2013 | Volume 4 | Article 59 | 11
Hertz Astrocytes, glutamine, glutamate, and GABA
FIGURE 12 | Developmental changes in glutamate content in
parietal/occipital gray matter measured by MRI in young children. On
percentage scale 100% refers to ages between 2 and 18 years. From Blüml
et al. (2013).
Sullivan, 1991). In odor learning, pairing with an aversive stimu-
lus has no negative effect until postnatal day 10–12 (Haroutunian
and Campbell, 1979; Camp and Rudy, 1988; Raineki et al., 2010).
This coincides with incipient glutamine–glutamate (GABA) cycle
function in the brain. It would be interesting to know if disruption
of glutamate production, and thus of glutamine supply to neu-
rons, by the inhibitor of glycogenolysis DAB would prevent the
effect of pairing with an aversive stimulus, but not odor learning
as such. Moreover, in the chick learning task, memory formation
can also be inhibited by pharmacological disruption of astrocytic
gap junction permeability, and trafficking of glutamine through
the astrocytic syncytium might play a role in connecting the visual
stimulus with the aversive gustatory signal. Would an inhibitor
of astrocytic gap junction affect odor learning as such and odor
learning paired with an aversive stimulus differently in 12-day-old
rats?
In human brain cortex neurogenesis is also completed at birth
in most brain regions (Bhardwaj et al., 2006), but as in many other
animals, cortical gliogenesis occurs peri- and postnatally (Marn-
Padilla, 2011). Recently glutamate content of different brain areas
was studied in human children of different ages by magnetic res-
onance spectroscopy (MRS) in several brain regions (Blüml et al.,
2013). Consistent with the late gliogenesis, the content of gluta-
mate quadrupled between birth and 2 years of age (Figure 12)
and subsequently remained stable at the adult value of about
12 mmol/kg. About one half of the change occurred during the
first 3 months of life and most of the rest between 3 and 12 months
of age.
Human learning may also provide a hint of functional gains
that are time-wise, and perhaps causally, related to the change
from a purely neuronal cerebral cortex to a cerebral cortex with
the costly neuronal–astrocytic interaction, such as the glutamine–
glutamate (GABA) cycle. Although disagreeing about mecha-
nisms involved, both Rovee-Collier and Giles (2010) and Bauer
and Nelson (Bauer et al., 2003; Bauer, 2006) similarly describe
how early-maturing memory functions support gradual and exu-
berant learning of perceptual and motor skills, but memories
are fragile and short-lived, as exemplified by our inability to
recall events from early life. Only later-maturing modification(s)
can support long-lasting representations of contextually specific
events, relationships, temporal orders, and associations. Bauer
(2006, 2008) as well as Nelson (1995) consider these differ-
ences as due to the operation of two different memory systems,
implicit and explicit, with implicit memory being the uncon-
scious memory function involved in motor skills. The develop-
ment of explicit memory may occur via a pre-explicit system,
and Nelson (1995) suggested that the development of explicit
memory from implicit and pre-explicit memory may be associ-
ated with recruitment of additional specific brain structures. The
simultaneous development and maturation of astrocytic func-
tions and glutamine–glutamate (GABA) cycle operation together
with the importance of astrocytic metabolic processes for aver-
sive learning in the chick brain might suggest that the newly
established, ubiquitous metabolic interactions between neurons
and astrocytes could also play an important role in human
learning.
CONCLUDING REMARKS
The importance of the neuronal–astrocytic glutamine–glutamate
(GABA) cycle in cerebral cortex is shown by the magnitude of this
flux, equaling total rate of neuronal glucose oxidation. Although
85% of the cycle “only” serves to return previously released neu-
rotransmitter glutamate and GABA, astrocytes contribute actively
by accumulating the transmitters and converting them to gluta-
mine, which can travel through gap junction-coupled astrocytes
before its release. The astrocytic participation in the remain-
ing 15% of the flux is even greater. They maintain an equilib-
rium between astrocytic biosynthesis and oxidative degradation,
which perhaps also is astrocytic and is capable of establishing
net synthesis or net degradation according to glutamate needs.
The requirement of aspartate for maximum glutamate synthesis
indicates that glutamate synthesis from α-ketoglutarate probably
occurs by transamination, and pathway models for interaction
between biosynthesis and oxidative degradation of both glutamate
and GABA suggest that transamination may also be the default
reaction for initiation of glutamate oxidation. Whether or not
glutamate oxidation mainly occurs in astrocytes is perhaps the
most important question, both for the viability of the suggested
models (Figures 3B and 7) and for the functional importance of
the glutamine–glutamate (GABA) cycle. Although the late devel-
opment and maturation of the cycle has been realized for more
than 40 years, the full consequences of this late and profound
alteration in brain metabolism remain to be fully understood.
They represent a development from purely neuronal processes to
integrated neuronal and astrocytic activities, which may be func-
tionally extremely important in brain function, including human
mental activities. In practical terms it also means that mature
brain function should not be investigated until full maturation
has occurred, which in mice and rats is at a postnatal age of
3–4 weeks.
ACKNOWLEDGMENT
Mr. Goncalo Vargas, Medicine Production Office, Frontiers is
thanked for his great help with the Figures.
Frontiers in Endocrinology | Cellular Endocrinology May 2013 | Volume 4 | Article 59 | 12
Hertz Astrocytes, glutamine, glutamate, and GABA
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Conflict of Interest Statement: The
authors declare that the research was
conducted in the absence of any com-
mercial or financial relationships that
could be construed as a potential con-
flict of interest.
Received: 28 March 2013; accepted: 02
May 2013; published online: 27 May
2013.
Citation: Hertz L (2013) The glutamate–
glutamine (GABA) cycle: importance
of late postnatal development and
potential reciprocal interactions
between biosynthesis and degrada-
tion. Front. Endocrinol. 4:59. doi:
10.3389/fendo.2013.00059
This article was submitted to Frontiers
in Cellular Endocrinology, a specialty of
Frontiers in Endocrinology.
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