A.C.H. Yu, L. Hertz, M.D.Norenberg. E. Sykova and S.G. Waxman (Eds.) Progress in Brain Research, Vol. 94 0 1992 Elsevier Science Publishers B.V. All rights reserved.

283

CHAPTER 24

Effects of monoamine transmitters on neurons and astrocytes: correlation between energy metabolism and intracellular messengers Leif Hertz and Liang Peng Department of Pharmacology, University of Saskatchewan, Saskatoon, Sask., S7N 0 WO Canada

Introduction

In the central nervous system (CNS), monoaminergic transmitters act on both neurons and glial cells, affecting energy metabolism, turnover of transmitter-related amino acids, potassium homeostasis, and/or calcium signaling. Glial cells outnumber neurons in the mammalian brain and constitute a considerable fraction of the volume in the cerebral cortex (Pope, 1978). The typical glial cell of the brain cortex is the astrocyte. Astrocytes are metabolically very active (Hertz and Peng, 1992), and metabolic and functional interactions between neurons and astrocytes seem to play a major role in brain function. In the brain, monoaminergic transmitters are released mainly from noradrenergic, serotonergic and dopaminergic fibers, extending from the brainstem over the entire cerebrum and cerebellum (e.g., Hertz, 1992b). A similar system seems to exist for histamine-releasing neurons, extending from the posterior hypothalamus (Schwartz et al., 1991; Steinbusch, 1991; Wada et al., 1991). Many of the fibers in these systems do not display regular synapses, but “varicosities”, from which the released transmitters reach their target cells: glial cells, microvessels and/or other neurons by diffusion (for references, see Schwartz et al., 1991; Hertz, 1992b). Since the transmitters are released from neurons, the effects on glial cells, and probably first and

foremost astrocytes, represent, by definition, a neuronal-astrocytic interaction. Many of the actions on astrocytes will, in turn, influence neuronal function. The general trend seems to be that the functional alterations induced in astrocytes, in turn, affect mainly energy metabolism, glutamatergic or GABAergic impulse transmission, and potassium homeostasis. It is likely that a similar chain of events can be initiated by cholinergic or purinergic effects on astrocytic function, but less information is presently available about such interactions. In the following, effects of monoamines on astrocytic metabolism, on turnover of glutamate and its precursors and metabolites, and on ion homeostasis will be discussed separately. Although mainly normal functional interactions will be described, some effects by drugs supposed to interact with binding sites for monoaminergic transmitters will be included, and a few examples will be given of pathological conditions where monoaminergic dysfunction appears to play a major role. Thereafter, some of the possible mechanisms by which these compounds affect cell function will be discussed. Effects

Energy metabolism Monoamines. Intravenous perfusion with adrena-

284

line and, under some conditions also noradrenaline, is able to stimulate energy metabolism in brain (King et al., 1952; Berntmanet al., 1978). Thelowpermeability of the blood-brain barrier to catecholamines, combined with the rapid metabolism of these compounds, makes this effect quite variable. However, intrathecal application of noradrenaline leads to a considerable increase in brain energy metabolism (MacKenzie et al., 1976a). Fibers supplying the entire cerebrum and cerebellum with noradrenergic innervation extend from locus coeruleus, a small brain-stem nucleus, and destruction of this nucleus influences some aspects of energy metabolism (Hertz, 1989a). Studies by the Sokoloff group on energy metabolism, measured by the aid of the 2deoxyglucose method (Sokoloff et al., 1977) in the rat brain in vivo, have shown that the a-adrenergic blockers phentolamine (both al- and a2antagonists), phenoxybenzamine (both al-and a2antagonists), and yohimbine (a2-antagonist) are all able to produce a depression of glucose utilization in many brain areas, especially in the neocortex. Phenoxybenzamine generally produces the greatest reduction in metabolism, but regional differences exist (Savaki et al., 1982). A few regions, including locus coeruleus, exhibit an increase in glucose utilization. Inoueet al. (1991) observedadecrease of glucose utilization in normal rats after administration of prazosin, an al-antagonist, but Mickley and Teitelbaum (1979) found that an increased deoxyglucose utilization produced by electrical stimulation of the lateral hypothalamus in rats is blocked by pre-treatment with yohimbine, an a2-antagonist. However, MacKenzie et al. (1976b) observed a decreased energy metabolism in the monkey brain as a result of application of propranolol, a @adrenergic antagonist. A recent report by Rogers et al. (1989), which also showed an increase in cerebral oxygen consumption in vivo by adrenergic stimulation, similarly concluded that this stimulation was mediated by @-receptors rather than a-receptors. These studies were carried out on newborn pigs, and species or age differences might exist. In spite of the unanimous finding of an enhancing effect of

noradrenaline on glucose utilization in normal brain, a functional decrease in glucose utilization in a whole hemisphere after localized cerebral damage (Pappius, 1991) can be prevented by pre-treatment with a-adrenergic antagonists (Inoue et al., 1991). In order to measure effects of noradrenaline on energy metabolism in different types of neural cells, several parameters have been studied in astrocytes,

GLUCOSE

T o r7

HALATE

VI

ACETYL CoA

PLURIPOTENT. NON-EXISTING. BRAIN CELL

Fig. 1. Metabolic pathways involved in glucose metabolism and formation of amino acid transmitters of the glutamate family (glutamate (Glu), y-aminobutyric acid (GABA), and aspartate (Asp)) in a non-existing "pluripotent brain cell". Glucose is degraded glycolytically to pyruvate (PYR),which under aerobic conditions can either enter the tricarboxylic acid (TCA) cycle via acetyl-CoA (formed by dehydrogenation, decarboxylation and condensation with coenzyme A) to be metabolized to CO, and water, or be condensed with CO, to provide net formation of oxaloacetate (OAA). I4CO2 production from [ l-'4C]pyruvate specifically indicates formation of acetyl-CoA, whereas [2-I4C] or [3-14C]pyruvateleads to formation of I4CO2 in the TCA cycle. Also indicated are interconversions between a-ketoglutarate (a-KG) and glutamate (by transamination or reductive aminatiodoxidative deamination) and formation of glutamine (Gln) from glutamate and hydrolysis of glutamine to glutamate. Note that within the central nervous system no single cell type is able to perform all reactions shown, since formation of GABA is restricted to GABAergic neurons and production of glutamine from glutamate or of oxaloacetate from pyruvate does not occur in neurons. Therefore, neuronal-astrocytic interactions are essential to maintain and regulate amino acid transmission in the CNS. (From Hertz and Peng, 1992.)

285

cerebellar granule cell neurons (a glutamatergic preparation), and cerebral cortical interneurons (a mainly GABAergic preparation) in primary cultures, which in our laboratory were obtained from the mouse brain. Production of labeled carbon dioxide was measured after application of several different substrates, labeled with I4C, and the net amount of lactate produced was used as an indication of the rate of glycolysis. Quantitative determination of metabolic fluxes are encumbered with more uncertainties, the more intermediates exist between the labeled substrate and the formation of I4CO, (unless the specific activities of these intermediates can be determined). In order to investigate effects of noradrenaline specifically on oxidative metabolism, i.e., tricarboxylic acid (TCA) cycle activity (Fig. l), we therefore measured the production of 14C0, from [U-I4C]aspartate rather than from glucose, which was added to the medium in its unlabeled form. Aspartate is rapidly taken up into astrocytes (Drejer et al., 1982) and transaminated to oxaloacetate, a tricarboxylic acid cycle constituent. This process represents a transamination between aspartate and oxaloacetate, which is accompanied by another transamination between glutamate and aketoglutarate. Therefore net oxidative metabolism is not affected, but the rate of CO, formation from aspartate is an indication of TCA cycle activity. In contrast, a stimulation of 14C02 production from labeled glucose (which is increased by noradrenaline) (Hertz, 1989a; Subbarao and Hertz, 1991) would not have distinguished between effects on aerobic glycolysis and on TCA cycle activity. Moreover, I4CO, production from labeled glucose is relatively slow and not rectilinear with time, reflecting the large pool of glucose derivatives in the cells and in the culture medium, into which the radioactivity is initially diluted. From Fig. 2 it can be seen that, in astrocytes, I4CO2 production from labeled aspartate is rectilinear with time and averages approximately 100 nmol aspartate/h per mg protein, corresponding to 400 nmol C02 production/h per mg protein (since one molecule aspartate contains four carbon

T

400

c 2

1

3

TIME (hr)

Fig. 2. Effect of noradrenaline (50 pM) on the formation of 14c0, from aspartate arta ate in primary cultures of astrocytes (.D; a.), cerebellar neurons (0; .a-)and cerebral cortical neurons (-A-; -A- ). Control values are shown by open symbols and CO, formation in the presence of noradrenaline by closed symbols. Each point represents the average & S.E.M. (From Subbarao and Hertz, 1990a.)

atoms). Noradrenaline stimulates the 14C0, formation by approximately 100% at all time periods ( P < 0.01). A dose-response curve showed an EC,, value for noradrenaline of approx. 5 x lo-’ M (Subbarao and Hertz, 1990a). The rate of CO, formation from aspartate in cerebellar neurons and cortical neurons is 1/2 and 1/10, respectively, of that in astrocytes (Fig. 2). These results are in agreement with earlier findings showing a higher rate of oxygen consumption and of CO, formation (Hertz et al., 1988; Hertz and Peng, 1992)and higher activities of many enzymes involved in energy metabolism (Rush et al., 1991) in astrocytes than in neurons. Noradrenaline caused no stimulation of CO, production in either cortical neurons or cerebellar granule cells, indicating the absence of any stimulatory effect of adrenergic agonists on oxidative metabolism in these cells. Although this probably indicates that neuronal metabolism is not affected, it cannot be ruled out that the neuronal cultures may be less intact metabolically than the astrocytic cultures (Hertz and Peng, 1992). Moreover, very transient effects might have been overlooked. By using [ l-14C]glutamate instead of labeled

286

--g

150

.z. c

za U

t

T

100

d

k

50

0

-U

5

0

AGONIST

Fig. 3. Effect of adrenergic agonists on CO, formation from [ I ''~]g~utarnatein primary cultures of astrocytes. Cultures were incubated with 5 pM of either noradrenaline (NA), isoproterenol (ISO), phenylephrine (PHE), or clonidine (CLON) or without any adrenergic agonist (NONE) for 1 h, and the I4CO2 formation was measured. Values are expressed as percentages of control CO, formation in the absence of any added adrenergic agonists. Eachvaluerepresentsthe average S.E.M. Thevalues indicated by asterisks are significantly different from the control ( P < 0.05). (From Subbarao and Hertz, 1991.)

aspartate, some information can be obtained about the localization of the stirnulatory effect within the TCA cycle. This is because formation of I4CO2 from [ ~ - W ] g ~ u t a m a in t e astrocytes is an unequivocal indication of decarboxylation of aketoglutarate (Fig. l), since it is specifically the carbon atom in the C-1 position which is oxidized in this reaction. From Fig. 3 it can be seen that 14C02 formation from [ l-14C]glutarnateis, indeed, increased in the presence of noradrenaline (NA). This shows that the a-ketoglutarate dehydrogenase step (which seems to be the rate-limiting reaction in the TCA cycle (Lai et al., 1977)) is affected, but does not exclude additional points of action. Fig. 3 also shows that phenylephrine (PHE), an al-agonist, and clonidine (CLON), an a2-agonist, exert a similar stimulation, whereas isoproterenol (ISO), a P-adrenergic agonist, has no effect. This subtype specificity was confirmed by investigating the effect of noradrenaline in the absence of any antagonist (NONE in Fig. 4) and in the presence of alprenolol (ALP), a 0-adrenergic antagonist, prazosine (PRAZ), an al-adrenergic antagonist,

and yohimbine (YOH), an a2-adrenergic antagonist. From Fig. 4 it can be seen that alprenolol did not affect the response, whereas the stimulation by noradrenaline was abolished in the presence of either prazosine or yohimbine. Thus, the conclusion reached on the basis of the effects of the agonists was confirmed. In addition to noradrenaline effects on the TCA cycle, the effect of noradrenaline on glycolysis (measured as lactate production), glycogenolysis (measured as release of radioactivity from [ 14C]glycogen) and hydrolysis of glutamine to glutamate (measured as incorporation rate of radioactivity from [14C]glutamine into glutamate) were also studied and found to be stimulatory (Table I). These reactions are all involved in energy production. In contrast, the rate of synthesis of glutamine from glutamate, an astrocyte-specific process, in-

150r

-50 -8""

t ~

T

T

~~

NONE

ALP

PRAZ

YOH

ANTAGONIST

Fig. 4. Effect of adrenergic antagonists on NA-stimulated CO, formation from [ ~ - ' ~ ~ l g l u t a r n ain t e primary cultures of astrocytes. Cultures were incubated for 1 h with 5 FM NA in the absence (NONE) and presence of an adrenergic antagonist (10 pM), and CO, formation was measured. Alprenolol (ALP) (a P-antagonist), prazosin (PRAZ) (an a,-antagonist), and yohimbine (YOH) (an a,-antagonist) were used in this study. The values are expressed as percentage of maximal effect (i.e., noradrenaline-stimulated CO, formation minus CO, formation in control cultures without NA is indicated as 100% effect). Each value represents the average S.E.M. The values indicated by asterisks are significantly different from the values obtained in the absence of any added adrenergic antagonists (P < 0.05), but not significantly different from zero, i.e., lack of any stirnulatory effect. (From Subbarao and Hertz, 1991.)

287

TABLE I Stimulation by noradrenaline (NA) and subtype specific agonists in primary cultures of astrocytes

Glycolysisa Pyruvate > acetyl C O A ~ Glycogenolysis' TCA cycle activitya Na' , K t ATPased Clutamine > glutamatee Glutamate uptake' GABA uptake'

+ + + + +

+ +

+ + +

+ +

+ + + + +

+

+ +

Subbarao and Hertz (1991); J.C.K. Lai, Y. Chen and L. Hertz (unpublished experiments); Subbarao and Hertz (1990b); I . Hajek and L. Hertz (unpublished experiments); R. Huang and L. Hertz (unpublished experiments); Hansson and Ronnback (1988). (Modified from Hertz, 1992b.)

a

volved in. the return to neurons of the carbon skeleton of accumulated glutamate (seep. 289), was not enhanced by noradrenaline (R. Huang and L. Hertz, unpublished experiments). From Table I it can be seen that different subtypes were involved in stimulation of different reactions. It might also be worthwhile to notice that noradrenaline stimulates

0

1

2 Concentratlon

3

4

5

( VM)

Fig. 5 . Dose-response curve for noradrenaline (NA) (W), isoproterenol (ISO) (0) and clonidine (CLON) ( 0 ) stimulated glycogenolysis in primary cultures of astrocytes. Each value represents the average k S.E.M. (From Subbarao and Hertz, 1990b.)

glycogenolysis more potently than does isoproterenol (0-adrenergic agonist), and that clonidine (cu2-adrenergicagonist) has less maximum effect than the two other compounds (Fig. 9, perhaps because it only acts on a subpopulation of the cells (see p. 294). The metabolism of pyruvate is a specially pivotal step in glucose degradation. Pyruvate can be converted to lactate or enter the TCA cycle via either oxidative decarboxylation and condensation with coenzyme A to form acetyl-CoA or by CO, fixation, leading to formation of oxaloacetate (Fig. 1). Lactate production, acetyl-CoA formation and further metabolism of acetyl-CoA in the TCA cycle occur in both neurons and astrocytes (Hertz et al., 1988; Hertz and Peng, 1992). Since utilization of glucose-derived acetyl-CoA in the TCA cycle is by far the most important pathway for energy production in brain (Sokoloff, this volume), it is important that formation of acetyl-CoA from pyruvate also is enhanced by noradrenaline (Tables I and 11). This stimulation isabolished by deletion of calciumfrom the incubation medium with a simultaneous increase of the magnesium concentration ( Y . Chen, J.C.K. Lai and L. Hertz, unpublished experiments). The incorporation of acetate does not lead to a net increase in the amount of TCA cycle intermediates, because each time one molecule acetate (containing two carbon atoms) from acetyl-CoA is incorporated, the TCA cycle has to turn once, leading to CO, production from two carbon atoms (Fig. l), before another molecule acetate can enter the TCA cycle. In order for net synthesis of TCA cycle intermediates to occur (which may be especially important in brain (see p. 289)), a reaction leading to CO, fixation must take place. The only CO, fixation reaction which appears to be of quantitative importance in brain is carboxylation of pyruvate (Patel, 1974), catalyzed by pyruvate carboxylase. Studies in both intact brain and cultured cells have shown that this enzyme is absent in neurons (both glutamatergic and GABAergic), but present in astrocytes (Yu et al., 1983; Shank et al., 1985; Kaufman and Driscoll, 1992). Unfortunately, it is not yet known whether

288 TABLE I1 Effect of noradrenaline (0.3 pM) on rate of I4CO2production from [l-'4C]pyruvate under control condition and in a high Mg2+/no Ca2+ medium Pyruvate oxidation (nmol/min per mg protein)

Control Control + noradrenaline

Control medium

No C a 2 +, 10 mM Mg"

2.36

2.29 f 0.35 2.25 t 0.28

f 0.34 3.18 k 0.35*

* Although not significant in this experiment, a similar degree of stimulation (approx. 35%) was highly significant, when measured in a large population. (Unpublished experiments by Y. Chen, J.C.K. Lai and L. Hertz.) Results are means f S.E.M. of six individual experiments.

pyruvate carboxylation in astrocytes is stimulated by noradrenaline. However, it has been established that this reaction is very much enhanced by an increase in the extracellular potassium concentration (Kaufman and Driscoll, 1992). In contrast, formation of acetyl-CoA from pyruvate in intact cells is, at least when bicarbonate is absent from the medium, not stimulated by excess potassium (Y. Chen, L. Hertz and J.C.K. Lai, unpublished experiments). In addition to noradrenaline, also serotonin (Cambray-Deakin et al., 1988; Magistretti, 1988) and histamine (Magistretti, 1988; Arbones et al., 1990) have a glycogenolytic effect in primary cultures of astrocytes (in the case of histamine probably resulting from an increase in cyclic AMP accumulation (Agullo et al., 1990)), as they do in brain slices (Quach et al., 1978). In contrast to noradrenaline, serotonin decreases glucose utilization and oxygen consumption in the brain in vivo (MacKenzie et al., 1977; Grome and Harper, 1985). Serotonin is also involved in a functional ipsilateral decrease in glucose utilization after cerebral damage (Pappius, 1988,1990,1991), which occurs in spite of adequate glucose supply (Buczek

et al., 1991), may be exerted on a 5-HT2 receptor (Pappius, 1991) and is prevented by administration of a serotonin synthesis inhibitor (Pappius et al., 1988). It is indicative of an involvement of astrocytes that quinolinic acid, a neurotoxic tryptophan metabolite, formed in astrocytes (Whetsell et al., 1988), accumulates in the traumatized hemisphere (Pappius, 1990, 1991). In spite of the fact that noradrenaline enhances and serotonin decreases the rate of glucose utilization in the brain in vivo, both compounds have a mainly inhibitory action on neuronal activity (e.g., Bloom et al., 1972; Waterhouse and Woodward, 1980). Evidence is found that quinolinic acid may be involved in the pathogenesis of tissue damage in brains of patients suffering from Huntington's disease (Whetsell et al., 1988). A possible causal correlation between noradrenergic and serotonergic deficiency and the development of Alzheimer's disease has also been suggested (Hertz, 1989a, 1992a). The possible role of monoamines in hepatic encephalopathy is discussed in this volume by Norenberg.

Drugs. Chronic, but not acute exposure to toxicologically relevant concentrations of cocaine on the one hand causes an increase in production of labeled C 0 2 from [l-14C]glutamatein astrocytes in primary cultures (Table 111) and, on the other hand, abolishes the stimulation normally exerted by noradrenaline (L. Peng and L. Hertz, unpublished experiments). This effect has been studied in more

TABLE I11 Production of labeled CO, from [l-14C]glutamatein primary cultures of astrocytes exposed acutely or chronically to 3 gM cocaine CO, production (070 of control) Acute Control Cocaine

100 f 84.6 f

Chronic

6.3 (14) 8.5 (8)

100 f 4.9 (3) 171.0 k 1.2 (3)

L. Peng and L. Hertz (unpublished experiments).

289

detail in very young cultures of astrocytes (exposure to cocaine from day 3 in cultures which have been obtained from neonatal mice). Since the human newborn at term in many metabolic aspects corresponds to approx. 1-week-old mice, this corresponds to drug exposure of the human fetus during the last one-half of the third trimester. In these cultures, the cocaine effects were also studied through a prolonged withdrawal (i.e., cessation of exposure to cocaine) period. After chronic exposure to 3 phi cocaine f o r 24 days, no normalization of either unstimulated energy metabolism or metabolism in the presence of noradrenaline was found even after 36 days of withdrawal (Peng and Hertz, 1992). The potential conclusions which could be drawn from these findings are frightening. Recent in vivo determinations of glucose utilization in the brain by the deoxyglucose method has shown alterations in energy metabolism during exposure to cocaine (London et al., 1986, 1990; Porrino et al., 1988; Kornetskyet al., 1991) and after its withdrawal (Volkow et al., 1991) in man and in experimental animals. This further supports the hypothesis of noradrenergic abnormalities, since administration of noradrenergic antagonists affects glucose utilization in the brain in vivo (see p. 284). It should also be mentioned that behavioral studies by Spear et al. (1989) led the authors to suggest a long-term, specifically catecholaminergic deficiency in rats after exposure to cocaine during a period of development that roughly corresponds to that during which the astrocytic cultures were treated with cocaine. This is also the period during which most of the noradrenergic fibers from locus coeruleus reach their targets in the cerebral cortex. That the ensuing noradrenergic innervation of astrocytes is of functional importance for astrocytic development can be seen from many observations of morphological and/or functional changes in astrocytes in primary cultures treated with either dibutyryl cyclic AMP (mimicking @-adrenergic stimulation) or phorbol esters (mimicking a l adrenergic stimulation) (for review and references, see Hertz, 1990b). This treatment has profound morphologic and physiologic effects, including an

enhanced response to the glycogenolytic effect of noradrenaline (K.V. Subbarao and L. Hertz, unpublished experiments), process formation and induction of the L-channel for calcium. Chronic treatment of astrocytic cultures with antidepressants causes, in contrast, a “downregulation” of @-adrenergic receptor function in astrocytes (Hertz and Richardson, 1983), but it is unknown if any of the responses to noradrenaline become altered by this treatment. Evidence is found that the receptor site which is affected is not a usual @-adrenergic receptor type (Manier and Sulser, 1990).

Amino acid transmitters The major excitatory transmitter in the CNS is the amino acid glutamate (Fonnum, 1984). A potassium-induced, calcium-dependent release of glutamate, i.e., a transmitter release, has been studied both in cerebellar granule cells in primary cultures (Peng et al., 1991), in vivo, and in brain slices (for review and references, see Hertz and Schousboe, 1986; Hertz et al., 1992). Extracellular glutamate is more efficiently accumulated into astrocytes than into neurons (McLennan, 1976; Hertz and Schousboe, 1986; Schousboe et al., 1988). The astrocytic uptake is further enhanced by noradrenaline (Table I), acting on an al-receptor (Hansson and Ronnback, 1988). In astrocytes, glutamate is partly used as a metabolic substrate and partly converted to glutamine, which can be returned t o neurons as a precursor for glutamate and GABA. Since glutamate is partly oxidized in astrocytes, other transmitter precursors are also used, mainly TCA cycle constituents formed in astrocytes (which express pyruvate carboxylase activity), but not in neurons (Hertz and Schousboe, 1986, 1988; Hertz et al., 1992). Oxidative metabolism of glutamate occurs in the TCA cycle and is enhanced by noradrenaline as previously described, whereas glutamine formation is not (R. Huang and L. Hertz, unpublished experiments). The potassium-induced glutamate release is potently inhibited by serotonin in brain slices (Maura et al., 1986, 1989) and in cerebellar granule

290

l2

GABA is accumulated into astrocytes and metabolized. This uptake is enhanced by stimulation of a 0-adrenergic receptor (Hansson and Ronnback, 1988).

r

3

4

a

9 1 3 1 4 1 8 1 9 2 2 2 3 2 4 25 2 6 FRACTION NO.

Fig. 6. Release of glutamate (nmol/min per mg protein) from 8day-old cultures of cerebellar granule cells during superfusion in a saline medium with glutamine as the glutamate precursor. The superfusion was for a total of 26 1-min periods, during each of which the superfusate was collected and its glutamate content determined by HPLC. During the periods 0- 5, 11 - 15, and 21 -23 min (open bars), the K f concentration of the superfusion medium was 5 mM, and during the periods 6 - 10, 16 - 20 and 24 - 26 min (closed bars) the K + concentration was raised to 50 mM; 1 nM serotonin (5-HT) was present during the time period I1 - 20 min (indicated by horizontal bar). All values are the averaees of two individual experiments. (From Hertz, 1989a.)

cells in primary cultures (Fig. 6). Glutamate release from astrocytes is also enhanced during exposure to an elevated concentration of potassium (Drejer et al., 1985; Nicholls and Attwell, 1990). This release differs from the neuronal release by not being calcium-dependent but, as in neurons, it is inhibited by serotonin (Hertz et al., 1989). The major inhibitory transmitter in the central nervous system is y-aminobutyric acid (GABA), a transmitter amino acid which is formed in GABAergic neurons from glutamate by decarboxylation (Fig. 1). The potassium-induced neuronal release is calcium-dependent, but, unlike glutamate, there is no potassium-induced release of GABA from astrocytes (Schousboe et al., 1988). A relatively larger fraction of released GABA than of released glutamate is probably reaccumulated into neurons (Hertz and Schousboe, 1986, 1987). Nevertheless, a substantial amount of

Potassium homeostasis Like glutamate, potassium is released from excited neurons (all types of neurons, not restricted to glutamatergic neurons) and possibly also from other cell types, leading to an increase in extracellular potassium concentration in the central nervous system (Sykova, 1983; Walz and Hertz, 1983; Walz, 1989). This increase is measureable during neuronal excitation, occurs to a larger extent (up to 10- 12 mM) during seizures, and is exceedingly high during anoxia or hypoglycemia (Hansen, 1985). Like glutamate, potassium ions are to a considerable extent actively accumulated into astrocytes. A potassium uptake, catalyzed by the Na+,K+-ATPase, occurs in both astrocytes and neurons but it is only stimulated by above-normal extracellular potassium levels in astrocytes (Grisar et al., 1979; Walz and Hertz, 1982; K.V. Subbarao, I. Hajek and L. Hertz, unpublished experiments). The Na+ ,K+-ATPase activity in brain slices and in astrocytes is increased by exposure to noradrenaline and to the &specific agonist isoproterenol (Fig. 7) (Hajek and Hertz, 1992), but it is disputed whether this is a genuine transmitter ef-

Fig. 7. Na' ,K+-ATPase activity in astrocytes (open bars) and cerebral cortical neurons (hatched bars) in the presence of noradrenaline (NA), isoproterenol (ISO), phenylephrine (PHE) and clonidine (CLON). (From Hajek and Hertz, 1992.)

29 I

fect. There is no indication of any increase in the presence of the a2-adrenergic agonist clonidine, and the apparent small stimulation by the a l agonist phenylephrine was not statistically significant. This subtype distribution is strikingly different from that observed for the stimulation of TCA cycle activity (Table I), suggesting that the enhanced oxidative metabolism in the presence of noradrenaline is probably not a simple consequenceof an increased ADP/ATP ratio. Noradrenaline also stimulates Na+ ,K -ATPase activity in neurons but isoproterenol causes a significant reduction (Fig. 7). In addition to accumulating potassium by active uptake, astrocytes contribute to potassium homeostasis by passive, current-carried redistribution of potassium ions through an astrocytic syncytium (Orkand et al., 1966; Hertz, 1986, 1990a; Walz, 1989). This mechanism depends upon extensive electrical (and dye) coupling between astrocytes as well as upon a high potassium conductance in the astrocytic cell membrane. Patch-clamp analysis has repeatedly demonstrated several different potassium channels in astrocytes, including a delayed rectifier channel, allowing a sustained potassium current (Barres et al., 1990). Radioisotope experiments have also shown a very intense channel-mediated potassium transport in primary cultures of mouse astrocytes (Hertz, 1986, 1990a). This passive system for potassium redistribution, often called the spatial buffer, is capable of transporting potassium ions from an extracellular location with a locally elevated potassium concentration through an astrocytic syncytium to other extracellular localizations. If the path ways traveled by potassium ions could be regulated, e.g., by transmitter signals, this system might play an important role in information processing (Hertz, 1990a, 1992b). Attempts to demonstrate an effect of either serotonin or noradrenaline (or any of its subtype-specific agonists) on channel-mediated potassium transport in astrocytes have until now not been successful. However, dye coupling between astrocytes is inhibited by al-agonists (Giaume et al., 1991), and exposure to active phorbol esters, which mimic the action of transmitters operating via the phosphoinositol system (e.g., 5-HT2-, al-, mus+

PHORBOL 12-MYAISTATE 13 ACETATE (!iM)

Fig. 8. Inhibition of K + uptake into astrocytes in primary cultures by phorbol 12-myristate 13-acetate at steady state conditions (U), i.e., reflecting mainly K t permeability, and in previously K+-depleted conditions (M), i.e., reflecting mainly active uptake. (From Hertz, 1989b.)

carinic or purinergic agonists), causes a potent inhibition of channel-mediated transport in both astrocytes (Fig. 8) and oligodendrocytes (Hertz et al., 1990). Since potassium conductance appears to be much higher in astrocytes than in oligodendrocytes (Hertz et al., 1990), it is probably mainly the effect on astrocytes which can be expected to be of functional importance.

Mechanisms Cyclic AMP Beta-adrenergic receptors appear predominantly to be found on astrocytes (see Stone et al., this volume), which have a high density of mainly plreceptors (Harden and McCarthy, 1982). This receptor activates the cyclic AMP second messenger system. Although such a stimulation is well established in astrocytes (see Stone et al., this volume), relatively little information is found regarding the functional significance of adenylate cyclase activation in mature astrocytes. The only effects described in this review were a stimulation of glycogenolysis and of Naf ,Kf -ATPase activity. In addition, stimulation of 0-adrenergic regulators has been found to cause release of taurine (Shain and Martin, 1984), release of nerve growth factor (Schwartz and Costa, 1977) and increased phosphorylation of intermediate filament protein

292

(McCarthy et al., 1985, 1988) in astrocytes. Very interestingly, synergistic effects between a l - and PIagonists on free intracellular calcium level (see below) have been described (Delumeau et al., 1991). Beta-adrenergic stimulation of cultured astrocytes also leads to early response gene activation, but it is doubtful whether this response plays any role during normal function in the adult brain (see Arenander and DeVellis, this volume). All of these effects are probably mediated via protein kinase A which in turn leads to phosphorylation of other proteins. It has already been mentioned that 0-adrenergic agonists seem to play a major role as differentiating agents during development (e.g., Hertz, 1990b, 1992a). Beta-adrenergic receptor activation may also play a major role for the establishment of memory during “imprinting” (Ng et al., this volume). It might be significant that this is a process occurring in developing brain. Moreover, it is the O1-receptor which is down-regulated during chronic exposure to antidepressants in vivo (Minneman et al., 1979). As already mentioned, a similar down-regulation has been observed in astrocytes in primary cultures (Hertz and Richardson, 1983; Richelson, 1990) although there is evidence that this does not represent down-regulation of a “classical” &receptor (Manier and Sulser, 1990). Very little information is available about stimulation of the cyclic AMP secondary messenger system in neurons. However, both forskolin and vasoactive intestinal peptide (VIP) have been found to increase the level of cyclic AMP in cultured chick sympathetic neurons (Przywara et al., 1991).

Calcium Calciumasan intracellular messenger. In virtually all cells, the cytosolic concentration of free calcium ions plays an important role as an intracellular signal. Normally, this concentration is very low (0.1 ~LM) but it can rapidly increase many-fold either by entry of calcium through one of several voltagesensitive or transmitter-operated calcium channels across the cell membrane or by release of bound

calcium from intracellular organelles; subsequently it can be lowered by calcium extrusion via calcium carriers or binding to intracellular organelles (Rasmussen, 1989). Release of calcium from its intracellular binding sites can be achieved by stimulation with the second mesenger inositol-triphosphate (IPJ, which is released to the cytoplasm from membrane-bound phosphatidylinositol-4-5-biphosphate(PIP,) as the result of appropriate transmitter receptor stimulation, e.g., by noradrenaline acting on an a l receptor. Hydrolysis of PIP, also leads to the formation of another messenger, diacylglycerol (DG) which, in the presence of an elevated intracellular calcium concentration, stimulates protein kinase C (PKC) (Berridge, 1987). Activation of PKC regulates a multitude of events on the cell membrane (enhancement or inhibition of ion channel and ion carrier activity) and in the cell interior (e.g., protein synthesis and phosphorylation). Mitochondria are surrounded by an outer and an inner mitochondrial membrane. The inner mitochondrial membrane is essentially impermeable to all charged molecules, unless specific carriers or channels are found. Among the second messengers, only calcium appears to be able to enter the mitochondrial matrix from the cytoplasm (Denton and McCormack, 1990). Mitochondria1 uptake of calcium occurs via a uniporter and is driven by the large electrical potential across the membrane, which is established as a result of proton extrusion in the respiratory chain (e.g., Rasmussen and Barrett, 1984; Denton and McCormack, 1985). It can be inhibited by ruthenium red (McCormack and England, 1983) or depletion of calcium from the medium together with elevated concentrations of magnesium (McCormack et al., 1989). In heart and several other tissues, the transfer of calcium out of the mitochondria mainly occurs by an electroneutral exchange of Ca2+ for 2 Na+ (Denton and McCormack, 1990). Initially, it was assumed that this system contributed to the regulation of the calcium concentration in the cytoplasm. However, Denton et al. (1980) have pointed out “that this general emphasis on the possible role of mitochondria in the

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regulation of cytoplasmic Ca2+ was probably misplaced’’ (Denton and McCormack, 1990), and that “the main function of the Ca2+ transport system in the inner mitochondrial membrane is to regulate the concentration of Ca2+ within mitochondria”. This view followed the recognition that three important dehydrogenases within mitochondria are sensitive to activation by Ca2+ in the approximate range of 0.1 - 10 pM (McCormack et al., 1989; Denton and McCormack, 1990). These include the pyruvate dehydrogenase and the aketoglutarate dehydrogenase together with the isocitrate dehydrogenase. In the former, the active, non-phosphorylated form of the enzymeis increased through stimulation of a phosphatase (Denton et al., 1972), whereas the two latter enzymes are activated by a direct effect on the enzymes, enhancing the affinity for their substrates (McCormack and Denton, 1979; Denton and McCormack, 1990). Other enzymes, such as glutaminase appear to be activated secondarily as a result of mitochrondrial swelling due to potassium uptake (McGivan et al., 1985; Halestrap, 1989). It should be noted that the pyruvate dehydrogenase, the a-ketoglutarate dehydrogenase and theglutaminase are the enzymes catalyzing exactly those metabolicfluxes which were found to be enhanced by noradrenaline (Table I)

-100

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$ 0

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EXTRAMITOCHONDRIAL [ca’’]

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Fig. 9. Effect of extramitochrondrial calcium concentration on intramitochrondrial calcium concentration ( 0 )and on activation of the pyruvate dehydrogenase complex (A) and the aketoglutarate dehydrogenase complex )(. (From Denton and McCormack, 1990.)

JNorpdrenPlin.1

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Increased mitochondria1

Activation of

Increased response

Pyruvnte Dehydmgenase

(Potassium Uptake Glutamate Uptake)

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Fig. 10. Proposed role of Ca2+ in the coupling of noradrenaline effects on energy-requiring responses and enhanced formation of ATP in intact astrocytes in primary cultures. (Modified from Denton and McCormack, 1990.)

and that it could not be excluded that TCA cycle activity was stimulated at additional points. This would include a possible stimulation of the isocitrate dehydrogenase. The important concept of regulation of mitochondrial activity by the intramitochondrial calcium concentration is primarily based on experiments using heart or liver mitochondria (e.g., Denton and McCormack, 1990), but isolated hepatocytes have also been used (Quinlan and Halestrap, 1986). Fig. 9 shows the correlation between extracellular calcium concentration, intramitochondrial free calcium concentration and calcium-dependent activation of pyruvate dehydrogenase and a-ketoglutarate dehydrogenase activity in rat heart mitochondria (Denton and McCormack, 1990). It can be seen that the EC,, for the activation is approx. 1.0 pM. Approximately

294

similar results have been obtained by MorenoSanchez and Hansford (1988) and Lukacs et al. (1988). From Fig. 9 it can also be seen that the rise in intramitochondrial calcium concentration is considerably steeper than that in the cytosolic calcium concentration when the latter is increased from its resting level. These findings led to the proposal (e.g., McCormack and Denton, 1986, 1990) that hormones and other external stimuli, which increase the cytosolic concentration of Ca2+, may enhance rates of mitochondria1 NADH formation, and hence ATP synthesis, by secondarily increasing also the intramitochondrial concentration of C$+ . Such a mechanism may play a central role in ATP homeostasis since, as illustrated in Fig. 10, energy supply may be increased to meet a greater energy demand by a direct effect on the TCA cycle, without necessarily depending upon a concomitant decrease in the ATP/ADP ratio as a requirement for an increased metabolic rate as envisaged in the classical Chance-Williams (Chance and Williams, 1956) hypothesis (Hansford, 1988). This mechanism does in no way invalidate the concept of a regulatory role by the ADP/ATP ratio, but represents an additional mechanism. Since it is evoked by stimulation of dehydrogenases it will lead to an increased NADH/NAD+ ratio. Before it can be concluded that the same mechanism is involved in metabolic regulation in other cell types it is of criticial importance to verifr the potential role of the intramitochondrial calcium concentration in these tissues. Such verification ultimately will include at least three requirements: (1) measurements of calcium in mitochondria of intact cells; (2) determination of fluxes from pyruvate to acetyl-CoA (catalyzed by the pyruvate dehydrogenase) and from a-ketoglutarate (or glutamate) to succinate in intact cells, as well as measurements of key metabolic parameters, such as ratios between ATP/ADP and NADH/NAD+; and (3) studies of the effect of compounds like ruthenium red, an inhibitor of calcium uptake into mitochrondria (McCormack and England, 1983) or of conditions like a high magnesium concentration in the absence of calcium in the medium, which likewise blocks

mitochondrial calcium uptake (Hansford, 1988; Denton and McCormack, 1990). Criterion (2) has been dealt with in the first one-half of this review. It has also been demonstrated that deletion of calcium from the incubation medium together with an increase in the magnesium concentration, abolishes the stimulation of CO, production from [l-'4C]pyruvate by noradrenaline (Table 11). Intramitochondrial calcium concentrations have as yet not been investigated in brain cells, although there is a substantial amount of information about effects of monoaminergic transmitters on free intracellular, i.e., cytosolic calcium concentrations, especially in astrocytes in primary cultures, as well as information about an apparent lack of correlation between intramitochondrial calcium concentration and metabolic activity in synaptosomes during potassiuminduced depolarization. These two topics will be discussed below.

Effects of monoaminergic transmitters on free intracellular calcium concentrations in astrocytes. A series of studies by McCarthy and coworkers (Salm and McCarthy, 1990; McCarthy and Salm, 1991) have shown that noradrenaline and other adrenergic agonists stimulate increases in free intracellular calcium concentration in over 80% of astrocytes in primary cultures. Although more cells appeared to react to a,-adrenergic than to a2adrenergic receptor activation, some cells responded equally well to activation of either subtype receptor agonists, and there was a subpopulation of cells which responded only to stimulation of a2receptors. Thisappearstobeconsistentwithourfinding (Fig. 5 ) that the a,-agonist clonidine caused a smaller maximum increase in glycogenolysis, measured in a whole culture dish, than did noradrenaline. We have recently confirmed an increase in average free intracellular calcium concentration in cultured astrocytes (measured in a population of, maybe, 50 cells), evoked by the extremely subtype-specific a2-agonist dexmedetomidine (Fig. 11). No corresponding response was evoked in similar cultures of neurons (Zhao et al., 1992). A noradrenaline-induced increase in free intracellular

295

calcium concentration and participation of a2receptors in this response in astrocytes in primary cultures has been confirmed by Nilsson et al. (1991). Serotonin, acting on a 5-HT, receptor (Nilsson et al., 1991)as well as histamine (McCarthy and Salm, 1991) are also able to increase free intracellular calcium concentration in astrocytes. All these experiments were performed using intact cultured cells and no specific information is available regarding intramitochondrial calcium concentrations. It is also not known to what extent, if at all, increases in mitochondrial calcium concentration will influence the average free intracellular calcium concentration in these cells. It appears likely that this, to a considerable extent, will depend on the precise cytosolic localization of the increased calcium concentration.

Correlation between metabolic rate and intramitochondrial calcium Concentration in synaptosomes. In synaptosomes, treatment with veratri-

dine, which is considered to depolarize neuronal preparations by a maintained opening of Na+ channels, causes a distinct increase in free intracellular calcium concentration (Hansford and Castro, 1985; Hansford, 1988). Veratridine also caused an increase in the active, dephosphorylated form of the pyruvate dehydrogenase. These two findings are consistent with the concept that the increase in the active form of the pyruvate dehydrogenase, as in

heart mitochondria, is a result of the increased intracellular, and thus presumably also intramitochondrial, calcium concentration. However, i f the extracellular medium was depleted of calcium, the

veratridine-induced increase in free intracellular concentration of calcium was abolished, whereas the activation of the enzyme was not (Hansford, 1988). Ruthenium red, which blocks calcium uptake into mitochondria (McCormack and England, 1983), neither diminished the rise in free intracellular (cytosolic) calcium after application of veratridine (Hansford and Castro, 1985), nor did it abolish the veratridine-induced increase in oxygen consumption in synaptosomes (Erecinska et al., 1991). Also, Kauppinen et al. (1989) found that synaptosoma1 oxidation of [ 1-14C]pyruvate increased in response to elevated ATP consumption (increased ADPIATP ratio) induced by either veratridine, elevated potassium concentration or a metabolic uncoupler, but that no elevation in free intracellular calcium concentration was required to adjust [ 114C]pyruvate oxidation to accommodate the increased energy demand. However, Pate1et al. (1988) have obtained data indicating an obligatory role of calcium in depolarization-induced stimulation of oxygen consumption in synaptosomes. Most, but

not all, presently available evidence therefore appears to suggest that increases in synaptosomal energy metabolism induced by depolarization are predominantly regulated by the A D P / A TP ratio, in accordance with the classical Chance-Williams model (Chance and Williams, 1956), with little additional

regulatory effect by the intramitochondrial calcium concentration.

b

P+

* f 20

w w 50

20mM K'

Fig. 11. Effect of the subtype specific a2-agonist dexmedetomidine (20 or 50 nM) on tracings of free intracellular calcium concentration in astrocytes (a) and in neurons (b) in primary cultures. (From Zhao et al., 1992.)

Since the correlation between free intracellular calcium concentration and energy metabolism in synaptosomes was studied using potassium-induced depolarization or exposure to veratridine or an uncoupler, it should be noted that an elevated

potassium concentration does also not increaseflux through eitherthepyruvatedehydrogenasecatalyzed step (CO, production from [l-14C]pyruvate)or the a-ketoglutarate dehydrogenase catalyzed step (CO, production from glutamate) in intact astrocytes (Y. Chen., J.C.K. Lai and L. Hertz, unpublished ex-

296

periments). Nevertheless, excess potassium does increase free intracellular calcium concentration (in differentiated primary cultures of astrocytes, treated with dibutyryl cyclic AMP, mainly by a calcium uptake through an L-channel (Code et al., 1991)), lactate production, oxygen consumption and glycogenolysis (for details and references, see Hertz and Peng, 1992), as well as CO, fixation, a process which does not occur in neurons (Kaufman and Driscoll, 1992). Thus, the apparent difference between synaptosomes and astrocytes observed above may rather represent a difference between metabolic reactions to a monoamine-induced increase in free intracellular calcium concentration, which may predominantly or exclusively be an astrocytic phenomenon, and a depolarizationinduced increase in free intracellular calcium concentration, occurring in both neurons and astrocytes. Nevertheless, monoaminergic agonists do exert other effects in at least some neurons, indicating the presence of receptor sites. For example, they influence calcium currents (e.g., Gray and Johnston, 1987; Bean, 1989; Fukui et al., 1991) and increase turnover of inositol phospholipids (Kanterman et al., 1990). Also, both depolarizing stimuli and certain neurotransmitters activate protein kinase C in sympathetic neurons, but they utilize separate pathways (Wakade et al., 1991).

Concluding remarks The findings reviewed in this paper strongly suggest that noradrenergic agonists increase the rate of oxidative metabolism in astrocytes by causing an increase in free intracellular calcium concentration in the cytosol, which subsequently triggers a rise in intramitochondrial calcium concentration and, thereby, increases metabolic fluxes. The correlation between the fluxes, which were found to be stimulated, i.e., the reactions catalyzed by the pyruvate dehydrogenase complex, the aketoglutarate dehydrogenase complex and phosphate-activated glutaminase, and the enzymatic reactions known to be enhanced, albeit in

other systems, by an increased intramitochondrial calcium concentration, is remarkable. Further evidence for a correlation between metabolic stimulation and intramitochondrial concentration of calcium is that omission of extracellular calcium, together with an elevation in the magnesium content, a procedure known to inhibit mitochondria1 calcium uptake, abolished the stimulation of 1 4 ~ 0 , formation from [ I ''C]pyruvate (Table 11), and that the same noradrenergic transmitter subtypes which stimulated metabolic activity also enhanced free intracellular calcium concentration. The role of drugs like cocaine on these systems requires further investigation, but it should be noted that this drug was found to exert profound effects on a homogeneous population of astrocytes in the absence of any neurons. Thus, these effects cannot be secondary to drug effects on neuronal (or astrocytic) transmitter uptake. It should be emphasized that not any increase in free intracellular calcium concentration is correlated with a metabolic increase due to direct calcium effects on the dehydrogenases, since most evidence indicated that the depolarization-induced stimulation of energy metabolism in both synaptosomes and astrocytes is not caused by calciuminduced intramitochondrial effects, but rather were the result of an altered ADP/ATP ratio, as suggested in the original Chance-Williams theory. In astrocytes, the increase in CO, fixation caused by an elevated potassium concentration (Kaufman and Driscoll, 1992) may perhaps also lead to an increase in oxidative metabolism, correlated with a decreased NADH/NAD+ ratio (Hertz and Peng, 1992). There appear to be fundamental differences regarding the functional consequences of the increased metabolism in response to, respectively, adrenergic stimulation and potassium-induced stimulation: the potassium-induced stimulation facilitates neuronal-astrocytic interactions by increasing the release of both transmitters and potassium from neurons and the production of transmitter precursors in astrocytes (by stimulation

297

of pyruvate carboxylation). In contrast, the noradrenaline and serotonin effects may rather interrupt andprevent neuronal-astrocytic interactions by enhancing cellular uptake of transmitters and potassium and by inhibiting glutamate release. The

Buczek, M., Ratcheson, R.A., Lust, W.D., McHugh, M. and Pappius, H.M. (1991) Effects of focal cortical freezing lesion on regional energy metabolism. J. Cereb. Blood Flow Metab., 11: 845-851. Cambray-Deakin, M., Pearce, B., Morrow, C. and Murphy, S. (1988) Effects of neurotransmitters on astrocytic glycogen stores in vitro. J. Neurochem., 51: 1852- 1857. apparent paradox that both noradrenergic and Chance, B. and Williams, G.R. (1956) The respiratory chain and serotonergic activation appears to have an inoxidative phosphorylation. Adv. Enzymol., 17: 65 - 134. hibitory influence on neuronal activity, in spite of Code, W.E., White, H.S. and Hertz, L. (1991) The effect of noradrenaline enhancing, and serotonin decreasing midazolam on calcium signaling in astrocytes. Ann. N.Y. glucose utilization, may possibly be explained by Acad. Sci., 625: 430 - 432. Delumeau, J.C., Marin, P., Cordier, J., Glowinski, J. and Preassuming that the neuronal inhibition in both cases mont, J. (1991) Synergistic effects in the alpha 1- and beta 1is a result of the dissociation between neuronal and adrenergic regulation of intracellular calcium level in striatal astrocytic function. However, it appears that astrocytes. Cell. Mol. Neurobiol., 11: 263 -267. serotonin acts by passively inhibiting release of exDenton, R.M. and McCormack, J.G. (1985) Ca2+ transport by citatory compounds such as glutamate, whereas mammalian mitochondria and its role in hormone action. Am. . I Physiol., . 249: E553 - E554. noradrenaline enhances active uptake of glutamate Denton, R.M. and McCormack, J.G. (1990) Ca2+ as a second and potassium into astrocytes. messenger within mitochondria of the heart and other tissues. Annu. Rev. Physiol.. 52: 451 -466. Acknowledgements Denton, R.M., Randle, P.J. and Martin, B.R. (1972) Stimulation by calcium ions of pyruvate dehydrogenase phosphatase. Biochem. J., 128: 161 - 163. The Medical Research Council of Canada is thankDenton, R.M., McCormack, J.G. and Edgell, N.J. (1980) Role ed for financial support to the authors' research and of calcium ions in the regulation of intramitochondrial for a Post Doctoral Fellowship to L. Peng. metabolism. Effects of Na', M$+ and ruthenium red on the Ca2'-stimulated oxidation of oxoglutarate and on pyruvate dehydrogenase activity in intact rat heart mitochondria. References Biochem. J., 190: 107-117. Drejer, J., Larsson, O.M. and Schousboe, A. (1982) Agullo, L., Picatoste, F. and Garcia, A. (1990) Histamine Characterization of uptake and release processes for Dstimulation of cyclic AMP accumulation in astrocyte-enriched aspartate in primary cultures of astrocytes and cerebellar and neuronal primary cultures from rat brain. J . Neurochem., granule cells. Neurochem. Res., 8: 231 - 243. 55: 1592 - 1598. Drejer, J., Benveniste, H., Diemer, N.H. and Schousboe, A. Arbones, L., Picatoste, F. and Garcia, A. (1990) Histamine (1985) The cellular origin of ischemia-induced glutamate stimulates glycogen breakdown and increases 45CaZ+ release from brain tissue in vivo and vitro. J. Neurochem., 45: permeability in rat astrocytes in primary culture. Mol. Pharmacol., 37: 921 - 927. 145- 151. Barres,B.A.,Chun,L.L.Y.andCorey,D.P.(1990)Ionchannels Erecinska, M . , Nelson, D. and Chance, B. (1991) Depolarization-induced changes in cellular energy production. Proc. in vertebrate glia. Annu. Rev. Neurosci., 13: 441 - 474. Natl. Acad. Sci. U.S.A., 88: 7600-7604. Bean, B.P. (1989) Multiple types of calcium channels in heart Fonnum, F. (1984) Glutamate: a neurotransmitter in mammuscle and neurons. Modulation by drugs and neurotransmitmalian brain. J. Neurochem., 42: 1 - 11. ters. Ann. N. Y. Acad. Sci., 560: 334 - 345. Fukui, H., Inagaki, N., Ito, S., Kubo, A., Kondoh, H., Berntman, L., Dahlgren, N. and Siesjo, B.K. (1978) Influenceof Yamatodani, A. and Wada, H. (1991) Histamine HIintravenously administered catecholamine on cerebral oxygen receptors on astrocytes in primary cultures: a possible target consumption and blood flow in the rat. Acta Physiol. Scand., for histaminergic neurons. Agents Actions (Suppl.), 33: 104: 101 - 108. 161 - 180. Berridge, M. J. (1987) Inositol tnphosphate and diacylglycerol: Giaume, C., Marin, P., Cordier, J., Glowinski, J. and Premont, two interacting second messengers. Annu. Rev. Biochem., 56: 159- 193. J . (1991) Adrenergic regulation of intercellular communications between cultured striatal astrocytes from the mouse. Bloom, F.E., Hoffer, B.J., Siggins, G.R., Barker, J.L. and Proc. Natl. Acad. Sci. U.S.A., 88: 5577 - 5581. Nicoll, R.A. (1972) Effect of serotonin on central neurons: Gray, R. and Johnston, D. (1987) Noradrenaline and betamicroiontophoretic administration. Fed. Proc., 31: 97 - 106.

298 adrenoceptor agonists increase activity of voltage-dependent calcium channels in hippocampal neurons. Nature, 327: 620 - 622. Grisar, T., Frere, J.M. and Franck, G. (1979) Effect of K + ions on kinetic properties of the (Na+,K+)-ATPase(EC. 3.6. 1.3) of bulk isolated glia cells, pericarions and synaptosomes from rabbit brain cortex. Brain Res., 165: 87 - 103. Grome, J.J. and Harper, A.M. (1985) Serotonin depression of local cerebral glucose utilisation after monoamine oxidase inhibition. J . Cereb. Blood Flow Metab., 5 : 473 - 475. Hajek, 1. and Hertz, L. (1992)Effects of monoaminesand amino acid transmitters on Na’, K+-ATPase activity in cultured mouse cortical astrocytes compared to spinal cord astrocytes and to cortical neurons. (Submitted). Halestrap, A.P. (1989) The regulation of the matrix volume of mammalian mitochondria in vivo and in vitro and its role in the control of mitochondrial metabolism. Biochim. Biophys. Acta, 973: 355 - 382. Hansen, A.J. (1985) Effects of anoxia on ion distribution in the brain. Physiol. Rev., 65: 101 - 148. Hansford, R. (1988) Relationship between cytosolic free calcium ion concentration and the control of pyruvate dehydrogenase in isolated cardiac myocytes and synaptosomes. In: D.R. Pfeiffer, J.B. McMillin and S. Little (Eds.), Cellular C&+ Regulation, Plenum, New York, London, pp. 230-242. Hansford, R.G. and Castro, F. (1985) Roles of Ca2+ in pyruvate dehydrogenase interconversion in brain mitochondria and synaptosomes. Biochem. J., 227: 129- 153. Hansson, E. and Ronnback, L. (1988) Regulation of glutamate and GABA transport by adrenoceptors in primary astroglial cell cultures. Life Sci., 44: 27 - 34. Harden, T.K. and McCarthy, K.D. (1982) Identification of the beta-adrenergic receptor subtype on astroglia purified from rat brain. J. Pharmacol. Exp. Ther., 222: 600 - 605. Hertz, L. (1986) Potassium transport in astrocytes and neurons in primary cultures. Ann. N. Y. Acad. Sci., 481: 318 - 333. Hertz, L. (1989a) Is Alzheimer’s disease an anterograde neuronal-glial degeneration, originating in the brain-stem, and disrupting metabolic and functional interactions between neurons and glial cells? Brain Res. Rev., 14: 335 - 353. Hertz, L. (1989b) Functional interactions between neurons and glial cells. In: S. Govoni, F. Battani, M.S. Magnoni and M. Trabbuchi (Eds.), Regulatory Mechanisms of Neuron to Vessel Communication in Brain, Springer, Heidelberg, pp. 271 - 303. Hertz, L. (1990a) Regulation of potassium homeostasis by glial cells. In: G. Levi (Ed.), Development and Function of Glial Cells, Wiley-Liss, New York, pp. 225 - 234. Hertz, L. (1990b) Dibutyryl cyclic AMP treatment of astrocytes in primary cultures as a substitute for normal morphogenic and “functiogenic” transmitter signals. In: A. Privat, E. Giacobini, P. Timiras and A. Vernadakis (Eds.), Molecular Aspects of Development and Aging in the Nervous System, Plenum, New York, pp. 227-243.

Hertz, L. (1992a) Neuronal-astrocytic interactions in brain development, brain function and brain disease. In: P.S. Timiras, A. Privat, E. Gacobini, J . Lauder and A. Vernadakis (Eds.), Plasticity and Regulation of the Nervous System, Plenum, New York, pp. 143 - 159. Hertz, L. (1992b) Autonomic control of neuronal-astrocytic interactions, regulating metabolic activities and ion fluxes in the CNS. Brain Res. Bull., 29, in press. Hertz, L. and Peng, L. (1992) Energy metabolism at the cellular level of the CNS. Can. J . Physiol. Pharmacol., in press. Hertz, L. and Richardson, J.S. (1983) Acute and chronic effects of antidepressant drugs on beta-adrenergic function in astrocytes in primary cultures - an indication of glial involvement in affective disorder? J. Neurosci. Res., 9: 173 - 183. Hertz, L. and Schousboe, A. (1986) Role of astrocytes in compartmentation of amino acid and energy metabolism. In: S. Fedoroff and A. Vernadakis (Eds.), Astrocytes, Vol. 2, Academic Press, New York, pp. 179 - 208. Hertz, L. and Schousboe, A. (1987) Primary cultures of GABAergic and glutamatergic neurons as model systems to study neurotransmitter functions. I. Differentiated cells. In: Model Systems of Development and Aging of the Nervous System, Martinus Nijhoff, Boston, MA, pp. 19-31. Hertz, L. and Schousboe, A. (1988) Metabolism of glutamate and glutamine in neurons and astrocytes in primary cultures. In: E. Kvamme (Ed.), Glutamineand Glutamate in Mammals, CRC Press, Boca Raton, FL, pp. 39 - 5 5 . Hertz, L., Drejer, J . and Schousboe, A. (1988) Energy metabolism in glutamatergic neurons, GABAergic neurons and astrocytes in primary cultures. Neurochem. Res., 13: 605 -610. Hertz, L., Peng, L., Hertz, E., Juurlink, B.H. J. and Yu, P.H. (1989) Development of monoamine oxidase activity and monoamine effects on glutamate release in cerebellar neurons and astrocytes. Neurochem. Res., 10: 1039- 1096. Hertz, L., Soliven, B., Hertz, E., Szuchet, S. and Nelson, D.J. (1990) Channel-mediated and carrier mediated uptake of K + into cultured ovine oligodendrocytes. Glia, 3: 547 - 550. Hertz, L., Peng, L., Westergaard, N., Yudkoff, M. and Schousboe, A. (1992) Neuronal-astrocytic interactions in metabolism of transmitter amino acids of the glutamate family. In: A. Schousboe and N. Diemer (Eds.), AIfred Benzon Symposium, No. 32, Munksgaard, Copenhagen, pp. 30 - 50. Inoue, M., McHugh, M. and Pappius, H.M. (1991) The effect of alpha-adrenergic receptor blockers prazosin and yohimbine on cerebral metabolism and biogenic amine content of traumatized brain. J. Cereb. Blood Flow Metab., 11: 242 - 252. Kanterman, R.Y., Felder, C.C., Brenneman, D.E., Ma, A.L., Fitzgerald, S. and Axelrod, J . (1990) Alpha 1-adrenergic receptor mediates arachidonic acids release in spinal cord neurons independent of inositol phospholipid turnover. J . Neurochem., 54: 1225 - 1232. Kaufman, E.E. and Driscoll, B.F. (1992) CO, fixation in

299

neuronal and glial cells in culture. J . Neurochem., 58: 258 - 262. Kauppinen, R.A., Taipale, H.T. and Komulainen, H. (1989) Interrelationships between glucose metabolism, energy state, and the cytosolic free calcium concentration in cortical synaptosomes from the guinea pig. J. Neurochem., 53: 761 -766. King, B.D., Sokoloff, L. and Wechsler, R.L. (1952) Theeffects of I-epinephrine and I-nor-epinephrine upon cerebral circulation and metabolism in man. J. Clin. Invest., 31: 273 - 279. Kornetsky, C., Huston-Lyons, D. and Porrino, L.J. (1991) The role of the olfactory tubercle in the effects of cocaine, morphine and brain stimulation reward. Bruin Res., 541: 75 - 81. Lai, J.C.K., Walsh, J .M., Dennis, S.C. and Clark, J.B. (1977) Synaptic and non-synaptic mitochondria from rat brain: isolation and characterization. J. Neurochem., 28: 625 - 631. London, E.D., Wilkerson, G., Geddberg, S.R. andRisner, M.E. (1986) Effects of L-cocaine on local cerebral glucose utilization in the rat. Neurosci. Lett., 68: 73-78. London, E.D., Cascella, N.G., Wong, D.F., Phollips, R.L., Dannals, R.F., Links, J.M., Herning, R., Grayson, R., Jaffe, J.H. and Wagner, Jr., H.N. (1990) Cocaine-induced reduction of glucose utilization in human brain. A study using position emission tomography and [fluorine 181-fluorodeoxyglucose. Arch. Gen. Psychiatry, 47: 567 - 574. Lukacs, G.L., Kapus, A. and Fonyn, A. (1988) Parallel measurement of oxoglutarate dehydrogenase activity and matrix free Ca2+ in fura-2-loaded heart mitochondria. FEBS Lett., 229: 219-223. MacKenzie, E.T., McCulloch, J., O'Kean, M., Pickard, J.D. and Harper, A.M. (1976a) Cerebral circulation and norepinephrine: relevance of the blood-brain barrier. A m . J. Physiol., 231: 483-488. MacKenzie, E.T., McCulloch, J . and Harper, A.M. (1976b) Influence of endogenous norepinephrine on cerebral blood flow and metabolism. A m . J. Physiol., 231: 489- 494. MacKenzie, E.T., Young, A.R., Stewart, M. and Harper, A.M. (1977) Effect of serotonin on cerebral function, metabolism and circulation. Acta Neurol. Scand. (Suppl.), 64: 76 - 77. Magistretti, P.J. (1988) Regulation of glycogenolysis by neurotransmitters in the central nervous system. Diabete Metub., 14: 237 - 246. Manier, D.H. and Sulser, F. (1990) Chronic exposure of rat glioma C6 cells to oxaprotiline reduces the density of adrenoceptors. SOC.Neurosci. Abstr., 16: 385. Maura, G., Richetti, A. and Raiteri, M. (1986)Serotonin inhibits the depolarization-evoked release of endogenous glutamate from rat cerebellar nerve endings. Neurosci. Lett., 67: 21 8 - 222. Maura, G., Roccatagliata, E., Ulivi, M. and Raiteri, M. (1989) Serotonin-glutamate interaction in rat cerebellum: involvement of 5-HT, and 5-HT, receptors. Eur. J. Pharmacol., 145: 31 - 38. McCarthy, K.D. and Salm, A.K. (1991) Pharmacologicallydistinct subsets of astroglia can be identified by their calcium

response to neuroligands. Neuroscience, 41: 325 - 333. McCarthy, K.D., Prime, J., Harmon, T. and Pollenz, R. (1985) Receptor-mediated phosphorylation of astroglial intermediate filament proteins in cultured astroglia. J. Neurochem., 44: 723 - 730. McCarthy, K.D., Salm, A. and Lerea, L.S. (1988) Astroglial receptors and their regulation of intermediate filament protein phosphorylation. In: H.K. Kimelberg (Ed.), Glial Cell Receptors, Raven Press, New York, pp. 1 - 22. McCormack, J.G. and Denton, R.M. (1979) The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex. Biochem. J . , 180: 533 - 544. McCormack, J.G. and Denton, R.M. (1986) Ca2+ as a second messenger within mitochondria. Trans. Biochem. Sci., 11: 258 - 262. McCormack, J.G. and Denton, R.M. (1990) The role of mitochondrial Ca2+ transport and matrix Ca2' in signal transduction in mammalian tissues. Biochim. Biophys. Acta, 1018: 287-291. McCormack, J.G. and England, P.J. (1983) Ruthenium red inhibits the activation of pyruvate dehydrogenase caused by positive inotropic agents in the perfused rat heart. Biochem. J., 214: 581 -585. McCormack, J.G., Browne, H.M. and Dawes, N.J. (1989) Studies on mitochondrial Ca2+-transport and matrix CaZ+ using fura-2-loaded rat heart mitochondria. Biochim. Biophys. Acta, 973: 420 - 427. McGivan, J., Vadher, M., Lacey, J. andBradford, N. (1985) Rat liver glutaminase. Regulation by reversible interaction with the mitochondrial membrane. Eur. J. Biochem., 148: 323 - 327. McLennan, H. (1976) The autoradiographic localization of 1[3H]glutamate in rat brain tissue. Brain Res., 115: 139- 144. Mickley, G.A. and Teitelbaum, H. (1979) Yohimbine blocks lateral hypothalamus-mediated behaviors. Eur. J. Phurmacol., 60: 143- 1 5 1 . Minneman, K.P., Dibner, M.D., Wolfe, B.B. and Molinoff, P.B. (1979) Betal- and betd-adrenergic receptors in rat cerebral cortex are independently regulated. Science, 204: 866 - 868. Moreno-Sanchez, 2. and Hansford, R.G. (1988) Dependence of cardiac mitochondrial pyruvate dehydrogenase activity on intramitochondrial free Ca2+ concentration. Biochem. J., 256: 403-412. Nicholls, D.G. and Attwell, D.A. (1990) The release and uptake of excitatory amino acids. Trends Pharmacol. Sci., 11: 462 - 468, Nilsson, M., Hansson, E. and Ronnback, L.(1991) Adrenergic and 5-HT2 receptors on the same astroglial cell. A microspectrofluorimetric study on cytosolic Ca'+ responses in single cells in primary culture. Dev. Bruin Res., 63: 33 - 41. Orkand, R.K., Nicholls, J.G. andKuffler, S.W. (1966) Effect of nerve impulses on the membrane potential of glial cells in the

300

central nervous system of amphibia. J. Neurophysiol., 29: 788 - 806.

Pappius, H.M. (1988) Significance of biogenic amines in functional disturbances resulting from brain injury. Metab. Brain Dis., 3: 303-310. Pappius, H.M. (1990) Neurochemical approaches to the amelioration of brain injury. J. Neural Transm. (Suppl), 29: 49 - 56. Pappius, H.M. (1991) Brain injury: new insights into neurotransmitter and receptor mechanisms. Neurochem. Res., 16: 941 -949. Pappius, H.M., Dadoun, R. and McHugh, M. (1988)The effect of p-chlorophenylalanine on cerebral metabolism and biogenic amine content of traumatized brain. J. Cereb. Blood Flow Metab., 8: 324- 334. Patel, M.S. (1974) The relative significance of C02-fixing enzymes in the metabolism of rat brain. J. Neurochem., 22: 717-724.

Patel, T.B., Sambasivarao, D. and Rashed, H.M. (1988) Role of calcium in synaptosomal substrate oxidation. Arch. Biochem. BiOphyS., 264: 368 - 375. Peng, L. and Hertz, L. (1992) Long-lasting abolishment of noradrenaline-induced stimulation of oxidative metabolism after chronic exposure of developing mouse astrocytes to cocaine. Brain Res., 581: 334-338. Peng, L., Juurlink, B.H.J. and Hertz, L. (1991) Difference in transmitter release, morphology, and ischemia-induced cell injury between cerebellar granule cell cultures developing in the presence and in the absence of a depolarizing potassium concentration. Dev. Brain Res., 63: 1 - 12. Pope, A. (1978) Neuroglia: quantitative aspects. In: E. Schoffeniels, G . Franck, L. Hertz and D.B. Tower (Eds.), Dynamic Properties of Glia Cells, Pergamon, Oxford, pp. 13 - 20. Porrino, L.J., Domer, F.R., Crane, A.M. and Sokoloff, L. (1988) Selective alteration in cerebral metabolism within the mesocorticolimbic dopaminergic system produced by acute cocaine administration in rats. Neuropsychopharmacology,1 : 109- 118.

Przywara, D.A., Bhave, S.V., Bhave, A., Wakade, T.D. and Wakade, A.R. (1991) Dissociation between intracellular Ca2+ and modulation of ['H]noradrenaline release in chick sympathetic neurons. J. Physiol. (Lond.), 437: 201 - 220. Quach, T.T., Rose, C. and Schwartz, J.C. (1978) [3H]Glycogen hydrolysis in brain slices: responses to neurotransmitters and modulation of noradrenaline receptors. J. Neurochem., 30: 1335 - 1341.

Quinlan, P.T. and Halestrap, A.P. (1986) The mechanism of the hormonal activation of respiration in isolated hepatocytes and its importance in the regulation of gluconeogenesis. Biochem. J., 236: 789-800. Rasmussen, H. (1989) The cycling of calcium as an intracellular messenger. Sci. Am., October: 66- 73. Rasmussen, H. and Barrett, P.Q. (1984) Calcium messenger system: an integrated view. Physiol. Rev., 64: 938 -984.

Richelson, E. (1990) The use of cultured cells in the study of mood-normalizing drugs. Pharmacol. Toxicol., 66 (Suppl. 3): 69-75.

Rogers, A.H., Armstead, W.M., Mirro, R., Busija, D.W. and Leffler, C. W. (1989) Influence of intraarterial norepinephrine on cerebral hemodynamics of newborn pigs. Proc. SOC. Exp. Biol. Med., 191: 174- 178. Rush, Jr., R.S., Carter, J.G., Martin, D., Nerbonne, J.M., Lampe, P.A., Pusaterl, M.E. and Lowry, O.H. (1991) Enzyme levels in cultured astrocytes, oligodendrocytes and Schwann cells, and neurons from the cerebral cortex and superior cervical ganglia of the rat. Neurochem. Res., 16: 991 -999.

Salm, A.K. and McCarthy, K.D. (1990) Norepinephrine-evoked calcium transients in cultured cerebral type 1 astroglia. Glia, 3: 529- 538.

Savaki, H.E., Kadekaro, M., McCulloc, J. and Sokoloff, F. (1982) The central noradrenergic system in the rat: metabolic mapping with alpha-adrenergic blocking agents. Brain Res., 234: 65 - 79. Schousboe, A., Drejer, J. and Hertz, L. (1988) Uptake and release of glutamate and glutamine in neurons and astrocytes in primary cultures. In: E. Kvamme (Ed.), Glutamine and Glutamate in Mammals, Vol. 2, CRC Press, Boca Raton, FL, pp. 21 -38. Schwartz, J.C., Arrang, J.M., Garbarg, M., Pollard, H., and Ruat, M. (1991) Histaminergic transmission in the mammalian brain. Physiol. Rev., 71: 1-51. Schwartz, J.P. and Costa, E. (1977) Regulation of nerve growth factor content in C6 glioma cells by beta-adrenergic receptor stimulation. Naunyn-Schmiedeberg's Arch. Pharmacol., 300: 123 - 129. Shain, W.G. and Martin, D.L. (1984) Activation of betaadrenergic receptors stimulates taurine release from glial cells. Cell. Mol. Neurobiol., 4: 191 - 196. Shank, R.P., Bennett, G.S., Freytag, S.D. and Campbell, G.L. (1985) Pyruvate carboxylase: an astrocyte-specific enzyme implicated in the replenishment of amino acid neurotransmitter pools. Brain Res., 329: 364- 367. Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, M.H., Patlack, C.S., Pettigrew, K.D., Salcurada, 0.and Shinohara, M. (1977) The (I4C) deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure and normal values in the conscious and anesthetized albino rat. J. Neurochem., 28: 897-916. Spear, L.P., Kirstein, C.L. and Frambes, N.A. (1989) Cocaine effects on the developing central nervous system: behavioral, psychopharmacological, and neurochemical studies. Ann. N . Y. Acad. Sci,. 562: 290 - 307. Steinbusch, H.W. (1991) Distribution of histaminergic neurons and fibers in rat brain. Comparison with noradrenergic and serotonergic innervation of the vestibular system. Acta Otolaryngol. (Suppl.), 479: 12 - 23. Subbarao, K.V. and Hertz, L. (1990a) Noradrenaline-induced

301 stimulation of oxidative metabolism in astrocytes but not in neurons in primary cultures. Brain Res., 527: 346 - 349. Subbarao, K.V. and Hertz, L. (1990b) Effect of adrenergic agonists on glycogenolysis in primary cultures of astrocytes. Brain Rex, 536: 220 - 226. Subbarao, K.V. and Hertz, L. (1991) Stimulation of energy metabolism in astrocytes by alpha-adrenergic agonists in primary cultures of astrocytes. J. Neurosci. Res., 28: 399 - 405. Sykova, E. (1983) Extracellular K + accumulation in the central nervous system. Prog. Biophys., 42: 135 - 189. Volkow, N.D., Fowler, J.S., Wolf, A.P., Hitzemann, R., Dewey, S., Bendriem, B., Alpert, R. and Hoff, A. (1991) Changes in brain glucose metabolism in cocaine dependence and withdrawal. A m . J. Psychiatry, 148: 621 -626. Wada, H., Inagaki, N., Itowi, N. and Yamatodani, A. (1991) Histaminergic neuron system: morphological features and possible functions. Agents Actions (Suppl.), 33: 1 1 - 27. Wakade,T.D., Bhave,S.V.,Bhave,A.S..Malhotra, R.K.and Wakade, A.R. (1991) Depolarizing stimuli and neurotransmitters utilize separate pathways to activate protein kinase C in sympathetic neurons. J. Biol. Chem., 266: 6424- 6428. Walz, W. (1989) Role of glial cells in the regulation of the brain

ion microenvironment. Prog. Neurobiol., 33: 309- 333. Walz, W . and Hertz, L. (1982) Ouabain-sensitive and ouabainresistant net uptake of potassium into astrocytes and neurons in primary cultures. J. Neurochem., 39: 70-77. Walz, W. and Hertz, L. (1983) Functional interactions between neurons and astrocytes. 11. Potassium homeostasis at the cellular level. Prog. Neurobiol., 20: 133- 183. Waterhouse, B.D. and Woodward, D.J. (1980) Interaction of norepinephrine with cerebrocortical activity evoked by stimulation of somatosensory afferent pathways in the rat. Exp. Neurol., 67: 1 1 - 34. Whetsell, W.O., Kohler, Jr., C. and Schwarcz, R. (1988) Quinolinic acid: a glia-derived excitotoxin in the mammalian central nervous system. In: M.D. Norenberg, L. Hertz and A. Schousboe (Eds.), The BiochemicalPathology of Astrocytes, Alan R. Liss, New York, pp. 191 -202. Yu, A.C.H., Drejer, J., Hertz, L. and Schousboe, A. (1983) Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. J. Neurochem., 41: 1484- 1487. Zhao, Z., Code, W.E. and Hertz, L. (1992) Dexmedetomidine, a potent and highly specific a,-agonist, evokes free intracellular calcium surge in astrocytes but not in neurons. Neuropharmacology, in press.