5845 Journal of Phy8iology (1996), 497.3, pp.745-751 745 The source of physiologically stimulated glutamate efflux from the striatum of conscious rats

Maddalena Miele, Martyn G. Boutelle and Marianne Fillenz * University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK

1. Glutamate in the extracellular compartment of the striatum of freely moving rats was monitored at 5 min intervals using microdialysis and an -based assay. 2. Basal levels of dialysate glutamate were 3-6 + 05 1uM. Local infusion through the dialysis probe of tetrodotoxin (TTX), cadmium chloride or magnesium chloride produced no reduction in basal levels of glutamate; with the latter two there was, instead, an increase. 3. Neuronal activation stimulated by induced grooming was accompanied by an increase in total glutamate efflux of 47 5 + 25-0% above basal level; this increase was not reduced by local infusion of 'ITX. 4. We propose that the ITX-insensitive release of glutamate in response to physiological stimulation is derived from glial cells and is a Ca2+-dependent mechanism triggered by a receptor-mediated release of Ca2+ from internal stores that spreads through the network of astrocytes.

Glutamate is the main central excitatory transmitter in recovery (Miele, Berners, Boutelle, Kusakabe & Fillenz, the vertebrate nervous system. This function has been 1996). We have used the zero net flux method of Lbnnroth extensively investigated using electrophysiological (Lonnroth, Jansson & Smith, 1987), which gives the true techniques. However glutamate, as well as being a neuro- extracellular concentration of glutamate at regions transmitter, has an additional role in energy and nitrogen undisturbed by the probe. metabolism. As a result, it is present in relatively high concentration in the cytoplasm of all neurones, as well as glia. Measurements of monoamine transmitters with micro- Glutamatergic nerve terminals, in addition to cytoplasmic dialysis have shown that their presence in the extracellular glutamate, have a vesicular compartment from which compartment represents overflow of neuronally released impulse-dependent release into the synaptic cleft is derived transmitter from the synapse. Local infusion of tetrodotoxin (for review see Fillenz, 1995). (TTX) (Westerink, Tuntler, Damsma, Rollema & De Vries, 1987; Sharp, Bramwell & Grahame-Smith, 1990; Morari, Released glutamate is cleared from the extracellular O'Connor, Ungerstedt & Fuxe, 1993; Vahabzadeh & Fillenz, compartment by carrier-mediated uptake into both 1994), omission of Ca2P or substitution by Mg2+ (Westerink glutamatergic neurones and into glial cells. This amino & De Vries, 1989; Morari et al. 1993), or infusion of Cd2+ acid transporter, located in the plasma membrane of (Westerink & De Vries, 1989; Sharp et al. 1990; Morari et al. glutamatergic and GABAergic neurones as well as glial cells 1993; Vahabzadeh & Fillenz, 1994) all result in a substantial (Gundersen, Danbolt, Ottersen & Storm-Mathisen, 1993), is reduction of the level in the dialysate of these neuro- specific for acidic amino acids and carries L-glutamate, transmitters. There is conflicting evidence concerning the L- and D-aspartate as well as L-cysteate and L-- origin of the extracellular glutamate measured by micro- sulphinate (Erecinska, Wantorsky & Wilson, 1983; dialysis. High K+ solutions lead to a parallel increase in Erecinska & Troeger, 1986). Reversal of the transporter can both glutamate and aspartate, suggesting a cytoplasmic lead to release of amino acids from the cytoplasmic origin; however, the K+-evoked release is reduced by compartment (Attwell, Barbour & Szatkowski, 1993). replacement of Ca2P with Mg2+ (Semba, Kito & Toru, 1995), Microdialysis has been used to monitor physiological release which is a criterion for exocytotic release. Similarly, the of excitatory amino acids in vivo (for review see Fillenz, origin of basal glutamate is not clear. Experiments using 1995). This technique cannot measure changes occurring in local infusion of ITX have reported no change (Moghaddam, the synapse, but provides information about changes in 1993), an increase (Keefe, Sved, Zigmond & Abercrombie, the extracellular compartment. We have recently used 1993), or a decrease (Rowley, Martin & Marsden, 1995). Ca2+ quantitative microdialysis to determine the extracellular removal or replacement with Mg2+ has been found to concentration of glutamate in the striatum and its in vivo decrease (Rowley et al. 1995) or produce a small, non-

* To whom correspondence should be addressed. 746 M. Miele, M. G. Boutelle and M. Fillenz J Phy8iol.497.3 significant increase (Westerink, Hofsteede, Damsma & De to form the outlet. The total exposed fibre length was 4 mm and the Vries, 1988). external diameter of the sampling region of the probe was 300 ,um. In spite of several attempts to monitor physiologically Enzyme packed-bed assay for glutamate stimulated changes in glutamate, only a few of these have The dialysate was analysed for glutamate using a flow injection been successful. There are reports of increases in extra- enzyme-based assay (Boutelle, Fellows & Cook, 1992). In brief, glutamate oxidase and horseradish peroxidase were immobilized on cellular glutamate in response to relatively severe stress such silica beads, which were then packed into a small bed. A phosphate as restraint, forced swimming or tail shock (Itoh, Saito, buffer containing the electrochemical mediator species ferrocene Fujimara, Watanabe & Saito, 1993; Keefe et al. 1993; Lowy, (100 mm Na2HPO4, 1 mm EDTA and 2 mm ferrocene mono- Gault & Yamamoto, 1993; Moghaddam, 1993). carboxylic acid, adjusted to pH 7'0, and 0'05% Kathon CG added to prevent bacterial growth) was pumped through the bed at In the present study we have used microdialysis in the 0'3 ml min-1 by an HPLC pump (model 2248; Pharmacia LKB). striatum of freely moving rats to monitor both basal Glutamate was oxidized by glutamate oxidase, producing H202. glutamate levels and changes resulting from mild Horseradish peroxidase converted this to H20 and was itself physiological stimulation. regenerated by the oxidation of the ferrocene species present in the buffer. The ferrocinium produced was electrochemically reversible and was detected by reduction at a downstream electrode. The METHODS detection limit of the enzyme bed was 4 pmol, equivalent to 10 #1 Surgery of 0'4/sM. Male Sprague-Dawley rats weighing 200-300 g were anaesthetized, The preoxidation system following published guidelines (Wolfensohn & Lloyd, 1994), with To reduce interference by ascorbic acid and other electroactive the following mixture: (a) Hypnorm, a combination of the compounds to acceptable levels, the dialysate was passed through a neuroleptic analgesic fentanyl (0-318 mg ml-') and fluanisone preoxidation system consisting of tubular electrodes (Berners, (10 mg ml-'), and (b) Hypnovel, the benzodiazepine midazolam Boutelle & Fillenz, 1994). The working electrode (Pt) is kept at (5 mg ml-'), and (c) sterile water, mixed 1:1: 2. This mixture was 600 mV vs. Ag-AgCl. At a flow rate of 2 ,sl min-' this reduces injected i.P. at a volume of 3-3 ml kg-', giving a dose of ascorbate and other electroactive species normally present in the 0'25 mg kg-' fentanyl, 0'8 mg kg-' fluanisone and 0'4 mg kg-' dialysate by more than 95%. midazolam. Surgery typically lasted 35 min and anaesthesia was reversed by an i.P. injection of naloxone (0'1 mg kg-'). As animals Materials began to recover from the anaesthetic they were given Vetergesic, Glutamate oxidase (EC 1.4.3.11) was a gift from Yamasa Shoyu, the long-lasting narcotic partial agonist buprenorphine (0'1 mg kg-', Tokyo, Japan. Horseradish peroxidase (EC 1.11.1.7, grade I) was s.c.), for pain relief. Despite its long half-life, buprenorphine is from Boehringer Mannheim. The silica beads used as the enzyme eliminated within 24 h and therefore does not confound the results support were from Merck. Glutamate (monosodium salt), ferrocene of the experiments. Occasionally, when surgery took longer, the monocarboxylic acid, o-phenylenediamine (puriss.) TTX, cadmium, animal was given a further I.M. injection of 0'1 ml of Hypnorm magnesium chloride and naloxone were from Sigma; fentanyl- every 10 min beyond the initial 30 min. fluanisone (Hypnorm) was from Janssen Pharmaceuticals Ltd, Once surgical anaesthesia was established, animals were placed in Oxford, UK; midazolam (Hypnovel) was from Roche Products Ltd, a stereotaxic frame and concentric microdialysis probes were Welwyn Garden City, UK; buprenorphine (Vetergesic) was from implanted in the right striatum, using the following co-ordinates: Reckitt and Colman Pharmaceuticals, Hull, UK. Kathon CG was 1 mm antero-posterior from bregma, 2'5 mm medio-lateral and from Rohm and Haas, Croydon, UK. Drugs were dissolved in -8'5 mm below the dura (Paxinos & Watson, 1986). Probes were artificial cerebrospinal fluid (ACSF) which contained the following secured in position using dental screws and acrylic (Associated (mM): NaCl, 147; KCl, 4; CaCl, 1'2; MgCl, 1'0. Dental Products Ltd, Swindon, UK). Amperometric measurements were made using EMS BP 1 Animals were allowed 24 h recovery. Animals were assessed for potentiostats from Electrochemical and Medical Systems, Newbury, good health, using the guidelines of Morton & Griffiths (1985), after UK. recovery from anaesthesia and at the beginning of the next day. All Experimental protocol animals used in this study had a score of 2 or less, as defined by Following surgery, the animals were placed in large plastic bowls Morton & Griffiths; in cases where the score was 3 or more, the (50 cm x 55 cm) and maintained in a temperature- and light- experiment was terminated. This work was carried out under controlled environment, with free access to food and water. licence in accordance with the Animals (Scientific Procedures) Act, Twenty-four hours after surgery experiments were carried out with 1986. The position of the probe in the brain was checked post the animal in its home bowl, allowing free movement. During mortem and in all cases the sampling membrane was located experiments the microdialysis probe was perfused with ACSF (mM: entirely within the striatum. NaCl, 147; KCl, 4; CaCl2, 1'2; and MgCl2, 1 (de Boer, Damsma, Microdialysis probe construction Fibiger, Timmerman, de Vries & Westerink, 1990) at a flow rate of Concentric microdialysis probes were constructed by inserting a 2 4a1 min-' using a CMA microinfusion pump (CMA Microdialysis, plastic-coated silica tube (Scientific Glass Engineering, Milton Stockholm, Sweden). The pH of this fluid is typically 6'5. Keynes, UK) and a tungsten wire (Goodfellow Metals, Cambridge, The perfusion fluid is not buffered, since fixing the pH of the UK) into a polyacrylonitrile dialysis fibre (Hospal Industrie, extracellular fluid (ECF) may interfere with pH-dependent Meyzieu, France) and gluing the fibre into a 12 mm stainless steel physiological processes. The buffering capacity of the ECF is cannula (Goodfellow Metals, Cambridge, UK). The tip was sealed sufficient to cope with the slight acidity of the perfusion fluid, since with epoxy glue and a second silica tube inserted into the cannula the efflux dialysate has a pH close to physiological pH. Dialysate is J Phy8iol.497.3 Source of stimulated glutamate release 747 passed through a preoxidation system of tubular electrodes in order RESULTS to remove ascorbic acid and other electrochemical interferents (Berners et al. 1994). For flow injection analysis, 10 usl samples were Basal glutamate collected in 250 l#1 microcentrifuge tubes (Alpha Laboratories, The basal glutamate level in the dialysate was 3-6 + 0 5 FM. Eastleigh, Hants, UK) attached to the outlet of the preoxidation This value represents the overall mean of six samples system. The sample was injected onto the enzyme bed through a collected at 5 min intervals from each of eighteen rats. Since Rheodyne injection valve equipped with a 10416 sample loop basal glutamate fluctuates with the state of activity of the (Anachem, Luton, UK). animal all results are shown as a percentage of the mean of Basal perfusate was collected for 2 h. Grooming was induced by six baseline samples before drug or stimulus application. dropping water from a plastic pipette onto the animal's nose. This stimulus was generally able to provoke 3-5 min of activity. TTX, Effect of tetrodotoxin CdCd2 and MgCl2 were dissolved in ACSF and perfused through the In order to investigate the contribution to basal glutamate microdialysis probe. of neuronally released transmitter, we used local application Data analysis of 'PJX, which, by blocking the voltage-gated Nae Glutamate levels in the dialysate are expressed in micromolar units channels, abolishes nerve impulse traffic. Basal levels in as-means + S.E.M. (n, number of rats). Basal levels were defined as these experiments were 2-8 + 1P2 FM. TTX, added to the the mean of six 5 min samples collected over a period of 30-60 min perfusion medium to a final concentration of 1P /SM, was prior to drug administration or stimulus application. perfused for 30 min. The mean level of glutamate during The response to grooming was measured as the increase in this period was 2 7 + 1 1 /FM (n= 6) (Fig. 1A). This glutamate above the baseline. Such increases were expressed as the difference is not statistically significant (P> 0 05). No area under the curve (AUC) measured in picomoles; they were decrease was observed in any animal. calculated by integration over the time interval of the increase above baseline values. Statistical comparison of the data was Effect of calcium replacement carried out by either Student's paired t test or the Mann-Witney U Cat+ in the perfusion medium was replaced with Cd2+, a test. non-specific Ca2+ channel blocker, in order to determine the

A 20 B 1000

CD 151 o S SE 750 CCD * SE : -a1:1 ao 3 0 500

0 1 2 o 250 TTX o4-_ -30 -20 -10 0 10 20 30 40 Time (min) Time (min)

C 160

140 0 D S.E c Cg 120-

ZD g

a -o

0 80

MgCI2 An I1I -60 -30 0 30 60 90 120 Time (min)

Figure 1. Basal dialysate glutamate levels A, the effect of a 30 min infusion of 1-5 uM TTX. B, the effect of a 60 min infusion of 1'2 mm CdCl2. C, the effect of a 90 min infusion of 20 mM MgC12. 748 M. Miele, M. G. Boutelle and M. Fillenz J. Physiol.497.3 dependence of extracellular glutamate on Ca2+ influx through delay of 15-20 min glutamate levels showed an increased Ca2+ channels. The mean basal glutamate level in dialysate efflux of 50 + 18 pmol above baseline which was not collected during perfusion with normal ACSF over a period statistically significant (P < 0 07, paired t test). When of 1 h was 5-2 + 0 5 /M (n = 5). The perfusion fluid was perfusion was switched to normal ACSF, glutamate levels then switched to one in which CdCl2 (1P2 mM) replaced recovered to control values (Fig. 1 C). CaCl2 and the perfusion continued for a further 1 h. During the first 15 min of perfusion there was no change in the level Minor and transient behavioural changes (increased of glutamate (Fig. 1B). After this period, however, glutamate exploratory activity, sniffing) were observed during MgCl2 levels began to rise steeply. The increase persisted throughout perfusion. perfusion with CdCl2 and began to decrease as soon as Stimulated glutamate release perfusion with normal ACSF was resumed, although Effect of grooming. The mean basal concentration in the glutamate levels did not recover to basal values. dialysate was 3-5 + 0 9 ,UM (n = 9). Dropping water onto The rise in glutamate concentration was accompanied by the animal's nose from a pipette elicited grooming. Grooming marked stereotyped behaviour; the animals showed intense for 5 min caused an increase in glutamate which outlasted exploratory activity, rearing, sniffing, licking, and ipsiversive the period of activity (mean duration, 16-6 + 2-3 min). The turning. behavioural response usually began a few seconds after the application of the stimulus. Occasional spontaneous grooming Effect of magnesium was also accompanied by an increase in glutamate. The Because of the unexpected effects of Cd2+, we carried out a total efflux into the dialysate during the glutamate increase separate set of experiments on the dependence of basal (AUC) was 160 + 48 pmol, compared with 133 + 44 pmol glutamate on extracellular Ca2+, in which 20 mm MgCl2 was during the equivalent control period. The difference in efflux added to Ca2+-free perfusion medium. The mean dialysate gives the increase in glutamate which accompanies level during perfusion with normal ACSF collected over 1 h activation as 26-0 + 7-2 pmol, equivalent to an increase of was 2 3 + 02 /uM (n = 4). Perfusion was switched to ACSF 47-5 + 25-0% above baseline (P < 0.01, n = 9). Glutamate containing 20 mm MgCl2 for a period of 90 min. After a levels then returned to basal values (Fig. 2A).

A 300

0

0 2502

0 10 co 200 D 0 0 0) 0o15CD a a1004 s - . I -T 0 Figure 2. Changes in dialysate glutamate levels in 0) response to physiological stimulation 50 _ At 0 time a period of 3 min grooming was induced by -30 -20 -10 0 10 20 30 40 50 dropping water onto the rat's snout in the presence of Time (min) normal perfusion medium (A) and in the presence of B 300 1-5 /SM TTX in the perfusion medium (B).

0a) E 250

co ' 200

0 0) 0) co0 5 CL100' 0a0 50 ' -30 -20 -10 0 10 20 30 40 50 Time (min) J Phy8iol. 497.3 Source of stimulated glutamate release 749

Effect of tetrodotoxin. In five animals, grooming in the and/or glia (Attwell et al. 1993). Recently two additional presence of normal ACSF was followed by the addition of mechanisms have been described; one of these involves ITX to a final concentration of 1P5 FM in the perfusion release-regulating hetero-transporters (Bonanno & Raiteri, medium and the grooming stimulus was then repeated. The 1994) and the other is neuroligand-evoked release of mean basal dialysate level of glutamate in the presence of glutamate and aspartate from glial cells (Parpura, Liu, ACSF was 341 + 041 #m; the mean basal level after addition Jeftinija, Haydon & Jeftinija, 1995b). Under physiological of TTX was 2-8 + 141 /M (n = 5). The increase in glutamate conditions only exocytosis is TTX sensitive and dependent which accompanied grooming (AUC) was 29 + 10 pmol in on the influx of Ca2+ through voltage-gated ion channels. the presence of ACSF and 61 + 33 pmol in the presence of Reversal of the uptake carrier and hetero-transporter TTX (Fig. 2B); there is no statistically significant difference release are both Na+ but not Ca2+ dependent. The between the increases under these two conditions (P = 0-29, neuroligand-evoked release depends on a rise of [Ca2+]i paired t test). There was no observable difference in derived from intracellular stores following activation of grooming behaviour after administration of TTX. phospholipase C. This release is insensitive to TTX, blockers of Ca2+ channels and inhibitors of the glutamate transporter (Parpura, Basarsky, Liu, Jeftinija, Jeftinija & Haydon, DISCUSSION 1994; Parpura, Liu, Brethorst, Jeftinija, Jeftinija & This study was prompted by the conflicting experimental Haydon, 1995a). Of these four mechanisms, reversal of the results concerning the source of extracellular glutamate. uptake carrier is more likely to occur under pathological The experiments were designed to test the hypothesis that than physiological conditions since it requires substantial physiologically stimulated increases in glutamate depend, changes in membrane potential or ion gradients (Attwell like the release of classical , on nerve et al. 1993). impulse traffic and exocytosis. In the present study local infusion of 1S FuM TTX, which We carried out a range of tests under uniform experimental produces a profound decrease in the basal release of conditions, i.e. 24 h after surgery, using the same probes, (Westerink et al. 1987), 5-HT and noradrenaline perfusion rates and ACSF. Most previous microdialysis (Vahabzadeh & Fillenz, 1994), had no effect on basal levels studies of glutamate release have used pre-derivatization of glutamate. Similarly, there was no decrease in basal followed by high-performance liquid chromatography and glutamate when Ca2+ was replaced by either Mg2+ or Cd2+. either fluorescence, or more recently, electrochemical Mg2+ caused a small, non-significant increase. An increase in detection (Rowley et al. 1995). These techniques measure basal glutamate during infusion of either Mg2+ (Westerink both glutamate and aspartate, but because of the pre- et al. 1988) or TTX (Herrera-Marschitz et al. 1996) has been derivatization and the separation steps, sampling is usually reported by others. The delayed large increase of glutamate carried out at intervals of 10-15 min. Our recently developed caused by infusion of Cd2+ was very surprising, but seems enzyme-based assays (Boutelle et al. 1992; Berners et al. to be characteristic of the striatum. We have observed a 1994) using the enzyme glutamate oxidase, have a high time similar delayed increase in together with behavioural resolution, are on-line, but measure glutamate only. activation after infusion of Cd2+ into the striatum (A. E. We used a physiological stimulus that elicits a normal activity Fray, unpublished observations). Infusion of TTX into the of the rat. We chose the striatum because any stimulus that striatum also produces behavioural activation and an increase elicits motor activation causes neuronal activation in this in glucose and ascorbate, but this has a latency of 1 h or brain region, as indicated by the increase in the release of more (A. E. Fray & M. Miele, unpublished observations). dopamine (Boutelle, Zetterstrom, Pei, Svensson & Fillenz, Infusion of the same concentration of Cd2+ or ITX into the 1990). Furthermore, induced grooming causes an increase in hippocampus for periods of up to 3 h produces a profound local cerebral blood flow (Boutelle & Fillenz, 1996), a criterion reduction in noradrenaline and 5-HT and no behavioural generally accepted as indicating neuronal activation and one activation (Vahabzadeh & Fillenz, 1994). A possible that is the basis of positron emission tomography (PET). explanation is that infusion of both Cd2+ and TTX produces a large reduction in basal release of dopamine. Since there are Although reliable, the induced grooming response shows inhibitory dopamine receptors on glutamatergic nerve considerable variation in magnitude and time course. This is terminals (Yamamoto & Davy, 1992), the removal of this the reason for the large standard error in the size of the presynaptic inhibition may cause an increase in the increased efflux in response to stimulation. neuronal release of glutamate, which triggers the glial Glutamate is not only stored in a number of different cellular release. compartments, but is also released by a number of distinct The characteristics of basal extracellular glutamate clearly mechanisms. Exocytosis from the vesicular compartment indicate that it is not derived from exocytotic release releases only glutamate. Reversal of the Na+-dependent associated with basal impulse traffic. Ca2P-independent Nae- glutamate carrier leads to release of both glutamate and dependent GABA-mediated release of glutamate has been aspartate from the cytoplasmic compartment of neurones demonstrated in cortical and hippocampal synaptosomes 750 M. Miele, M. 0. Boutelle and M. Fillenz J Physiol.497.3

(Bonanno, Pittaluga, Fedele, Fontana & Raiteri, 1993). both glutamate and aspartate in striatal dialysate, which is GABA heterocarriers on striatal glutamatergic nerve an indication that the release is from the cytoplasmic terminals have not so far been reported and the presence of compartment. such heterocarriers on glial cells has not been investigated. The present findings are an illustration of the fact that The striatum has a rich GABAergic innervation and electrophysiological recordings and microdialysis provide heterocarrier-mediated release of glutamate provides a information about very different aspects of neuronal function. possible source for basal glutamate. Intracellular microelectrode recording provides information Induced grooming, elicited by a mild stimulus, produced a about synaptic mechanisms. In vivo chemical monitoring significant increase in glutamate; this is the first such report. techniques, which include microdialysis and voltammetry, The increase outlasted the end of the stimulus by 10 min provide information about changes in the extracellular and was not reduced by the infusion of 1P5 uM TTX. compartment, which parallel rapid synaptic transmission. Although the difference in release under control conditions They include overflow of transmitters from the synapse, and in the presence of TTX is not statistically significant, release of into the extracellular compartment and possibly because of the large standard error attributable to release from glial cells of lactate, glucose, ascorbate and the variation in the grooming response commented on excitatory amino acids. Such changes in the extracellular above, there is a suggestion that TTX actually increases the compartment are not involved directly in fast synaptic stimulated response. transmission, but have widespread effects mediated by extrasynaptic receptors. This stimulated TTX-insensitive release may be due to neuroligand-evoked release from glial cells. Release of glutamate and aspartate from cultured neocortical astrocytes and organotypic cultures of Schwann cells, which is ATTWELL, D., BARBOUR, B. & SZATKOWSKI, M. (1993). Nonvesicular unaffected by inhibitors of the glutamate transporter, has release of . Neuron 11, 401-407. been shown to be due to a rise in [Ca2+]i (Parpura et al. BERNERS, M. 0. M., BOUTELLE, M. G. & FILLENZ, M. (1994). On-line 1994). The rise in [Ca2P]1 follows receptor-mediated measurement of brain glutamate with an enzyme/polymer-coated activation of phosopholipase C and is derived from inositol tubular electrode. Analytical Chemistry 66, 2017-2021. trisphosphate (IP3)-sensitive intracellular stores (Parpura et BONANNO, G., PITTALUGA, A., FEDELE, E., FONTANA, G. & RAITERI, al. 1995 b). Although insensitive to blockers of Ca2P channels, M. (1993). and y-aminobutyric acid modulate each removal of Ca2+ leads to failure of the mechanism because of other's release through heterocarriers sited on the axon terminals of depletion of the intracellular Ca2+ stores. One class of rat brain. Journal of Neurochemistry 61, 222-230. metabotropic glutamate receptors activate phospholipase C BONANNO, G. & RAITERI, M. (1994). Release-regulating presynaptic (Pin & Duvoisin, 1995). There is now extensive evidence heterocarriers. Progress in Neurobiology 44, 451-462. that glutamate, acting on glial glutamate receptors, induces BOUTELLE, M. G., FELLOWS, L. K. & COOK, C. (1992). Enzyme packed propagating Ca2+ waves in astrocytes coupled by gap bed system for on-line measurement of glucose, glutamate, and lactate in brain microdialysate. Analytical Chemistry 64, junctions (Cornell-Bell, Finkbeiner, Cooper & Smith, 1990; 1790-1794. Finkbeiner, 1992; Kim, Rioult & Cornell-Bell, 1994). This BOUTELLE, M. G. & FILLENZ, M. (1996). A combined probe to study phenomenon is not restricted to astrocytes in culture, but the pharmacology of physiologically stimulated regional blood flow occurs in organotypically cultured slices of rat hippocampus in the rat brain. Journal of Physiology 491.P, 29P (Dani, Chernjavsky & Smith, 1992). Such propagation of BOUTELLE, M. G., ZETTERSTROM, T., PEI, Q., SVENSSON, L. & Ca2+ waves through networks of astrocytes, and the resulting FILLENZ, M. (1990). 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