Ceftriaxone attenuates acute cocaine-evoked dopaminergic neurotransmission in the nucleus accumbens of the rat.

BACKGROUND AND PURPOSE: Ceftriaxone is a ß-lactam antibiotic and glutamate transporter activator that reduces the reinforcing effects of psychostimulants. Ceftriaxone also reduces locomotor activation following acute psychostimulant exposure, suggesting that alterations in dopamine transmission in the nucleus accumbens contribute to its mechanism of action. In the present studies we tested the hypothesis that pretreatment with ceftriaxone disrupts acute cocaine-evoked dopaminergic neurotransmission in the nucleus accumbens. EXPERIMENTAL APPROACH: Adult male Sprague-Dawley rats were pretreated with saline or ceftriaxone (200 mg kg(-1) , i.p. × 10 days) and then challenged with cocaine (15 mg kg(-1) , i.p.). Motor activity, dopamine efflux (via in vivo microdialysis) and protein levels of tyrosine hydroxylase (TH), the dopamine transporter and organic cation transporter as well as α-synuclein, Akt and GSK3ß were analysed in the nucleus accumbens. KEY RESULTS: Ceftriaxone-pretreated rats challenged with cocaine displayed reduced locomotor activity and accumbal dopamine efflux compared with saline-pretreated controls challenged with cocaine. The reduction in cocaine-evoked dopamine levels was not counteracted by excitatory amino acid transporter 2 blockade in the nucleus accumbens. Pretreatment with ceftriaxone increased Akt/GSK3ß signalling in the nucleus accumbens and reduced levels of dopamine transporter, TH and phosphorylated α-synuclein, indicating that ceftriaxone affects numerous proteins involved in dopaminergic transmission. CONCLUSIONS AND IMPLICATIONS: These results are the first evidence that ceftriaxone affects cocaine-evoked dopaminergic transmission, in addition to its well-described effects on glutamate, and suggest that its ability to attenuate cocaine-induced behaviours, such as psychomotor activity, is due in part to reduced dopaminergic neurotransmission in the nucleus accumbens.

Mephedrone ("bath salt") pharmacology: insights from invertebrates.

Psychoactive bath salts (also called meph, drone, meow meow, m-CAT, bounce, bubbles, mad cow, etc.) contain a substance called mephedrone (4-methylcathinone) that may share psychostimulant properties with amphetamine and cocaine. However, there are only limited studies of the neuropharmacological profile of mephedrone. The present study used an established invertebrate (planarian) assay to test the hypothesis that acute and repeated mephedrone exposure produces psychostimulant-like behavioral effects. Acute mephedrone administration (50-1000 µM) produced stereotyped movements that were attenuated by a dopamine receptor antagonist (SCH 23390) (0.3 µM). Spontaneous discontinuation of mephedrone exposure (1, 10 µM) (60 min) resulted in an abstinence-induced withdrawal response (i.e. reduced motility). In place conditioning experiments, planarians in which mephedrone (100, 500 µM) was paired with the non-preferred environment during conditioning displayed a shift in preference upon subsequent testing. These results suggest that mephedrone produces three behavioral effects associated with psychostimulant drugs, namely dopamine-sensitive stereotyped movements, abstinence-induced withdrawal, and environmental place conditioning.

ß-lactamase inhibitors display anti-seizure properties in an invertebrate assay.

Antibiotics containing a beta-lactam ring (e.g. ceftriaxone) display anti-glutamate effects that underlie their efficacy in animal models of central nervous system (CNS) diseases [Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, Jin L, Dykes Hoberg M, Vidensky S, Chung DS, Toan SV, Bruijn LI, Su ZZ, Gupta P, Fisher PB (2005) Nature 433:73-77]. We hypothesized that the structurally related beta-lactamase inhibitors (clavulanic acid, tazobactam)--which also contain a beta-lactam ring--will mimic ceftriaxone efficacy in an invertebrate (planarian) assay designed to screen for anti-seizure activity [Rawls SM, Thomas T, Adeola M, Patil T, Raymondi N, Poles A, Loo M, Raffa RB (2009) Pharmacol Biochem Behav 93:363-367]. Glutamate or cocaine administration produced planarian seizure-like activity (pSLA). Glutamate- or cocaine-induced pSLA was inhibited by ceftriaxone, clavulanic acid, or tazobactam, but not by the non-beta-lactam antibiotic vancomyocin. The present findings indicate beta-lactamase inhibitors display efficacy, and mimic ceftriaxone activity, in an invertebrate anti-seizure screen. These results suggest beta-lactamase inhibitors--particularly ones such as clavulanic acid that display enhanced brain penetrability, oral bioavailability, and negligible anti-bacterial activity--might offer an attractive alternative to direct antibiotic therapy for managing CNS diseases caused by increased glutamate transmission and provide a solution to the growing concern that ceftriaxone will be of only limited utility as a CNS-active therapeutic because of its intolerable side effects.

The beta-lactam antibiotic, ceftriaxone, attenuates morphine-evoked hyperthermia in rats.

BACKGROUND AND PURPOSE: Beta-lactam antibiotics are the first practical pharmaceuticals capable of increasing the expression and activity of the glutamate transporter, GLT-1, in the CNS. However, the functional impact of beta-lactam antibiotics on specific drugs which produce their pharmacological effects by increasing glutamatergic transmission is unknown. One such drug is morphine, which causes hyperthermia in rats, mediated by an increase in glutamatergic transmission. Since drugs (e.g. antibiotics) that enhance glutamate uptake also decrease glutamatergic transmission, we tested the hypothesis that ceftriaxone, a beta-lactam antibiotic, would block the glutamate-dependent portion of morphine-evoked hyperthermia. EXPERIMENTAL APPROACH: A body temperature assay was used to determine if ceftriaxone decreased morphine-induced hyperthermia in rats by increasing glutamate uptake. KEY RESULTS: Body temperatures of rats treated with ceftriaxone (200 mg kg(-1), i.p. x 7 days) did not differ from rats receiving saline. Morphine (1, 4, 8 and 15 mg kg(-1), s.c.) caused significant hyperthermia. Pre-treatment with ceftriaxone, as described above, decreased the hyperthermic response to these doses of morphine. The effects of ceftriaxone were prevented by TBOA (0.2 micromol, i.c.v.), an inhibitor of glutamate transport. CONCLUSIONS AND IMPLICATIONS: Ceftriaxone attenuated the hyperthermia caused by morphine, an effect prevented by inhibiting glutamate transport. Thus this effect of ceftriaxone was most likely mediated by increased glutamate uptake. These data revealed a functional interaction between ceftriaxone and morphine and indicated that a beta-lactam antibiotic decreased the efficacy of morphine in conscious rats.

Icilin evokes a dose- and time-dependent increase in glutamate within the dorsal striatum of rats.

Icilin, the peripheral cold channel agonist, activates TRPM8 and TRPA1, localized on dorsal root ganglia and trigeminal neurons in rats. Icilin precipitates immediate wet-dog shakes in this species, which are antagonized by centrally acting mu and kappa opioid agonists, implicating the central nervous system in the behavioral response. We studied the effect icilin has on glutamate levels in the dorsal striatum, a brain region involved in movement. Icilin (0.25, 0.5 and 0.75 mg/kg, i.p.) elicited a dose- and time-dependent increase in glutamate within the striatum, indicative of icilin's neurochemical effect in rats.

GABAA receptors modulate cannabinoid-evoked hypothermia.

Cannabinoids evoke hypothermia by stimulating central CB(1) receptors. GABA induces hypothermia via GABA(A) or GABA(B) receptor activation. CB(1) receptor activation increases GABA release in the hypothalamus, a central locus for thermoregulation, suggesting that cannabinoid and GABA systems may be functionally linked in body temperature regulation. We investigated whether GABA receptors modulate the hypothermic actions of [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one] (WIN 55212-2), a selective cannabinoid agonist, in male Sprague-Dawley rats. WIN 55212-2 (2.5 mg/kg im) produced a rapid hypothermia that peaked 45-90 min postinjection. The hypothermia was attenuated by bicuculline (2 mg/kg ip), a GABA(A) antagonist. However, SCH 50911 (1-10 mg/kg ip), a GABA(B) blocker, did not antagonize the hypothermia. Neither bicuculline (2 mg/kg) nor SCH 50911 (10 mg/kg) by itself altered body temperature. We also investigated a possible role for CB(1) receptors in GABA-generated hypothermia. Muscimol (2.5 mg/kg ip), a GABA(A) agonist, or baclofen (5 mg/kg ip), a GABA(B) agonist, evoked a significant hypothermia. Blockade of CB(1) receptors with SR141716A (2.5 mg/kg im) did not antagonize muscimol- or baclofen-induced hypothermia, indicating that GABA-evoked hypothermia does not contain a CB(1)-sensitive component. Our results implicate GABA(A) receptors in the hypothermic actions of cannabinoids and provide further evidence of a functional link between cannabinoid and GABA systems.

L-NAME (N omega-nitro-L-arginine methyl ester), a nitric-oxide synthase inhibitor, and WIN 55212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one], a cannabinoid agonist, interact to evoke synergistic hypothermia.

Cannabinoids evoke profound hypothermia in rats by activating central CB(1) receptors. Nitric oxide (NO), a prominent second messenger in central and peripheral neurons, also plays a crucial role in thermoregulation, with previous studies suggesting pyretic and antipyretic functions. Dense nitric-oxide synthase (NOS) staining and CB(1) receptor immunoreactivity have been detected in regions of the hypothalamus that regulate body temperature, suggesting that intimate NO-cannabinoid associations may exist in the central nervous system. The present study investigated the effect of N(omega)-nitro-L-arginine methyl ester (L-NAME), a NO synthase inhibitor, on the hypothermic response to WIN 55212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one], a selective cannabinoid agonist, in rats. WIN 55212-2 (1-5 mg/kg, i.m.) produced dose-dependent hypothermia that peaked 45 to 90 min post-injection. L-NAME (10-100 mg/kg, i.m.) by itself did not significantly alter body temperature. However, a nonhypothermic dose of L-NAME (50 mg/kg) potentiated the hypothermia caused by WIN 55212-2 (0.5-5 mg/kg). The augmentation was strongly synergistic, indicated by a 2.5-fold increase in the relative potency of WIN 55212-2. The inactive enantiomer of WIN 55212-2, WIN 55212-3 [S-(-)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-napthanlenyl) methanone mesylate] (5 mg/kg, i.m.), did not produce hypothermia in the absence or presence of L-NAME (50 mg/kg), confirming that cannabinoid receptors mediated the synergy. The present data are the first evidence that drug combinations of NOS blockers and cannabinoid agonists produce synergistic hypothermia. Thus, NO and cannabinoid systems may interact to induce superadditive hypothermia.

N-methyl-D-aspartate antagonists and WIN 55212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one], a cannabinoid agonist, interact to produce synergistic hypothermia.

CB(1) cannabinoid receptors mediate profound hypothermia when cannabinoid agonists are administered to rats. Glutamate, the principal excitatory neurotransmitter in the central nervous system (CNS), is thought to tonically increase body temperature by activating N-methyl-D-aspartate (NMDA) receptors. Because NMDA antagonists block cannabinoid-induced antinociception and catalepsy, intimate glutamatergic-cannabinoid interactions may exist in the CNS. The present study investigated the effect of two NMDA antagonists on the hypothermic response to WIN 55212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one], a selective cannabinoid agonist, in rats. WIN 55212-2 (1-10 mg/kg i.m.) produced dose-dependent hypothermia that peaked 60 to 180 min postinjection. Dextromethorphan (5-75 mg/kg i.m.), a noncompetitive NMDA antagonist, or LY 235959 [(-)-6-[phosphonomethyl-1,2,3,4,4a,5,6,7,8,8a-decahydro-isoquinoline-2-carboxylate]](1-4 mg/kg i.m.), a competitive and highly selective NMDA antagonist, evoked hypothermia in a dose-sensitive manner, suggesting that endogenous glutamate exerts a hyperthermic tone on body temperature. A dose of dextromethorphan (10 mg/kg) that did not affect body temperature by itself potentiated the hypothermic response to WIN 55212-2 (1, 2.5, or 5 mg/kg). The enhancement was strongly synergistic, indicated by a 2.7-fold increase in the relative potency of WIN 55212-2. Similarly, a dose of LY 235959 (1 mg/kg) that did not affect body temperature augmented the hypothermia associated with a single dose of WIN 55212-2 (2.5 mg/kg), thus confirming that NMDA receptors mediated the synergy. We have demonstrated previously that CB(1) receptors mediate WIN 55212-2-evoked hypothermia in rats. The present data are the first evidence that NMDA antagonists exert a potentiating effect on cannabinoid-induced hypothermia. Taken together, these data suggest that interactions between NMDA and CB(1) receptors produce synergistic hypothermia.

Sigma sites mediate DTG-evoked hypothermia in rats.

1,3,-Di-o-tolylguanidine (DTG), a sigma agonist, produces hypothermia in rats, but the inability of purported sigma antagonists to block the hypothermia suggests that sites other than sigma may mediate the effect. Recently, N-[2-(3,4-dichlorophenyl) ethyl]-N-methyl-2-(dimethylamino) ethylamine (BD 1047) has been identified as a functional sigma antagonist in vivo because of its high selectivity for sigma sites and its ability to block DTG-induced dystonia and cocaine-evoked behaviors. Therefore, the present study investigated the effect of BD 1047 on DTG-evoked hypothermia. DTG (1, 10, 20 and 30 mg/kg sc) induced dose-dependent hypothermia. The onset of DTG-induced hypothermia was rapid, with a reduction in body temperature observed 15 min postinjection. To determine whether sigma sites mediated DTG-induced hypothermia, BD 1047 was injected 30 min prior to DTG. BD 1047 (1, 5, 7.5 and 10 mg/kg sc) attenuated the hypothermia in a dose-dependent fashion, thus revealing a sigma site mechanism. The injection of BD 1047 alone did not alter body temperature, suggesting that endogenous sigma systems do not play a tonic role in thermoregulation. The present experiments demonstrate for the first time that a selective sigma antagonist attenuates sigma agonist-induced hypothermia. Moreover, these data provide further evidence that BD 1047 is an effective antagonist for characterizing sigma-mediated effects in vivo.

Delta opioid receptors regulate calcium-dependent, amphetamine-evoked glutamate levels in the rat striatum: an in vivo microdialysis study.

Blockade of opioid receptors decreases amphetamine-induced behaviors and dopamine release in the striatum. Use of selective opioid receptor ligands has indicated that these effects are mediated by delta opioid receptors (DORs). However, the site of action of delta receptors and the influence of delta receptor antagonists on other neurotransmitters released by amphetamine are unknown. Therefore, the effect of reverse microdialysis of the selective delta opioid antagonist, naltrindole, on extracellular striatal glutamate levels evoked by amphetamine (2.5 mg/kg, i.p.) was investigated. Naltrindole (10-100 microM) decreased amphetamine-evoked glutamate levels in a concentration-dependent manner. The selective delta agonist, [D-Pen(2,5)]-enkephalin (100, 500 microM), reversed the effect of naltrindole, confirming that delta receptors mediated this effect. The amphetamine-evoked increase in extracellular glutamate levels was determined to be 39% calcium-sensitive by lowering the calcium concentration in the perfusate. Under these conditions, naltrindole had no effect on the calcium-independent component of amphetamine-evoked glutamate levels. These data indicate that intrastriatal DORs modulate a calcium-dependent, amphetamine-evoked component of extracellular glutamate levels that may depend on activation of a transsynaptic basal ganglia-thalamo-cortical loop.

Presynaptic kappa-opioid and muscarinic receptors inhibit the calcium-dependent component of evoked glutamate release from striatal synaptosomes.

In addition to cytosolic efflux, reversal of excitatory amino acid (EAA) transporters evokes glutamate exocytosis from the striatum in vivo. Both kappa-opioid and muscarinic receptor agonists suppress this calcium-dependent response. These data led to the hypothesis that the calcium-independent efflux of striatal glutamate evoked by transporter reversal may activate a transsynaptic feedback loop that promotes glutamate exocytosis from thalamo- and/or corticostriatal terminals in vivo and that this activation is inhibited by presynaptic kappa and muscarinic receptors. Corollaries to this hypothesis are the predictions that agonists for these putative presynaptic receptors will selectively inhibit the calcium-dependent component of glutamate released from striatal synaptosomes, whereas the calcium-independent efflux evoked by an EAA transporter blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), will be insensitive to such receptor ligands. Here we report that a muscarinic agonist, oxotremorine (0.01-10 microM), and a kappa-opioid agonist, U-69593 (0.1-100 microM), suppressed the calcium-dependent release of glutamate that was evoked by exposing striatal synaptosomes to the potassium channel blocker 4-aminopyridine. The presynaptic inhibition produced by these ligands was concentration dependent, blocked by appropriate receptor antagonists, and not mimicked by the delta-opioid agonist [D-Pen2,5]-enkephalin. The finding that glutamate efflux evoked by L-trans-PDC from isolated striatal nerve endings was entirely calcium independent supports the notion that intact basal ganglia circuitry mediates the calcium-dependent effects of this agent on glutamate efflux in vivo. Furthermore, because muscarinic or kappa-opioid receptor activation inhibits calcium-dependent striatal glutamate release in vitro as it does in vivo, it is likely that both muscarinic and kappa receptors are inhibitory presynaptic heteroceptors expressed by striatal glutamatergic terminals.

The kappa-opioid agonist, U-69593, decreases acute amphetamine-evoked behaviors and calcium-dependent dialysate levels of dopamine and glutamate in the ventral striatum.

The effects of a kappa-opioid receptor agonist on acute amphetamine-induced behavioral activation and dialysate levels of dopamine and glutamate in the ventral striatum were investigated. Amphetamine (2.5 mg/kg i.p.) evoked a substantial increase in rearing, sniffing, and hole-poking behavior as well as dopamine and glutamate levels in the ventral striatum of awake rats. U-69593 (0.32 mg/kg s.c.) significantly decreased the amphetamine-evoked increase in behavior and dopamine and glutamate levels in the ventral striatum. Reverse dialysis of the selective kappa-opioid receptor antagonist, nor-binaltorphimine, into the ventral striatum antagonized the effects of U-69593 on amphetamine-induced behavior and dopamine and glutamate levels. Reverse dialysis of low calcium (0.1 mM) into the ventral striatum decreased basal dopamine, but not glutamate, dialysate levels by 91% 45 min after initiation of perfusion. Strikingly, 0.1 mM calcium perfusion significantly reduced the 2.5 mg/kg amphetamine-evoked increase in dopamine and glutamate levels in the ventral striatum, distinguishing a calcium-dependent and a calcium-independent component of release. U-69593 did not alter the calcium-independent component of amphetamine-evoked dopamine and glutamate levels. These data are consistent with the view that a transsynaptic mechanism augments the increase in dopamine and glutamate levels in the ventral striatum evoked by a moderately high dose of amphetamine and that stimulation of kappa-opioid receptors suppresses the calcium-dependent component of amphetamine's effects.

Muscarinic receptors regulate extracellular glutamate levels in the rat striatum: an in vivo microdialysis study.

Regulation of extracellular glutamate levels by muscarinic receptors in the striatum of unanesthetized rats was investigated by microdialysis. Extracellular glutamate levels were elevated by intrastriatal perfusion of L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), a competitive substrate of plasma membrane excitatory amino acid transporters. The nonselective muscarinic agonist, oxotremorine (0.5-54 microM) significantly decreased L-trans-PDC-evoked glutamate levels in a concentration-dependent manner. Scopolamine (0.1-10 microM), a nonselective muscarinic receptor antagonist, reversed the effect of oxotremorine, which confirms that muscarinic receptor activation mediated the reduction of L-trans-PDC-evoked glutamate levels. In addition, scopolamine (10 microM) significantly elevated basal extracellular glutamate levels, an effect prevented by oxotremorine, which suggests that acetylcholine tonically regulates glutamatergic transmission in the striatum. Previous data from this laboratory have shown that L-trans-PDC-evoked glutamate levels are partially calcium-dependent. The present study demonstrated that attenuation of L-trans-PDC-evoked glutamate levels by reduced calcium was not altered by oxotremorine. Therefore, it is likely that muscarinic receptors regulate calcium-dependent glutamate release evoked by L-trans-PDC.

Kappa receptor activation attenuates L-trans-pyrrolidine-2,4-dicarboxylic acid-evoked glutamate levels in the striatum.

The effects of local kappa receptor activation and blockade on extracellular striatal glutamate levels evoked by reverse microdialysis of L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) were investigated. L-trans-PDC elevates extracellular glutamate levels in vivo by acting as a competitive substrate for plasma membrane excitatory amino acid transporters. The selective kappa-opioid receptor agonist U-69593 (1-100 nM) significantly attenuated L-trans-PDC-stimulated glutamate levels in a concentration-dependent manner. The selective kappa receptor antagonist nor-binaltorphimine (1-100 nM) reversed the U-69593-induced decrease in L-trans-PDC-evoked glutamate levels also in a concentration-dependent manner, indicating that the U-69593-induced reduction was mediated by kappa receptor activation. In addition, nor-binaltorphimine significantly elevated basal extracellular glutamate levels, implying that kappa receptors tonically regulate glutamate efflux in the striatum. Previous data from this laboratory have shown that L-trans-PDC-evoked extracellular glutamate levels are partially calcium-sensitive. The present study demonstrated that the inhibition of L-trans-PDC-evoked glutamate levels by reduced calcium perfusion was not altered by U-69593. Therefore, kappa receptors regulate the calcium-dependent component of L-trans-PDC-evoked extracellular glutamate levels in the striatum.

L-trans-pyrrolidine-2,4-dicarboxylic acid-evoked striatal glutamate levels are attenuated by calcium reduction, tetrodotoxin, and glutamate receptor blockade.

L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) reverses plasma membrane glutamate transporters and elevates extracellular glutamate levels in vivo. We investigated the possibility that L-trans-PDC-stimulated glutamate levels are mediated partially by increases in transsynaptic activity. Therefore, the degree to which L-trans-PDC-evoked glutamate levels depend on calcium, sodium-channel activation, and glutamate-receptor activation was investigated by infusing via reverse microdialysis (a) 0.1 mM calcium, (b) 1 microM tetrodotoxin, a selective blocker of voltage-dependent sodium channels, (c) R(-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), a selective NMDA-receptor antagonist, or (d) LY293558, a selective alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate antagonist. In separate experimental groups, L-trans-PDC-evoked glutamate levels were reduced significantly by 55% in the presence of 0.1 mM calcium and by 46% in the presence of tetrodotoxin. Additionally, CPP and LY293558 significantly attenuated L-trans-PDC-evoked glutamate levels without altering basal glutamate levels. These data suggest that glutamate transporter reversal by L-trans-PDC initially elevates extracellular glutamate levels enough to stimulate postsynaptic glutamate receptors within the striatum. It is proposed that glutamate-receptor stimulation activates a positive feedback loop within the basal ganglia, leading to further glutamate release from corticostriatal and thalamostriatal afferents. Therefore, either extracellular striatal calcium reduction or tetrodotoxin perfusion leads to decreased action potential-dependent glutamate release from these terminals. In addition, blocking glutamate receptors directly reduces medium spiny neuronal firing and indirectly attenuates corticostriatal and thalamostriatal activity, resulting in an overall depression of L-trans-PDC-stimulated glutamate levels.