Pharmacokinetics of Synthetic Cathinones Found in Bath Salts in Mouse Brain and Plasma Using High-Pressure Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND AND OBJECTIVES: Approximately 10 years ago, "bath salts" became popular as legal alternatives to the psychostimulants cocaine and the amphetamines. These products contained synthetic cathinones, including 3,4-methylenedioxypyrovalerone (MDPV), 4-methylmethcathinone (mephedrone), and 3,4-methylenedioxymethcathinone (methylone). Most preclinical investigations have only assessed the effects of these synthetic cathinones independently; however, case reports and Drug Enforcement Administration (DEA) studies indicate that bath salts contain mixtures of these substances. In this study, we examine the pharmacokinetic interactions of the drug combination. We hypothesized that combined exposure to MDPV, mephedrone, and methylone would result in increased drug concentrations and enhanced total drug concentrations when compared to individual administration. METHODS: Adolescent male Swiss-Webster mice were injected intraperitoneally with either 10 mg/kg MDPV, 10 mg/kg mephedrone, 10 mg/kg methylone, or 10 mg/kg combined MDPV, mephedrone, and methylone. Following injection, brains and plasma were collected at 1, 10, 15, 30, 60, and 120 min. Drugs were extracted via solid-phase extraction, and concentrations were determined using a previously published high-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method. RESULTS: All drugs crossed the blood-brain barrier quickly. For methylone, the maximal concentration (Cmax) and the total drug exposure [as represented by the area under the concentration-time curve (AUC)] were significantly higher when combined with mephedrone and MDPV in both matrices (2.89-fold increase for both Cmax and AUC with combined treatment). For mephedrone, the Cmax was unchanged, but the AUC in brain was increased when in combination by approximately 34%. Interestingly, for MDPV, the Cmax was unchanged, yet the AUC was higher when MDPV was administered individually (there was a 62% decrease in AUC with combined treatment). CONCLUSIONS: The pharmacokinetics of methylone, mepedrone, and MDPV are altered when the drugs are used in combination. These data provide insight into the consequences of co-exposure to synthetic cathinones in popular bath salt products.

Effect of Chronic Methylphenidate Treatment in a Female Experimental Model of Parkinsonism.

Methylphenidate (MPH) is the most commonly prescribed drug for the treatment of ADHD in males and females. However, a majority of previous studies investigated the effect of MPH in only males, and little is known regarding consequences of female exposure to MPH. This is unfortunate because the few studies that have been conducted indicate that females have a greater sensitivity to MPH. Previous research in male mice has shown that chronic exposure to MPH causes dopaminergic neurons within the nigrostriatal pathway to be more sensitive to the Parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, estrogen has been shown to protect dopaminergic neurons from MPTP neurotoxicity. Therefore, in this study, we test the hypothesis that chronic MPH exposure in female mice will render dopaminergic neurons in the nigrostriatal pathway more sensitive to MPTP, and that estrogen may play a protective role. Interestingly, proestrus females exhibited greater sensitivity to MPTP, with significantly reduced dopaminergic neurons in the SN and significant increases in DA quinone production. Chronic MPH exposure contributed to GSH depletion, but surprisingly, it did not increase dopamine quinone levels or dopaminergic cell loss. There were no significant differences in anestrus animals, with the exception of a depletion in GSH seen when animals received chronic high-dose (10 mg/kg) MPH followed by MPTP. Thus, estrogen may actually sensitize neurons to MPTP in this model, and chronic MPH may contribute to GSH depletion within the striatum. This study provides insight into how chronic psychostimulant use may affect males and females differently.

Chronic methylphenidate induces increased quinone production and subsequent depletion of the antioxidant glutathione in the striatum.

BACKGROUND: Methylphenidate (Ritalin®) is a psychostimulant used chronically to treat attention deficit hyperactivity disorder. Methylphenidate acts by preventing the reuptake of dopamine and norepinephrine, resulting in an increase in these neurotransmitters in the synaptic cleft. Excess dopamine can be autoxidized to a quinone that may lead to oxidative stress. The antioxidant, glutathione helps to protect the cell against quinones via conjugation reactions; however, depletion of glutathione may result from excess quinone formation. Chronic exposure to methylphenidate appears to sensitize dopaminergic neurons to the Parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We hypothesized that oxidative stress caused by the autooxidation of the excess dopamine renders dopaminergic neurons within the nigrostriatal pathway to be more sensitive to MPTP. METHODS: To test this hypothesis, male mice received chronic low or high doses of MPH and were exposed to saline or MPTP following a 1-week washout. Quinone formation in the striatum was examined via dot blot, and striatal GSH was quantified using a glutathione assay. RESULTS: Indeed, quinone formation increased with increasing doses of methylphenidate. Additionally, methylphenidate dose-dependently resulted in a depletion of glutathione, which was further depleted following MPTP treatment. CONCLUSIONS: Thus, the increased sensitivity of dopamine neurons to MPTP toxicity following chronic methylphenidate exposure may be due to quinone production and subsequent depletion of glutathione.

Faculty Applicants' Attempt to Inflate CVs Using Predatory Journals.

Recently, scientific publishing has experienced an expansion of journals and publishers whose primary goal is profit and whose peer review process is virtually non-existent. These "predatory" or "opportunistic" journals pose a threat to the credibility and integrity of legitimate scientific literature, and quality science. Unfortunately, many scientists choose to publish in these journals and/or serve on their editorial boards, either due to ease of rapid publication or naivety. Here, we highlight the extensive use of predatory publications or editorial board involvement by applicants applying for a faculty position in the Pharmaceutical Sciences department at the Bill Gatton College of Pharmacy at East Tennessee State University. We caution search committees at other pharmacy schools to thoroughly examine applicant curricula vitarum (CVs) for predatory publishing.

Dopaminergic Effects of Major Bath Salt Constituents 3,4-Methylenedioxypyrovalerone (MDPV), Mephedrone, and Methylone Are Enhanced Following Co-exposure.

Designer drug mixtures popularized as "bath salts" often contain the synthetic cathinones 3,4 methylenedioxypyrovalerone (MDPV), mephedrone, and methylone in various combinations. However, most preclinical investigations have only assessed the effects of individual bath salt constituents, and little is known about whether co-exposure to MDPV, mephedrone, and methylone produces significant neuropharmacological interactions. This study evaluated and compared how MDPV, mephedrone, and methylone influence discrete brain tissue dopamine (DA) levels and motor stimulant responses in mice when administered alone and as a ternary mixture. Male adolescent Swiss-Webster mice received intraperitoneal injections of saline or 1 or 10 mg/kg doses of MDPV, mephedrone, or methylone, or a cocktail of all three cathinones at doses of 1, 3.3, or 10 mg/kg each. The effect of each treatment on DA and DA metabolite levels in mesolimbic and nigrostriatal brain tissue was quantified 15 min after a single exposure using HPLC-ECD. Additionally, locomotor activity was recorded in mice after acute (day 1) and chronic intermittent (day 7) dosing. MDPV, mephedrone, and methylone produced dose-related increases in mesolimbic and nigrostriatal DA levels that were significantly enhanced following their co-administration. In addition, mice treated with the cathinone cocktail displayed decreased locomotor activity on day 1 that was exacerbated by day 7 and not observed with any of the drugs alone. Our findings demonstrate a significant enhanced effect of MDPV, mephedrone, and methylone on both DA, and these effects on DA result in significant alterations in locomotor activity.

Neurogenesis within the hippocampus after chronic methylphenidate exposure.

Methylphenidate is a psychostimulant used to treat attention deficit hyperactivity disorder. Neurogenesis occurs throughout adulthood within the dentate gyrus of the hippocampus and can be altered by psychoactive medications; however, the impact of methylphenidate on neurogenesis is not fully understood. We investigated the effects of chronic low (1 mg/kg) and high (10 mg/kg) intraperitoneal doses of methylphenidate on neurogenesis in mouse hippocampus following 28 days and 56 days of treatment. Interestingly, methylphenidate, at both doses, increased neurogenesis. However, if methylphenidate treatment was not continued, the newly generated cells did not survive after 28 days. If treatment was continued, the newly generated neurons survived only in the mice receiving low-dose methylphenidate. To investigate the mechanism for this effect, we examined levels of proteins linked to cell proliferation in the hippocampus, including brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), tropomyosin receptor kinase B (TrkB), and beta-catenin. BDNF or GDNF levels were not significantly different between groups. However, hippocampal VEGF, TrkB, and beta-catenin were significantly increased in mice receiving low-dose methylphenidate for 28 days compared to controls. Interestingly, high-dose methylphenidate significantly decreased beta-catenin after 28 days and decreased VEGF, beta-catenin, and TrkB after 56 days compared to controls. Thus, low-dose methylphenidate appears to increase cell proliferation and cell survival in the hippocampus, and these effects may be mediated by increase in VEGF, TrkB, and beta-catenin. While high dose methylphenidate may initially increase neuronal proliferation, newly generated neurons are unable to survive long-term, possibly due to decrease in VEGF, TrkB and beta-catenin.

The pharmacokinetic profile of synthetic cathinones in a pregnancy model.

In recent years, the abuse of synthetic cathinones or 'bath salts' has become a major public health concern. Although these compounds were initially sold legally and labeled "not for human consumption", the 'bath salts' are psychostimulants, with similar structures and pharmacologic mechanisms to cocaine, the amphetamines, and 3,4 methylendioxymethamphetamine (MDMA, Molly, or Ecstasy). The reported use of these substances by women of child-bearing age highlights the necessity of studies seeking to delineate risks of prenatal exposure. Three popular drugs of this type are methylone, mephedrone, and 3, 4-methylenedioxypyrovalerone (MDPV). Unfortunately, there is currently no information available on the teratogenicity of these compounds, or of the extent to which they cross the placenta. As such, the purpose of this study was to examine the pharmacokinetic profile of the 'bath salts' in a pregnancy model. Pregnant mice (E17.5 gestation) were injected intraperitoneally with a cocktail of 5mg/kg methylone, 10mg/kg mephedrone, and 3mg/kg (MDPV) dissolved in sterile saline. Maternal brain, maternal plasma, placenta, and fetal brain were collected at 30s, 1min, 5min, 10min, 15min, 30min, 1h, 2h, 4h, and 8h following injection. Methylone, mephedrone, and MDPV were extracted from tissue by solid phase extraction, and concentrations were determined using a previously validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Interestingly, all 3 cathinones reached measurable concentrations in the placenta, as well as the fetal brain; in fact, for MDPV, the maximal concentration (Cmax) was highest in fetal brain, while mephedrone's highest Cmax value was achieved in placenta. Additionally, the total drug exposure for all 3 compounds (as represented by area under the curve, AUC) was higher in fetal matrices (placenta and fetal brain) than in maternal matrices (maternal brain and plasma), and the half-lives for the drugs were longer. Given the extensive presence of methylone, mephedrone, and MDPV in the fetal brain following prenatal exposure, fetal risk is definitely a concern. As there are currently no prenatal studies available on the teratogenicity of these agents, pregnant patients should be informed about the potential risks that these substances may have.

Quantification of Synthetic Cathinones in Rat Brain Using HILIC-ESI-MS/MS.

The abuse of synthetic cathinones, formerly marketed as "bath salts", has emerged over the last decade. Three common drugs in this class include 3,4-methylenedioxypyrovalerone (MDPV), 4-methylmethcathinone (mephedrone), and 3,4-methylenedioxymethcathinone (methylone). An LC-MS/MS method has been developed and validated for the simultaneous quantification of MDPV, mephedrone, and methylone in brain tissue. Briefly, MDPV, mephedrone, methylone, and their deuterium-labeled analogs were subjected to solid phase extraction (SPE) and separated using an HILIC Silica Column. The HPLC was coupled to a Shimadzu IT-TOF (ion trap-time of flight) system with the electrospray source running in positive mode (+ESI). The method was validated for precision, accuracy, and extraction efficiency. All inter-day and intra-day % RSD (percent relative standard deviation) and % error values were less than 15% and extraction efficiency exceeded 80%. These conditions allowed for limits of detection of 1ng/mL for MDPV, and 5 ng/mL for both mephedrone and methylone. The limits of quantification were determined to be 5ng/mL for MDPV and 10 ng/mL for mephedrone and methylone. The method was utilized to evaluate the pharmacokinetics of these drugs in adult male rats following administration of a drug cocktail including MDPV, mephedrone, and methylone. All three compounds reached peak concentrations in the brain within 15 min. Although methylone and mephedrone were administered at the same dose, the peak concentration (Cmax) of mephedrone in the brain was significantly higher than that for methylone, as was the area under the curve (AUC). In summary, this quick and sensitive method for measuring synthetic cathinones may be used for future pharmacokinetic investigations of these drugs in target tissue.

The pharmacokinetic profile of methylphenidate use in pregnancy: A study in mice.

The purpose of this study was to quantify the amounts of the d- and l-threo enantiomers of methylphenidate in maternal plasma, placenta, and maternal and fetal brain tissue following prenatal exposure and to establish a pharmacokinetic profile for MPH during pregnancy. Due to increasing rates of use of methylphenidate amongst females of childbearing age, it is important to understand the extent of exposure to the fetus. Briefly, pregnant mice were injected with 5 mg/kg methylphenidate at 18 days gestation, and tissue was collected 1, 5, 10, 30, 60, and 120 min following injection. Methylphenidate was extracted from tissue via solid phase extraction, and concentrations were determined using liquid chromatography-mass spectrometry (LC-MS). Because methylphenidate is administered as a racemic mixture of d- and l-threo enantiomers and the d-enantiomer is more pharmacologically active, the enantiomers were quantified separately. Interestingly, we found that methylphenidate does cross the placenta and enter the fetal brain. Although the highest concentrations were achieved in maternal brain, the concentrations of d- and l-methylphenidate in fetal brain were comparable to those of maternal plasma. Additionally, both d- and l-methylphenidate had longer half-lives in placenta than in maternal or fetal brain. Interestingly, there was a bimodal peak in maternal brain concentrations, at 5 min and again at 60 min, which was not observed in maternal plasma. Finally, the total exposure (as represented by area under the curve) was statistically significantly higher for the active d-enantiomer than the l-enantiomer in maternal brain tissue. In conclusion, methylphenidate crosses the placenta and reaches measurable concentrations in fetal brain. Although long-term behavioral and developmental studies are needed to determine specific outcomes of prenatal exposure, discussion with pregnant patients on the potential risks of methylphenidate exposure is warranted.

Chromatographic and electrophoretic strategies for the chiral separation and quantification of d- and l-threo methylphenidate in biological matrices.

Commercially available methylphenidate (MPH) exists as a racemic mixture composed of the d- and l-threo enantiomers. Various pharmacokinetic studies of MPH have shown a greater pharmacological potency of the d-threo enantiomer. Furthermore, it was deduced that the stereoselective cleavage of MPH to produce ritalinic acid (RA) by human carboxylesterase results in a higher oral bioavailability of the d-threo enantiomer. As a requirement for pharmaceutical regulation authorities, efforts have been made to determine the differential biological distribution of d- and l-threo MPH and RA enantiomers. In support of these efforts, numerous analytical procedures have been developed for the chiral separation and quantification of MPH enantiomers in a variety of biological matrices. The available methodologies accomplish the enantioseparation and quantification of MPH using gas chromatography, liquid chromatography or capillary electrophoretic techniques coupled with a variety of detectors. The current review discusses the technical procedures involved, and the sensitivity and selectivity of these assays.

Quantitative determination of d- and l-threo enantiomers of methylphenidate in brain tissue by liquid chromatography-mass spectrometry.

Methylphenidate, a psychostimulant used for the treatment of attention deficit hyperactivity disorder and narcolepsy, is administered as a 50:50 racemic mixture, despite the fact that d-methylphenidate has been shown to have greater pharmacologic activity. This paper presents a validated LC-MS/MS approach to separation and quantification of methylphenidate enantiomers using a vancomycin column and triethylammonium acetate to enhance the chiral separation. The method is applicable to the monitoring of these enantiomers in mouse brain, with a limit of detection of 0.5 ng/mL and a lower limit of quantification of 7.5 ng/mL.

Sex and dose-related differences in methylphenidate adolescent locomotor sensitization and effects on brain-derived neurotrophic factor.

This study analyzed repeated methylphenidate (MPH) administration and its effects on brain-derived neurotrophic factor (BDNF) in the dorsal striatum and nucleus accumbens of male and female adolescent rats. In Experiment 1, rats were administered intraperitoneal (ip) saline, 1, 3, or 5 mg/kg dose of MPH every second day from postnatal day (P)33-P49. Locomotor activity was analyzed for 10 min after each administration. Results revealed that the 1 mg/kg dose of MPH produced locomotor suppression, however, the 5 mg/kg dose of MPH produced locomotor sensitization and robust behavioral activation in females as compared to males. In Experiment 2, animals were administered ip saline or the 5 mg/kg dose of MPH using an identical regimen but a 30 min behavioral test was employed. Dorsal striatum and nucleus accumbens tissue was assayed for BDNF at P50. Females demonstrated sensitization to MPH and increased locomotor activation compared to males. Interestingly, females given MPH demonstrated a significant 42% decrease of striatal BDNF whereas males administered MPH demonstrated a significant 50.4% increase of striatal BDNF compared to controls. There were no effects on accumbal BDNF. This report demonstrates robust sex differences in the behavioral response, but sex-dependent changes in striatal BDNF in response to MPH in adolescence.

Methylphenidate exposure induces dopamine neuron loss and activation of microglia in the basal ganglia of mice.

BACKGROUND: Methylphenidate (MPH) is a psychostimulant that exerts its pharmacological effects via preferential blockade of the dopamine transporter (DAT) and the norepinephrine transporter (NET), resulting in increased monoamine levels in the synapse. Clinically, methylphenidate is prescribed for the symptomatic treatment of ADHD and narcolepsy; although lately, there has been an increased incidence of its use in individuals not meeting the criteria for these disorders. MPH has also been misused as a "cognitive enhancer" and as an alternative to other psychostimulants. Here, we investigate whether chronic or acute administration of MPH in mice at either 1 mg/kg or 10 mg/kg, affects cell number and gene expression in the basal ganglia. METHODOLOGY/PRINCIPAL FINDINGS: Through the use of stereological counting methods, we observed a significant reduction (∼20%) in dopamine neuron numbers in the substantia nigra pars compacta (SNpc) following chronic administration of 10 mg/kg MPH. This dosage of MPH also induced a significant increase in the number of activated microglia in the SNpc. Additionally, exposure to either 1 mg/kg or 10 mg/kg MPH increased the sensitivity of SNpc dopaminergic neurons to the parkinsonian agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Unbiased gene screening employing Affymetrix GeneChip® HT MG-430 PM revealed changes in 115 and 54 genes in the substantia nigra (SN) of mice exposed to 1 mg/kg and 10 mg/kg MPH doses, respectively. Decreases in the mRNA levels of gdnf, dat1, vmat2, and th in the substantia nigra (SN) were observed with both acute and chronic dosing of 10 mg/kg MPH. We also found an increase in mRNA levels of the pro-inflammatory genes il-6 and tnf-α in the striatum, although these were seen only at an acute dose of 10 mg/kg and not following chronic dosing. CONCLUSION: Collectively, our results suggest that chronic MPH usage in mice at doses spanning the therapeutic range in humans, especially at prolonged higher doses, has long-term neurodegenerative consequences.

A case-based toxicology elective course to enhance student learning in pharmacotherapy.

OBJECTIVE: To assess the impact of a case-based toxicology elective course on student learning in related required courses and student performance on the Pharmacy Curriculum Outcomes Assessment (PCOA) examination. DESIGN: A case-based clinical toxicology elective course that contained topics from 2 required courses, Pharmacology III and Pharmacotherapy II, was offered in the spring 2009 to second- and third-year pharmacy students. ASSESSMENT: Scores on the Toxicology subsection of the PCOA of students enrolled in the elective were higher than those of students not enrolled (91.3% ± 4.1 vs. 67.2% ± 5.7). Enrollment in the elective was related to increased examination scores among Pharmacotherapy II students (89.5% ± 2.0 vs. 83.9% ± 1.8). Students indicated on course survey instruments that they were satisfied with the new elective offering. CONCLUSIONS: A toxicology elective provided a clinically relevant, active-learning experience for pharmacy students that addressed a curricular need within the college and increased examination scores.

The chloride transporter Na(+)-K(+)-Cl- cotransporter isoform-1 contributes to intracellular chloride increases after in vitro ischemia.

Ischemic episodes in the CNS cause significant disturbances in neuronal ionic homeostasis. To directly measure changes in intracellular Cl- concentration ([Cl-]i) during and after ischemia, we used Clomeleon, a novel ratiometric optical indicator for Cl-. Hippocampal slices from adult transgenic mice expressing Clomeleon in hippocampal neurons were subjected to 8 min of oxygen-glucose deprivation (OGD) (an in vitro model for ischemia) and reoxygenated in the presence of glucose. This produced mild neuronal damage 3 h later that was prevented when the extracellular [Cl-] was maintained at 10 mm during reoxygenation. OGD induced a transient decrease in fluorescence resonance energy transfer within Clomeleon, indicating an increase in [Cl-]i. During reoxygenation, there was a partial recovery in [Cl-]i, but [Cl-]i rose again 45 min later. To investigate sources of Cl- accumulation, we examined the effects of Cl- transport inhibitors on the rises in [Cl-]i during and after OGD. Bumetanide and furosemide, which inhibit Cl- influx through the Na(+)-K(+)-Cl- cotransporter isoform-1 (NKCC-1) and efflux through the K(+)-Cl- cotransporter isoform-2, were unable to inhibit the first rise in [Cl-]i, yet entirely prevented the secondary rise in [Cl-]i during reoxygenation. In contrast, picrotoxin, which blocks the GABA-gated Cl- channel, did not inhibit the secondary rise in [Cl-]i after OGD. [Cl-]i increases during reoxygenation were accompanied by an increase in phosphorylation of NKCC-1, an indication of increased NKCC-1 activity after OGD. We conclude that NKCC-1 plays an important role in OGD-induced Cl- accumulation and subsequent neuronal damage.

Chloride transport inhibitors influence recovery from oxygen-glucose deprivation-induced cellular injury in adult hippocampus.

Cerebral ischemia in vivo or oxygen-glucose deprivation (OGD) in vitro are characterized by major disturbances in neuronal ionic homeostasis, including significant rises in intracellular Na(+), Ca(2+), and Cl(-) and extracellular K(+). Recently, considerable attention has been focused on the cation-chloride cotransporters Na-K-Cl cotransporter isoform I (NKCC-1) and K-Cl cotransporter isoform II (KCC2), as they may play an important role in the disruption of ion gradients and subsequent ischemic damage. In this study, we examined the ability of cation-chloride transport inhibitors to influence the biochemical (i.e. ATP) and histological recovery of neurons in adult hippocampal slices exposed to OGD. In the hippocampus, 7 min of OGD caused a loss of ATP that recovered partially (approximately 50%) during 3 h of reoxygenation. Furosemide, which inhibits the NKCC-1 and KCC2 cotransporters, and bumetanide, a more specific NKCC-1 inhibitor, enhanced ATP recovery when measured 3 h after OGD. Furosemide and bumetanide also attenuated area CA1 neuronal injury after OGD. However, higher concentrations of these compounds appear to have additional non-specific toxic effects, limiting ATP recovery following OGD and promoting neuronal injury. The KCC2 cotransporter inhibitor DIOA and the Cl(-) ATPase inhibitor ethacrynic acid caused neuronal death even in the absence of OGD and promoted cytochrome c release from isolated mitochondria, indicating non-specific toxicities of these compounds.

Changes in intracellular chloride after oxygen-glucose deprivation of the adult hippocampal slice: effect of diazepam.

Ischemic injury to the CNS results in loss of ionic homeostasis and the development of neuronal death. An increase in intracellular Ca2+ is well established, but there are few studies of changes in intracellular Cl- ([Cl-]i) after ischemia. We used an in vitro model of cerebral ischemia (oxygen-glucose deprivation) to examine changes in [Cl-]i and GABA(A) receptor-mediated responses in hippocampal slices from adult rats. Changes in [Cl-]i were measured in area CA1 pyramidal neurons using optical imaging of 6-methoxy-N-ethylquinolinium chloride, a Cl--sensitive fluorescent indicator. Oxygen-glucose deprivation induced an immediate rise in [Cl-]i, which recovered within 20 min. A second and more prolonged rise in [Cl-]i occurred within the next hour, during which postsynaptic field potentials failed to recover. The sustained increase in [Cl-]i was not blocked by GABA(A) receptor antagonists. However, oxygen-glucose deprivation caused a progressive downregulation of the K+-Cl- cotransporter (KCC2), which may have contributed to the Cl- accumulation. The rise in [Cl-]i was accompanied by an inability of the GABA(A) agonist muscimol to cause Cl- influx. In vivo, diazepam is neuroprotective when given early after ischemia, although the mechanism by which this occurs is not well understood. Here, we added diazepam early after oxygen-glucose deprivation and prevented the downregulation of KCC2 and the accumulation of [Cl-]i. Consequently, both GABA(A) responses and synaptic transmission within the hippocampus were restored. Thus, after oxygen-glucose deprivation, diazepam may decrease neuronal excitability, thereby reducing the energy demands of the neuron. This may prevent the activation of downstream cell death mechanisms and restore Cl- homeostasis and neuronal function