Brooks B. Pond

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.

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 GABAA 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 GABAA 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 GABAA 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 GABAA 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.

GSTπ expression mediates dopaminergic neuron sensitivity in experimental parkinsonism

The cause of 95% of Parkinsons disease (PD) cases is unknown. It is hypothesized that PD arises from an interaction of free-radical-generating agents with an underlying genetic susceptibility to these compounds. Here we use the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of parkinsonism to examine the role of a dual function protein, GSTπ, in dopaminergic neuron death. GSTπ is the only GST family member expressed in substantia nigra neurons. GSTπ reduction by pharmacological blockade, RNA inhibition, and gene targeting increases sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, suggesting that differential expression of GSTπ contributes to the sensitivity to xenobiotics in the substantia nigra and may influence the pathogenesis of reactive oxygen species-induced neurological disorders including PD.

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.

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.

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.

A simple and sensitive high-performance liquid chromatography- electrochemical detection assay for the quantitative determination of monoamines and respective metabolites in six discrete brain regions of mice

A rapid, sensitive, and reproducible assay is described for the quantitative determination of the monoamine neurotransmitters dopamine, norepinephrine and serotonin, their metabolites, and the internal standard 3,4-dihydroxybenzlyamine hydro-bromide in mouse brain homogenate using high-performance liquid chromatography with electrochemical detection. The method was validated in the following brain areas: frontal cortex, striatum, nucleus accumbens, hippocampus, substantia nigra pars compacta and ventral tegmental area. Biogenic amines and relevant metabolites were extracted from discrete brain regions using a simple protein precipitation procedure, and the chromatography was achieved using a C18 column. The method was accurate over the linear range of 0.300-30 ng/mL (r = 0.999) for dopamine and 0.300-15 ng/mL (r = 0.999) for norepinephrine, 3,4-dihydroxybenzlyamine hydro-bromide, homovanillic acid and 5-hydroxyindolacetic acid, with detection limits of ~0.125 ng/mL (5 pg on column) for each of these analytes. Accuracy and linearity for serotonin were observed throughout the concentration range of 0.625-30 ng/mL (r = 0.998) with an analytical detection limit of ~0.300 ng/mL (12 pg on column). Relative recoveries for all analytes were approximately ≥90% and the analytical run time was <10 min. The described method utilized minimal sample preparation procedures and was optimized to provide the sensitivity limits required for simultaneous monoamine and metabolite analysis in small, discrete brain tissue samples.

Plagiarism Among Applicants for Faculty Positions

To the Editor. Recently, Dr. DiPiro published an article in the Journal1 that discussed several aspects pertinent to the process of faculty recruitment, emphasizing an individuals “fit” within the culture of the hiring institution. In the present article, we discuss another aspect of “fitness” that became evident to our search committee during the 2010-2011 academic year. Our search committee was established in May 2010 to hire a tenure-track faculty member at the level of assistant/associate professor in the department of pharmaceutical sciences. The committee consisted of faculty members in the departments of pharmaceutical sciences and pharmacy practice. Throughout the year, the search committee met 4 times and discussed 48 applicants. The committee members recommended several applicants to the department chair for screening calls and interviewed 2 candidates on site. During a review of applications, the search committee members discovered that several applicants submitted teaching philosophies that contained text that could be found verbatim elsewhere. Specifically, portions of these documents had been copied from online sources and pasted into the application. Obviously, our enthusiasm for these and other candidates with similar issues was significantly diminished and their applications were not pursued further. Plagiarism is defined as “to steal and pass off (the ideas or words of another) as ones own, to use (anothers production) without crediting the source, to commit literary theft, or to present as new and original an idea or product derived from an existing source.”2 Unfortunately, plagiarism is far too common. “Turnitin” reported 110 million instances of matched content found in 40 million papers submitted by students during a 10-month period.3 However, this problem is not limited to students. The occurrence of plagiarism in the scientific literature has been well documented.4,5 In fact, several publishers have implemented software programs to detect plagiarism in manuscripts submitted to their journals.5 Additionally, peer-reviewers for journals have been encouraged to be vigilant in searching for any matched content.6 Plagiarism in application materials is also not a new concept.7 Segal and colleagues reported plagiarism in medical residency application essays.8 In fact, this group found an evidence of plagiarism in more than 10% of essays submitted for a residency program at Brigham and Womens Hospital in Boston, Massachusetts. Although the rate of occurrence was higher among international students, plagiarism was observed in the application of US citizen as well. Surprisingly, students with academic honors were among those applicants with plagiarized application essays. A 2007 report in the journal Family Medicine also cites examples of plagiarism among applicants for medical fellowships, hypothesizing that the increased availability of content on the Web has led to a decreased awareness of the issue of plagiarism.9 The easy availability of technology may make plagiarism easier to commit, but it can also make it easier to detect.5,10 For instance, with some of our applicants, plagiarism was detected using a simple Google search. Faculty members are considered role models for students; their conduct has the potential to affect the reputation of the academic institution involved. As such, it is essential to check all application materials submitted by faculty applicants for any evidence of plagiarism. Faculty members have a responsibility to train students not only in their content areas, but also in areas such as research ethics. Additionally, because graduate students and postdoctoral trainees are the main group of applicants who apply for junior faculty positions, it would be helpful to educate graduate students and trainees about the negative impact of plagiarism on the educational system.10

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.