Analysis of 23andMe antidepressant efficacy survey data: implication of circadian rhythm and neuroplasticity in bupropion response.

Genetic predisposition may contribute to the differences in drug-specific, class-specific or antidepressant-wide treatment resistance. Clinical studies with the genetic data are often limited in sample sizes. Drug response obtained from self-reports may offer an alternative approach to conduct a study with much larger sample size. Using the phenotype data collected from 23andMe 'Antidepressant Efficacy and Side Effects' survey and genotype data from 23andMe's research participants, we conducted genome-wide association study (GWAS) on subjects of European ancestry using four groups of phenotypes (a) non-treatment-resistant depression (n=7795) vs treatment-resistant depression (TRD, n=1311), (b) selective serotonin reuptake inhibitors (SSRI) responders (n=6348) vs non-responders (n=3340), (c) citalopram/escitalopram responders (n=2963) vs non-responders (n=2005), and (d) norepinephrine-dopamine reuptake inhibitor (NDRI, bupropion) responders (n=2675) vs non-responders (n=1861). Each of these subgroups was also compared with controls (n ~ 190 000). The most significant association was from bupropion responders vs non-responders analysis. Variant rs1908557 (P=2.6 × 10(-8), OR=1.35) passed the conventional genome-wide significance threshold (P=5 × 10(-8)) and was located within the intron of human spliced expressed sequence tags in chromosome 4. Gene sets associated with long-term depression, circadian rhythm and vascular endothelial growth factor (VEGF) pathway were enriched in the bupropion analysis. No single-nucleotide polymorphism passed genome-wide significance threshold in other analyses. The heritability estimates for each response group compared with controls were between 0.15 and 0.25, consistent with the known heritability for major depressive disorder.

An inverse agonist selective for alpha5 subunit-containing GABAA receptors enhances cognition.

Alpha5IA is a compound that binds with equivalent subnanomolar affinity to the benzodiazepine (BZ) site of GABA(A) receptors containing an alpha1, alpha2, alpha3, or alpha5 subunit but has inverse agonist efficacy selective for the alpha5 subtype. As a consequence, the in vitro and in vivo effects of this compound are mediated primarily via GABA(A) receptors containing an alpha5 subunit. In a mouse hippocampal slice model, alpha5IA significantly enhanced the burst-induced long-term potentiation of the excitatory postsynaptic potential in the CA1 region but did not cause an increase in the paroxysmal burst discharges that are characteristic of convulsant and proconvulsant drugs. These in vitro data suggesting that alpha5IA may enhance cognition without being proconvulsant were confirmed in in vivo rodent models. Hence, alpha5IA significantly enhanced performance in a rat hippocampal-dependent test of learning and memory, the delayed-matching-to-position version of the Morris water maze, with a minimum effective oral dose of 0.3 mg/kg, which corresponded to a BZ site occupancy of 25%. However, in mice alpha5IA was not convulsant in its own right nor did it potentiate the effects of pentylenetetrazole acutely or produce kindling upon chronic dosing even at doses producing greater than 90% occupancy. Finally, alpha5IA was not anxiogenic-like in the rat elevated plus maze nor did it impair performance in the mouse rotarod assay. Together, these data suggest that the GABA(A) alpha5-subtype provides a novel target for the development of selective inverse agonists with utility in the treatment of disorders associated with a cognitive deficit.

Design and application of a novel brain slice system that permits independent electrophysiological recordings from multiple slices.

We describe a novel brain slice system 'SliceMaster' that allows electrophysiological recordings from eight brain slices independently. The system consists of two autonomous units each supporting four modular brain slice chambers enabling high signal-to-noise ratio recordings, each chamber has one stimulation electrode, one recording electrode, a twin camera system and a solution application system. The positioning of both electrodes and cameras are controlled from a remote user console. The software both acquires and performs on-line analysis of the data. We have demonstrated utility of this system in obtaining recordings of spontaneous firing activity and evoked synaptic activity from mouse hippocampal slices, with reduced variability within and between experiments. Furthermore, we show recordings of population spikes from the perirhinal cortex, indicating applicability of this system for further brain regions. In addition, stable recordings could be maintained until recording was terminated after 3 h, permitting investigation of the induction and maintenance of synaptic plasticity. Recordings of spontaneous and synaptic activity, and effects of pharmacological and electrophysiological manipulation, were consistent with reports using conventional methods. However, the described system permits concurrent and independent recordings from eight brain slices, thus improving throughput, statistical design, and reducing animal use.

Neurological phenotype and synaptic function in mice lacking the CaV1.3 alpha subunit of neuronal L-type voltage-dependent Ca2+ channels.

Neuronal L-type calcium channels have been implicated in pain perception and neuronal synaptic plasticity. To investigate this we have examined the effect of disrupting the gene encoding the CaV1.3 (alpha 1D) alpha subunit of L-type Ca2+ channels on neurological function, acute nociceptive behavior, and hippocampal synaptic function in mice. CaV1.3 alpha 1 subunit knockout (CaV1.3 alpha 1(-/-)) mice had relatively normal neurological function with the exception of reduced auditory evoked behavioral responses and lower body weight. Baseline thermal and mechanical thresholds were unaltered in these animals. CaV1.3 alpha 1(-/-) mice were also examined for differences in N-methyl-D-aspartate (NMDA) receptor-dependent (100 Hz tetanization for 1 s) and NMDA receptor-independent (200 Hz in 100 microM DL-2-amino-5-phosphopentanoic acid) long-term potentiation within the CA1 region of the hippocampus. Both NMDA receptor-dependent and NMDA receptor-independent forms of long-term potentiation were expressed normally. Radioligand binding studies revealed that the density of (+)[3H]isradipine binding sites in brain homogenates was reduced by 20-25% in CaV1.3 alpha 1(-/-) mice, without any detectable change in CaV1.2 (alpha 1C) protein levels as detected using Western blot analysis. Taken together these data indicate that following loss of CaV1.3 alpha 1 subunit expression there is sufficient residual activity of other Ca2+ channel subtypes to support NMDA receptor-independent long-term potentiation and some forms of sensory behavior/function.

Chronic substance P (NK1) receptor antagonist and conventional antidepressant treatment increases burst firing of monoamine neurones in the locus coeruleus.

The mechanism of action of conventional antidepressants (e.g. imipramine) has been linked to modulation of central monoamine systems. Substance P (NK1) receptor antagonists may have antidepressant and anxiolytic effects in patients with major depressive disorder and high anxiety but, unlike conventional antidepressants, are independent of activity at monoamine reuptake sites, transporters, receptors, or monoamine oxidase. To investigate the possibility that substance P receptor antagonists influence central monoamine systems indirectly, we have compared the effects of chronic administration of imipramine with that of the substance P receptor antagonist L-760735 on the spontaneous firing activity of locus coeruleus neurones. Electrophysiological recordings were made from brain slices prepared from guinea-pigs that had been dosed orally every day for 4 weeks with either L-760735 (3 mg/kg), imipramine (10 mg/kg), or vehicle (water), or naive animals. Chronic, but not acute, treatment with the substance P receptor antagonist L-760735, induced burst firing of neurones in the locus coeruleus. This effect resembles that of the conventional antidepressant imipramine. However, their effects are dissociable since, in contrast to chronic imipramine treatment, chronic L-760735 treatment does not cause functional desensitisation of somatic alpha2 adrenoceptors. The mechanism by which chronic substance P receptor antagonist or conventional antidepressant treatment influences the pattern of firing activity of norepinephrine neurones remains to be elucidated. However, an indirect action in the periphery or distant brain nuclei has been excluded by the use of the in vitro slice preparation, suggesting a local site of action in the locus coeruleus.

Role of the histidine residue at position 105 in the human alpha 5 containing GABA(A) receptor on the affinity and efficacy of benzodiazepine site ligands.

1. A histidine residue in the N-terminal extracellular region of alpha 1,2,3,5 subunits of the human GABA(A) receptor, which is replaced by an arginine in alpha 4 and alpha 6 subunits, is a major determinant for high affinity binding of classical benzodiazepine (BZ)-site ligands. The effect of mutating this histidine at position 105 in the alpha 5 subunit to an arginine (alpha 5H105R) on BZ-site pharmacology has been investigated using radioligand binding on HEK293 and L(tk-) cells and two electrode voltage clamp recording on Xenopus oocytes in which GABA(A) receptors of subtypes alpha 5, alpha 5H105R, alpha 4 and alpha 6 were co-expressed with beta 3 gamma 2s. 2. The classical BZs, diazepam and flunitrazepam (full agonists on the alpha 5 receptor) showed negligible affinity and therefore negligible efficacy on alpha 5H105R receptors. The beta-carbolines DMCM and beta CCE (inverse agonists on the alpha 5 receptor) retained some affinity but did not exhibit inverse agonist efficacy at alpha 5H105R receptors. Therefore, the alpha 5H105R mutation confers an alpha 4/alpha 6-like pharmacology to the classical BZs and beta-carbolines. 3. Ro15-4513, flumazenil, bretazenil and FG8094, which share a common imidazobenzodiazepine core structure, retained high affinity and were higher efficacy agonists on alpha 5H105R receptors than would be predicted from an alpha 4/alpha 6 pharmacological profile. This effect was antagonized by DMCM, which competes for the BZ-site and therefore is likely to be mediated via the BZ-site. 4. These data indicate that the conserved histidine residue in the alpha subunit is not only a key determinant in the affinity of BZ-site ligands on alpha 5 containing GABA(A) receptors, but also influences ligand efficacy.

PECAM-1/IgG attenuates peroxynitrite-mediated extremity reperfusion injury.

OBJECTIVE: Neutrophil transendothelial migration, a key feature of skeletal muscle ischemia and reperfusion (I/R) injury, is mediated by the platelet endothelial cell adhesion molecule-1 (PECAM-1). Peroxynitrite anion, a toxic product of neutrophil superoxide anion and nitric oxide, contributes to oxidative skeletal muscle injury and can be quantified by measurement of protein tyrosine nitration after I/R. This study hypothesizes that administration of the PECAM-1/IgG antibody chimera will inhibit peroxynitrite-mediated injury after I/R. METHODS: The study was composed of five groups: an I/R group (n = 4), a sham treatment group anesthetic control (n = 3), a treatment group receiving the PECAM-1/immunoglobulin G (IgG) antibody chimera with I/R (n = 9), a treatment group receiving human IgG with I/R as an antibody control (n = 6), and a treatment group receiving normal saline solution with I/R as a vehicle control (n = 5). The right hind limb in male New Zealand white rabbits was rendered ischemic by occluding the iliac and femoral arteries for 3 hours, followed by 2 hours of reperfusion (I/R). Sham-treated rabbits underwent arterial dissection without arterial occlusion. PECAM-1/IgG-treated rabbits and IgG-treated rabbits received an infusion of 1 mg/kg in normal saline solution 20 mL via an ear vein catheter during the last 5 minutes of ischemia and the first 15 minutes of reperfusion. Saline solution-treated rabbits similarly received normal saline solution 20 mL. The anterior tibialis muscle was harvested after reperfusion. Immunohistochemical staining for nitrotyrosine was performed with monoclonal antinitrotyrosine antibodies and fluorescently labeled secondary antibodies. Computed morphometric study was performed to calculate relative fluorescence scores for each histologic section. Averaged fluorescence scores were analyzed by one-way analysis of variance with Bonferroni post hoc comparison. RESULTS: The averaged fluorescence scores (mean +/- SEM) for the sham-treated (2.88 +/- 0.78) and PECAM-1/IgG-treated (6.16 +/- 0.43) groups demonstrated a significant reduction in quantitative fluorescence compared with the IgG- (15.17 +/- 2.01) and saline solution-treated (17.46 +/- 3.71) control groups, and the I/R-treated (18.52 +/- 3.00) group, (P <.05). CONCLUSIONS: These results suggest that PECAM-1/IgG diminishes peroxynitrite-mediated oxidative skeletal muscle injury by inhibiting neutrophil transendothelial migration and may therefore prove a useful therapeutic agent in the treatment of reperfusion injury.

Age-related impairment of synaptic transmission but normal long-term potentiation in transgenic mice that overexpress the human APP695SWE mutant form of amyloid precursor protein.

We have studied synaptic function in a transgenic mouse strain relevant to Alzheimer's disease (AD), overexpressing the 695 amino acid isoform of human amyloid precursor protein with K670N and M671L mutations (APP(695)SWE mice), which is associated with early-onset familial AD. Aged-transgenic mice had substantially elevated levels of Abeta (up to 22 micromol/gm) and displayed characteristic Abeta plaques. Hippocampal slices from 12-month-old APP(695)SWE transgenic animals displayed reduced levels of synaptic transmission in the CA1 region when compared with wild-type littermate controls. Inclusion of the ionotropic glutamate receptor antagonist kynurenate during preparation of brain slices abolished this deficit. At 18 months of age, a selective deficit in basal synaptic transmission was observed in the CA1 region despite treatment with kynurenate. Paired-pulse facilitation and long-term potentiation (LTP) were normal in APP(695)SWE transgenic mice at both 12 and 18 months of age. Thus, although aged APP(695)SWE transgenic mice have greatly elevated levels of Abeta protein, increased numbers of plaques, and reduced basal synaptic transmission, LTP can still be induced and expressed normally. We conclude that increased susceptibility to excitotoxicity rather than a specific effect on LTP is the primary cause of cognitive deficits in APP(695)SWE mice.

Lessons learned in adopting endovascular techniques for treating abdominal aortic aneurysm.

HYPOTHESIS: Endovascular exclusion of abdominal aortic and common iliac aneurysms can be performed safely, and in the short term represents a feasible alternative to traditional, open aneurysm repair. PATIENTS AND METHODS: Forty-one patients were treated with endovascular grafts for 39 abdominal aortic and 2 common iliac artery aneurysms. RESULTS: All devices were successfully deployed. The size of the abdominal aortic aneurysms varied from 4.9 to 11.9 cm (average, 6.13 cm). The median procedure time was 195 minutes. There was one iliac artery rupture, which required celiotomy for repair. The hospital stay varied from 2 to 39 days (average, 6.7 days). The perioperative mortality rate was 2.4%. Sixteen patients (39%) had groin wound complications. Ten patients (24%) had evidence of contrast (endoleak) within the aneurysm sac on completion of the procedure. There were no obvious direct leaks from either the point of proximal or distal fixation. Seven of these endoleaks have resolved spontaneously. Two patients required additional procedures in the postoperative period to treat endoleak. The final patient has evidence of persistent endoleak on 3-month surveillance computed tomography scan. Major late problems occurred in 3 patients. CONCLUSION: Patients with large abdominal aortic aneurysms and considerable cardiac comorbidity can safely undergo endovascular aneurysm repair. Femoral groin wound complications resulting in prolonged hospitalization remain the major cause of perioperative morbidity. In contradistinction to open aneurysm repair, long-term surveillance is essential to detect migration of the device and identify flow within the residual aneurysm sac-complications that could lead to aneurysm rupture following endovascular repair.

Substance P stimulates inhibitory synaptic transmission in the guinea pig basolateral amygdala in vitro.

To determine the physiological role of tachykinin NK1 receptors in the basolateral nucleus of the amygdala (BLN) we have studied the electrophysiological effects of substance P (SP) in the absence and presence of selective tachykinin receptor antagonists in guinea pig brain slices. Recordings were made from two populations of neurones; spiny pyramidal and stellate neurones, both thought to be projection neurones. Activation of NK1 receptors with SP increased the frequency of spontaneous inhibitory postsynaptic potentials in the majority of cells. This effect was blocked by bicuculline or tetrodotoxin, but not ionotropic glutamate receptor antagonists. The enhanced synaptic activity induced by SP was antagonised by the NK1 receptor antagonist L-760,735 but not by the less active enantiomer L-781,773 or the NK3 receptor antagonist L-769,927. Thus in the basolateral nucleus of the guinea pig amygdala, NK1 receptor activation preferentially stimulates inhibitory synaptic activity. Consistent with this observation, immunohistochemistry revealed NK1 receptor immunoreactivity to be largely restricted to a subset of GABA interneurones. These studies support a physiological role for SP in the regulation of pathways involved in the control of emotional behaviour.

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor.

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.

Regulation of vascular surgery by the federal government.

Governmental regulation of medicine seeks to protect patients and employees providing health care and to insure fair reimbursement for services. This report outlines workplace regulation of bloodborne pathogens and ergonomics. The investigation of potential Medicare fraud and abuse is reviewed. Mechanisms that control physician payment policy, including the Relative Base Resource Value System, the Correct Coding Initiative, and Current Procedural Terminology, are described.

Determinates of functional disability after complex upper extremity trauma.

This is a retrospective chart review of 71 patients who were operated on for presumed upper extremity arterial trauma between June 1992 and June 1998. Penetrating trauma occurred in 50 (70%) patients, and blunt trauma in 21 (30%). There were 2 innominate, 6 subclavian, 13 axillary, 26 brachial, 5 radial, 6 ulnar, and 6 multiple arterial injuries. There were 7 negative explorations (4 venous injuries, 2 false-positive angiograms, and 1 branch artery injury). In addition to the vascular injury, 44 patients (69%) had another injury in the extremity, including 8 (12.5%) orthopedic injuries, 12 (19%) nerve injuries, and 24 (37.5%) combination nerve and orthopedic injuries. There were three arterial thromboses, one arterial disruption, and four amputations, resulting in a patency rate and limb salvage rate of 94%. Persistent disability was more common in those patients with blunt injury (p = 0.02) and in those patients with associated neurologic and orthopedic injuries (p < 0.05). Full functional recovery was seen in 21 (33%) patients, while some form of disability was noted in the remaining 67%. The magnitude of the concomitant neurologic injury was the major determinate of functional outcome in this patient population.

Biophysical properties, pharmacology, and modulation of human, neuronal L-type (alpha(1D), Ca(V)1.3) voltage-dependent calcium currents.

Voltage-dependent calcium channels (VDCCs) are multimeric complexes composed of a pore-forming alpha(1) subunit together with several accessory subunits, including alpha(2)delta, beta, and, in some cases, gamma subunits. A family of VDCCs known as the L-type channels are formed specifically from alpha(1S) (skeletal muscle), alpha(1C) (in heart and brain), alpha(1D) (mainly in brain, heart, and endocrine tissue), and alpha(1F) (retina). Neuroendocrine L-type currents have a significant role in the control of neurosecretion and can be inhibited by GTP-binding (G-) proteins. However, the subunit composition of the VDCCs underlying these G-protein-regulated neuroendocrine L-type currents is unknown. To investigate the biophysical and pharmacological properties and role of G-protein modulation of alpha(1D) calcium channels, we have examined calcium channel currents formed by the human neuronal L-type alpha(1D) subunit, co-expressed with alpha(2)delta-1 and beta(3a), stably expressed in a human embryonic kidney (HEK) 293 cell line, using whole cell and perforated patch-clamp techniques. The alpha(1D)-expressing cell line exhibited L-type currents with typical characteristics. The currents were high-voltage activated (peak at +20 mV in 20 mM Ba2+) and showed little inactivation in external Ba2+, while displaying rapid inactivation kinetics in external Ca2+. The L-type currents were inhibited by the 1,4 dihydropyridine (DHP) antagonists nifedipine and nicardipine and were enhanced by the DHP agonist BayK S-(-)8644. However, alpha(1D) L-type currents were not modulated by activation of a number of G-protein pathways. Activation of endogenous somatostatin receptor subtype 2 (sst2) by somatostatin-14 or activation of transiently transfected rat D2 dopamine receptors (rD2(long)) by quinpirole had no effect. Direct activation of G-proteins by the nonhydrolyzable GTP analogue, guanosine 5'-0-(3-thiotriphospate) also had no effect on the alpha(1D) currents. In contrast, in the same system, N-type currents, formed from transiently transfected alpha(1B)/alpha(2)delta-1/beta(3), showed strong G-protein-mediated inhibition. Furthermore, the I-II loop from the alpha(1D) clone, expressed as a glutathione-S-transferase (GST) fusion protein, did not bind Gbetagamma, unlike the alpha(1B) I-II loop fusion protein. These data show that the biophysical and pharmacological properties of recombinant human alpha(1D) L-type currents are similar to alpha(1C) currents, and these currents are also resistant to modulation by G(i/o)-linked G-protein-coupled receptors.

Shear force regulates matrix metalloproteinase activity in human saphenous vein organ culture.

BACKGROUND: Development of vein graft intimal hyperplasia has been related both to shear force and to the activity of matrix metalloproteinases (MMPs). Little data are available regarding the effects of shear on MMP expression and activity. The aim of this study was to examine the relationship among shear force, metalloproteinase activity, and intimal thickening in human saphenous vein segments maintained in organ culture. MATERIALS AND METHODS: Segments of human saphenous vein were cultured under static conditions, or perfused under low-flow and high-flow conditions in a perfusion apparatus for 7 days. Metalloproteinase levels and activities were measured using ELISA and substrate gel zymography, respectively. Intimal thickening was determined by morphometric analysis. Results were compared with control vein tissue, which was not subjected to organ culture, using a one-way ANOVA. RESULTS: A 13% increase in proteolytic activity was noted on substrate gel zymography at 68-72 kDa in high-flow vein tissue. The protein content of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 was increased in high-flow vein tissue by 21%, 126%, more than 100-fold, and 86%, respectively. In culture media bathing the outside of the vein, TIMP-2 was increased in high-flow specimens, while TIMP-1 was inversely related to flow rate. Intimal thickening was directly related to flow rates, and was progressively increased in the low-flow and high-flow groups by 3-fold and 4-fold, respectively. CONCLUSIONS: Metalloproteinase levels in human saphenous vein cultures are related to shear force. MMP levels and activity correlate with the degree of intimal thickening. This model may provide a valuable tool for the analysis of physical forces and their influence on intimal thickening in human saphenous vein.

The importance of beta-lactamase resistance in surgical infections.

Substantial costs are associated with the treatment of nosocomial infections, 2 million cases of which occur annually in the United States. Hospital-acquired, gram-negative infection has become an increasing problem, particularly in the intensive care unit where up to 40% of the most frequently isolated strains of Enterobacteriaceae are resistant to standard beta-lactam antibiotics. Among several mechanisms of acquisition of resistance, beta-lactamase production accounts for a high percentage of treatment failures and relapses. By the end of the 1980s, some 10-30% of all nosocomial infections were caused by type-1 beta-lactamase-producing gram-negative isolates, and Enterobacter species had emerged as a major resistant pathogen. The beta-lactam/beta-lactamase inhibitor combinations, such as ampicillin/sulbactam, represent an innovative approach to the problem of beta-lactamase-mediated resistance. Clinical use of these agents has been associated with low rates of resistance and new data suggest they may have a specific role in controlling the emergence and spread of nosocomial infections.

Reduced long-term potentiation in hippocampal slices prepared using sucrose-based artificial cerebrospinal fluid.

Sucrose-based artificial cerebrospinal fluid (aCSF) is sometimes used to prepare brain slices for in vitro electrophysiological experiments. This study compared the effect of preparing brain slices using chilled sucrose-based aCSF versus the conventional method using chilled aCSF on hippocampal synaptic plasticity. Brain slices from each treatment group were transferred to normal aCSF before electrophysiological recordings were made. The stimulus-response relationship of field excitatory postsynaptic potentials (fEPSPs) in the CA1 region was indistinguishable between the two treatment groups. However, the amount of LTP induced by either a θ-burst (four stimuli at 100 Hz repeated ten times at 200 ms intervals) or tetanic stimulation (100 Hz for 1 s) was significantly reduced in slices that had been prepared using sucrose-based aCSF. This was associated with reduced facilitation of the fEPSPs during the high frequency stimulus, reduced post-tetanic potentiation and short-term potentiation. In sucrose-cut slices the fEPSPs were slightly shorter in duration (29%, P<0.01), and during paired-pulse stimulation the broadening of the second fEPSP was enhanced. The LTP deficit in sucrose-cut slices was reversed by blocking GABA(A) receptor function with picrotoxin. These data suggest that the use of sucrose based aCSF better preserves GABA-mediated synaptic transmission, which limits the induction of LTP in hippocampal brain slices.

All-trans-retinoic acid decreases cell proliferation and increases apoptosis in an animal model of vein bypass grafting.

BACKGROUND: We have previously demonstrated a decrease in intimal hyperplasia in vein bypass grafts from animals treated with all-trans-retinoic acid (atRA). The purpose of this study was to examine the effect of atRA on proliferation and apoptosis rates in healing vein bypass grafts. METHODS: Interposition jugular vein bypass grafts were placed in the carotid artery of 30 New Zealand white rabbits. Animals received either atRA (10 mg/kg/d) or vehicle (corn oil) for a period of 2 weeks. Animals were killed at 3, 7, or 28 days after graft placement after having received 3 doses of 5-bromo-2'-¿Deoxyuridine (BRDU, 35 Mg/KG). Animals Were Perfusion Fixed, And Vein Grafts Were Prepared For Immunohistochemistry By Using Antibodies To Brdu, Proliferating Cell Nuclear Antigen, And Bcl-XL. Apoptosis Was Measured By Using The Tunel Assay. Histologic Sections Were Analyzed By A Pathologist Blinded To The Study, And An Index Of Positively Stained Cells Was Generated For Each Layer Of The Vein Graft Wall. RESULTS: All-trans-retinoic acid reduced the proliferation index in the neointima of vein grafts during the first week after surgery. Apoptotic rates were higher in the intima of vein grafts from animals treated with atRA, which could not be explained by changes in bcl-xl expression. No differences were noted in the media or adventitia between the groups. CONCLUSIONS: atRA decreased cell proliferation and increased apoptosis in the intima of healing vein bypass grafts. These effects contribute to decreased intimal hyperplasia, which has been previously noted.

Pharmacology of recombinant human GABA(A) receptor subtypes measured using a novel pH-based high-throughput functional efficacy assay.

To facilitate the discovery of novel compounds that modulate human GABA(A) receptor function, we have developed a high throughput functional assay using a fluorescence imaging system. L(tk-) cells expressing combinations of human GABA(A) receptor subunits were incubated with the pH-sensitive dye 2',7'bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein, then washed and placed in a 96-well real-time fluorescence plate reader. In buffer adjusted to pH 6.9 there was a robust and persisting acidification response to addition of GABA, which was antagonised by the GABA(A) receptor antagonist bicuculline. The concentration-response relationship for GABA was modulated by allosteric ligands, including benzodiazepine (BZ) site agonists and inverse agonists. The effects of BZ site ligands on the pH response to GABA for receptors containing alpha1beta3gamma2, alpha3beta3gamma2 or alpha5beta3gamma2 subunits were well correlated with results from electrophysiological studies on the same receptor subunit combinations expressed in Xenopus oocytes. Most modulatory compounds tested were found to be relatively unselective across the three subunit combinations tested; however, some showed subtype-dependent efficacy, such as diazepam, which had highest agonist effects on the alpha3beta3gamma2 subtype, substantial but lesser agonism on alpha1beta3gamma2 and still substantial but the least agonism on alpha5beta3gamma2. This indicates that the alpha subunit within the recombinant receptor expressed in L(tk-) cells can affect the efficacy of the response to some BZ compounds. Inhibitors of Na(+)/Cl(-) cotransport, anion/anion exchange and the gastric type of H(+)/K(+) ATPase potently inhibited GABA-evoked acidification, indicating that multiple transporters are involved in the GABA-evoked pH change. This novel fluorescence-based high throughput functional assay allows the rapid characterization of allosteric ligands acting on human GABA(A) receptors.

Similar levels of long-term potentiation in amyloid precursor protein -null and wild-type mice in the CA1 region of picrotoxin treated slices.

Although mutations in amyloid precursor protein (APP) are known to be involved in the development of Alzheimer's disease in some individuals, the role of this protein in normal brain function is poorly understood. We have reported previously that in APP-null mice long-term potentiation (LTP) in the CA1 region of the hippocampus is present but its magnitude is reduced compared to wild-type littermate controls. In the present study, we have confirmed this deficit using a different theta burst induction protocol. Significantly, however, we find that this deficit is no longer apparent when LTP experiments are performed following blockade of gamma-aminobutyric acid(A) receptors. These results suggest that the LTP process per se is not altered by the absence of APP. The deficit may therefore be an indirect consequence of other changes in the hippocampus that occur in the APP-null animal.