Prediction of Postoperative Major Complications in Pancreaticoduodenectomy -a Prospective Comparative Analysis of Braga and WHipple-ABACUS Scores.

Background Major complications following pancreaticoduodenectomy have a severely deleterious effect on postoperative course, rather than just occurrence of pancreatic fistula. Surgical risk stratification with Braga and WHipple-ABACUS have been proposed and validated. Objective The study aimed at comparing the Braga and WHipple-ABACUS scores for prediction of major complications following pancreaticoduodenectomies. Method This was a prospective observational study at the Tribhuvan University Teaching Hospital from February 2018 to April 2019. After ethical approval, all consecutive 41 patients who underwent pancreaticoduodenectomies were included. Each patient was graded in Braga and WHipple-ABACUS scores. Perioperative events occurring over 30 days were graded as per Clavien -Dindo complications for pancreatic surgery. The predictive value of the scores were assessed using a receiver operating characteristic curve analysis. The categorical data were compared using the Pearson χ2 test or Fisher's exact test. Result Over period of 14 months, total of 41 patients (M:F=2.15:1) with median age of 58 years (range, 21-86) underwent pancreatoduodenectomy. The mean scores were Braga (4.6±3.1) and WHipple-ABACUS (1.8±1.6). Major complications over 30 days were developed in 11 patients with five mortality. There were significant differences in mean values of Braga score (7.0±3.4 vs 3.7±2.6, p-value=0.02) and WHippleABACUS score (3.2±1.8 vs 1.3±1.3, p-value=0.01) in patients with major complications to those without respectively. The area under curves for Braga and WHipple-ABACUS scores were 0.800 and 0.779 respectively. Conclusion Both WHipple-ABACUS and Braga scores are easy to calculate and predict the development of major complications significantly in patients undergoing pancreatoduodenectomy.

Successful medical management of acute mesenteric ischemia due to superior mesenteric and portal vein thrombosis in a 27-year-old man with protein S deficiency: a case report.

BACKGROUND: Acute mesenteric ischemia poses a diagnostic challenge due to nonspecific clinical clues and lack of awareness owing to its rarity. Ischemia due to mesenteric venous thrombosis has a good prognosis compared to arterial cause and can be managed conservatively with early diagnosis. The portomesenteric venous system is an unusual site of thrombosis in patients with protein S deficiency, and its thrombosis is an uncommon cause of acute mesenteric ischemia. CASE PRESENTATION: We present a case of a 27-year-old Mongolian man who presented with acute abdominal pain increasing in severity, and refractory to repeated attempts at treatment with a misdiagnosis of acute peptic ulcer disease. Contrast-enhanced computed tomography of his abdomen detected complete occlusion of the superior mesenteric vein, an extension of acute thrombus into the portal vein, and ischemic mid-jejunal loops. Early diagnosis and immediate anticoagulation with continuous intravenous infusion of unfractionated heparin prevented subsequent consequences. On further workup, our patient was diagnosed with isolated protein S deficiency. We started lifelong thromboprophylaxis with warfarin to prevent recurrence and our patient was asymptomatic on the latest follow-up 5 months after discharge. CONCLUSION: Despite accurate detection of acute mesenteric ischemia by contrast-enhanced computed tomography, high index of suspicion is indispensable for its early diagnosis. Early diagnosis and immediate anticoagulation will prevent subsequent complications and need for surgical intervention. Young patients without known risk factors presenting with venous thrombosis in atypical sites should be investigated for prothrombotic diseases.

Effect of the 2014/2015 Ebola outbreak on reproductive health services in a rural district of Guinea: an ecological study.

Background: The 2014/2015 Ebola outbreak was the most sustained in history. In Guinea, we compared trends in family planning, antenatal care, and institutional deliveries over the period before, during and after the outbreak. Methods: We carried out an ecological study involving all the health facilities during pre-Ebola (1 March 2013 to 28 February 2014), intra-Ebola (1 March 2014 to 28 February 2015) and post-Ebola (1 March to 31 July 2016) periods in Macenta district. Results: Utilization of family planning declined from a monthly average of 531 visits during the pre-Ebola period to 242 visits in the peak month of the Ebola outbreak (51% decline) but recovered in the post-Ebola period. From a monthly average of 2053 visits pre-Ebola, antenatal care visits declined by 41% during Ebola and then recovered to only 63% of the pre-Ebola level (recovery gap of 37%, p<0.001). From a monthly average of 1223 deliveries pre-Ebola, institutional deliveries also declined during Ebola and then recovered to only 66% of the pre-Ebola level (p<0.001). Conclusions: All services assessed were affected by Ebola. Family planning recovered post-Ebola; however, shortfalls were observed in recovery of antenatal care and institutional deliveries. We call for stronger political will, international support and generous funding to change the current state of affairs.

Effect of Ebola virus disease on maternal and child health services in Guinea: a retrospective observational cohort study.

BACKGROUND: The 2014 west African epidemic of Ebola virus disease posed a major threat to the health systems of the countries affected. We sought to quantify the consequences of Ebola virus disease on maternal and child health services in the highly-affected Forest region of Guinea. METHODS: We did a retrospective, observational cohort study of women and children attending public health facilities for antenatal care, institutional delivery, and immunisation services in six of seven health districts in the Forest region (Beyla, Guéckédou, Kissidougou, Lola, Macenta, and N'Zérékoré). We examined monthly service use data for eight maternal and child health services indicators: antenatal care (≥1 antenatal care visit and ≥3 antenatal care visits), institutional delivery, and receipt of five infant vaccines: polio, pentavalent (diphtheria, tetanus, pertussis, hepatitis B virus, and Haemophilus influenzae type b), yellow fever, measles, and tuberculosis. We used interrupted time series models to estimate trends in each indicator across three time periods: pre-Ebola virus disease epidemic (January, 2013, to February, 2014), during-epidemic (March, 2014, to February, 2015) and post-epidemic (March, 2015, to Feb, 2016). We used segmented ordinary least-squares (OLS) regression using Newey-West standard errors to accommodate for serial autocorrelation, and adjusted for any potential effect of birth seasonality on our outcomes. FINDINGS: In the months before the Ebola virus disease outbreak, all three maternal indicators showed a significantly positive change in trend, ranging from a monthly average increase of 61 (95% CI 38-84) institutional deliveries to 119 (95% CI 79-158) women achieving at least three antenatal care visits. These increasing trends were reversed during the epidemic: fewer institutional deliveries occurred (-240, 95% CI -293 to -187), and fewer women achieved at least one antenatal care visit (-418, 95% CI -535 to -300) or at least three antenatal care visits (-363, 95% CI -485 to -242) per month (p<0·0001 for all). Compared with the negative trend during the outbreak, the change in trend during the post-outbreak period showed that 173 more women per month (95% CI 51-294; p=0·0074) had at least one antenatal care visit, 257 more (95% CI 117-398; p=0·0010) had at least three antenatal care visits and 149 more (95% CI 91-206; p<0·0001) had institutional deliveries. However, although the numbers for these indicators increased in the post-epidemic period, the trends for all stagnated. Similarly, the increasing trend in child vaccination completion during the pre-epidemic period was followed by significant immediate and trend reductions across most vaccine types. Before the outbreak, the number of children younger than 12 months who had completed each vaccination ranged from 5752 (95% CI 2821-8682) for tuberculosis to 8043 (95% CI 7621-8464) for yellow fever. Immediately after the outbreak, significant reductions occurred in the level of all vaccinations except for yellow fever for which the reduction was marginal. The greatest reductions were noted for polio and tuberculosis at -3594 (95% CI -4811 to -2377; p<0·0001) and -3048 (95% CI -5879 to -216; p=0·0362) fewer vaccines administered, respectively. Compared with pre-Ebola virus disease outbreak trends, significant decreases occurred for all vaccines except polio, with the trend of monthly decreases in the number of children vaccinated ranging from -419 (95% CI -683 to -155; p=0·0034) fewer for BCG to -313 (95% CI-446 to -179; p<0·0001) fewer for pentavalent during the outbreak. In the post-Ebola virus disease outbreak period, vaccination coverage for polio, measles, and yellow fever continued to decrease, whereas the trend in coverage for tuberculosis and pentavalent did not significantly differ from zero. INTERPRETATION: Most maternal and child health indicators significantly declined during the Ebola virus disease outbreak in 2014. Despite a reduction in this negative trend in the post-outbreak period, the use of essential maternal and child health services have not recovered to their pre-outbreak levels, nor are they all on a course that suggests that they will recover without targeted interventions. FUNDING: University of Conakry and Centre National de Formation et Recherche de Maferinyah (Guinea).

Maternal and Child Health Services in the Context of the Ebola Virus Disease: Health Care Workers' Knowledge, Attitudes and Practices in Rural Guinea.

The objective of this study was to document maternal and child health care workers' knowledge, attitudes and practices on service delivery before, during and after the 2014 EVD outbreak in rural Guinea. We conducted a descriptive cross-sectional study in ten health districts between October and December 2015, using a standardized self-administered questionnaire. Overall 299 CHWs (94% response rate) participated in the study, including nurses/health technicians (49%), midwives (23%), managers (16%) and physicians (12%). Prior to the EVD outbreak, 87% of CHWs directly engaged in managing febrile cases within the facility, while the majority (89% and 63%) referred such cases to another facility and/or EVD treatment centre during and after the EVD outbreak, respectively. Compared to the period before the EVD outbreak when approximately half of CHWs (49%) reported systematically measuring body temperature prior to providing any care to patients, most CHWs reported doing so during (98%) and after the EVD outbreak (88%). The main challenges encountered were the lack of capacity to screen for EVD cases within the facility (39%) and the lack of relevant equipment (10%). The majority (91%) of HCWs reported a decrease in the use of services during the EVD outbreak while an increase was reported by 72% of respondents in the period following the EVD outbreak. Infection prevention and control measures established during the EVD outbreak have substantially improved self-reported provider practices for maternal and child health services in rural Guinea. However, more efforts are needed to maintain and sustain the gain achieved.

Maternal and child health services in the context of the ebola virus disease: health care workers' knowledge, attitudes and practices in rural Guinea

The objective of this study was to document maternal and child health care workers' knowledge, attitudes and practices on service delivery before, during and after the 2014 EVD outbreak in rural Guinea. We conducted a descriptive cross-sectional study in ten health districts between October and December 2015, using a standardized self-administered questionnaire. Overall 299 CHWs (94% response rate) participated in the study, including nurses/health technicians (49%), midwives (23%), managers (16%) and physicians (12%). Prior to the EVD outbreak, 87% of CHWs directly engaged in managing febrile cases within the facility, while the majority (89% and 63%) referred such cases to another facility and/or EVD treatment centre during and after the EVD outbreak, respectively. Compared to the period before the EVD outbreak when approximately half of CHWs (49%) reported systematically measuring body temperature prior to providing any care to patients, most CHWs reported doing so during (98%) and after the EVD outbreak (88%). The main challenges encountered were the lack of capacity to screen for EVD cases within the facility (39%) and the lack of relevant equipment (10%). The majority (91%) of HCWs reported a decrease in the use of services during the EVD outbreak while an increase was reported by 72% of respondents in the period following the EVD outbreak. Infection prevention and control measures established during the EVD outbreak have substantially improved self-reported provider practices for maternal and child health services in rural Guinea. However, more efforts are needed to maintain and sustain the gain achieved

EGFRvIII promotes glioma angiogenesis and growth through the NF-κB, interleukin-8 pathway.

Sustaining a high growth rate requires tumors to exploit resources in their microenvironment. One example of this is the extensive angiogenesis that is a typical feature of high-grade gliomas. Here, we show that expression of the constitutively active mutant epidermal growth factor receptor, ΔEGFR (EGFRvIII, EGFR*, de2-7EGFR) is associated with significantly higher expression levels of the pro-angiogenic factor interleukin (IL)-8 in human glioma specimens and glioma stem cells. Furthermore, the ectopic expression of ΔEGFR in different glioma cell lines caused up to 60-fold increases in the secretion of IL-8. Xenografts of these cells exhibit increased neovascularization, which is not elicited by cells overexpressing wild-type (wt)EGFR or ΔEGFR with an additional kinase domain mutation. Analysis of the regulation of IL-8 by site-directed mutagenesis of its promoter showed that ΔEGFR regulates its expression through the transcription factors nuclear factor (NF)-κB, activator protein 1 (AP-1) and CCAAT/enhancer binding protein (C/EBP). Glioma cells overexpressing ΔEGFR showed constitutive activation and DNA binding of NF-κB, overexpression of c-Jun and activation of its upstream kinase c-Jun N-terminal kinase (JNK) and overexpression of C/EBPß. Selective pharmacological or genetic targeting of the NF-κB or AP-1 pathways efficiently blocked promoter activity and secretion of IL-8. Moreover, RNA interference-mediated knock-down of either IL-8 or the NF-κB subunit p65, in ΔEGFR-expressing cells attenuated their ability to form tumors and to induce angiogenesis when injected subcutaneously into nude mice. On the contrary, the overexpression of IL-8 in glioma cells lacking ΔEGFR potently enhanced their tumorigenicity and produced highly vascularized tumors, suggesting the importance of this cytokine and its transcription regulators in promoting glioma angiogenesis and tumor growth.

Vaginal hysterectomy for pelvic organ prolapse in Nepal.

BACKGROUND: UVP is a significant Public Health Problem in Nepal. This problem is mainly prevalent in rural areas where the women are socio--economically less privileged and cannot afford the costs of treatment. OBJECTIVE: An analysis of peri operative and post operative complications of vaginal hysterectomies for pelvic organ prolapse. MATERIALS AND METHODS: A hospital based prospective study was carried out in the department of obstetrics and gynaecology, NGMC followed up from the time of operation to time of discharge. RESULTS: 632 cases underwent vaginal hysterectomy with financial support from UNFPA. There were no operative complications. The most common post operative complications as noted were retention of urine, pelvic infection & pelvic abscess. In two cases laparotomy was done for haemoperitoneum. Pelvic abscess was drained vaginally. Mortality was nil. CONCLUSION: Proper screening before operation is the key to reduce operative as well as peri operative complications.

Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits.

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin (Htt) gene. HD is autosomal dominant and, in theory, amenable to therapeutic RNA silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1-400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.

GFRalpha3 is expressed predominantly in nociceptive sensory neurons.

Activation of the RET receptor tyrosine kinase by glial-derived neurotrophic factor family members is dependent on a family of coreceptors, GFRalpha1-4. GFRalpha3 preferentially binds the newest member of the glial-derived neurotrophic factor family of ligands, artemin. The major site of GFRalpha3 expression is in the dorsal root ganglion; however, the class of sensory neurons that expresses GFRalpha3 has not been reported previously. Using immunohistochemical methods, we show that the majority of dorsal root ganglion cells that express GFRalpha3 also express vanilloid receptor type 1, peripherin, RET, trkA and calcitonin gene-related peptide. In addition, a significant subpopulation of GFRalpha3-expressing cells also binds the lectin IB4. We demonstrate that GFRalpha3 artemin neurons are immunopositive for markers expected of nociceptors and include a subset of neurons distinct from the GDNF-responsive population. Our results indicate artemin may exert selective effects on pain sensation.

TA1/LAT-1/CD98 light chain and system L activity, but not 4F2/CD98 heavy chain, respond to arginine availability in rat hepatic cells. Loss Of response in tumor cells.

Tumor associated gene-1/L amino acid transporter-1 (TA1/LAT-1) was recently identified as a light chain of the CD98 amino acid transporter and cellular activation marker. Our previous studies with primary rat hepatocyte cultures demonstrated that TA1 RNA levels were responsive to media amino acid concentrations, suggesting adaptive regulation. High level TA1 expression associated with transformed cells also suggested a role in tumor progression. The present study examined the relationship of TA1/CD98 expression, adaptive response, and associated amino acid transport to neoplastic transformation using a panel of well characterized rat hepatic cell lines. We found 1) increased expression of TA1 in response to amino acid depletion, specific for arginine but not glutamine; 2) loss of TA1 response to arginine in gamma-glutamyl transpeptidase-positive transformed and tumorigenic cells; 3) no appreciable response of 4F2/CD98 heavy chain to arginine levels; and 4) correlation of system L amino acid transport activity in response to arginine with changes in TA1/LAT-1 mRNA but not total immunoreacting protein. Our results suggest this CD98 light chain may act as an environmental sensor, responding to amino acid availability and that its regulation is complex. We hypothesize that altered TA1 expression is an early event in hepatocarcinogenesis giving neoplastic cells a growth or survival advantage, particularly under conditions of limited amino acid availability.

Motoneuron differentiation of immortalized human spinal cord cell lines.

Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties.

A renewable source of human sensory neurons would greatly facilitate basic research and drug development. We had established previously conditionally immortalized human CNS cell lines that can differentiate into functional neurons (). We report here the development of an immortalized human dorsal root ganglion (DRG) clonal cell line, HD10.6, with a tetracycline-regulatable v-myc oncogene. In the proliferative condition, HD10.6 cells have a doubling time of 1.2 d and exhibit a neuronal precursor morphology. After differentiation of clone HD10.6 for 7 d in the presence of tetracycline, v-myc expression was suppressed, and >50% of the cells exhibited typical neuronal morphology, stained positively for neuronal cytoskeletal markers, and fired action potentials in response to current injection. Furthermore, this cell line was fate-restricted to a neuronal phenotype; even in culture conditions that promote Schwann cell or smooth muscle differentiation of neural crest stem cells, HD10.6 differentiated exclusively into neurons. Moreover, differentiated HD10.6 cells expressed sensory neuron-associated transcription factors and exhibited capsaicin sensitivity. Taken together, these data indicate that we have established an immortalized human DRG cell line that can differentiate into sensory neurons with nociceptive properties. The cell line HD10.6 represents the first example of a human sensory neuronal line and will be valuable for basic research, as well as for the discovery of novel drug targets and clinical candidates.

Bipotent progenitor cell lines from the human CNS.

Human central nervous system (CNS) cell lines would substantially facilitate drug discovery and basic research by providing a readily renewable source of human neurons. We isolated clonal human CNS cell lines that had been immortalized with a tetracycline (Tc)-responsive v-myc oncogene; addition of Tc to the growth medium suppressed the oncoprotein rapidly and virtually completely, allowing differentiation to proceed. Two classes of bipotent precursor cells were immortalized: the first class had a default differentiation pathway of neurons only, and the second class had a default differentiation pathway of neurons and astrocytes. We found that after exposure to different external signals in vitro, the environment is capable of redirecting the fate of a particular cell, even in the case of the bipotent precursor cell whose default differentiation pathway was neurons only. These data suggest that extrinsic cues can prevail over intrinsic determinants in directing cell fate in the human CNS.

Regulation of voltage- and ligand-gated currents in rat hippocampal progenitor cells in vitro.

Growth medium and substrate regulate the proliferation and differentiation of neural progenitor cells in vitro and the expression of cell type-specific histochemical markers. An important question is whether neural progenitor cells exhibit voltage- and ligand-gated currents, features characteristic of neurons, and whether these currents are regulated differentially by growth conditions. Another issue of interest is whether passaged progenitor cells, after expansion with basic fibroblast growth factor (FGF-2), exhibit the same degree of plasticity as their primary counterparts, or whether they are more committed to a particular phenotype. In primary cultures of embryonic rat hippocampal progenitor cells, growth in proliferative conditions (FGF-2) was associated with low levels of sodium, calcium, N-methyl-D-aspartate (NMDA), and kainate currents compared with other growth conditions. After multiple passages in the continued presence of FGF-2, sodium, calcium, and NMDA, responses declined further; interestingly, kainate and gamma-aminobutyric acid (GABA) responses remained substantial. Moreover, the expression of functional channels and receptors in primary cultures of progenitor cells is up-regulated strongly by growth factors such as BDNF, and NT-3, whereas sodium and calcium currents in passaged cultures respond to such growth conditions to a lesser extent. Kainate and GABA responses were present to a significant extent in passaged cultures, independent of growth condition. We conclude that environmental cues regulate different channels and receptors in distinct ways in neural progenitor cells.

Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene.

A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.

Human fetal central neurons in culture: voltage- and ligand-gated currents.

1. The functional properties of sodium, potassium, calcium, N-methyl-D-aspartate (NMDA), kainate, and gamma-aminobutyric acid (GABA) currents were studied in dissociated monolayer cultures of fetal human brain neurons, using the whole cell patch-clamp technique. 2. Sodium currents were characterized with respect to the following properties: current density, voltage dependence of activation, voltage dependence of inactivation, and sensitivity to tetrodotoxin (TTX). All sodium currents exhibited voltage dependencies of activation and inactivation, and sensitivities to TTX that are characteristic of the neuronal form of the sodium current. 3. At least two types of potassium current were present, resembling the delayed rectifier and fast-inactivating potassium current. These two types of potassium current were distinguishable by their different kinetics, voltage dependencies of activation and inactivation, and sensitivities to 4-aminopyridine and tetraethylammonium. 4. High-voltage-activated calcium channel currents were present and were characterized with respect to current density, voltage dependencies of activation and inactivation, and sensitivity to cadmium. Low-voltage-activated calcium channel currents were also present. 5. NMDA- and kainate-gated currents were studied with respect to current density, time course, and current-voltage relationship. Kainate currents were also characterized with respect to inhibition by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In addition, NMDA and kainate responses were compared for cortical versus cerebellar neurons. NMDA responses, which are only found in neurons, were present, confirming the neuronal phenotype suggested by the presence of the neuronal form of the sodium current. Nondesensitizing kainate currents were also present, with a half-maximally effective concentration (EC50) of approximately 200 microM for kainate; CNQX inhibited the kainate current with a half-inactivating concentration of 0.55 microM. 6. GABA-gated currents were characterized with respect to current density, time course, receptor subtype, desensitization, dose response, current-voltage relationship, ionic selectivity, pharmacology, and potentiation by the neurosteroid 5 alpha-pregnan-3 alpha-ol-11,20-dione (alfaxalone). Desensitizing GABAA currents were selective for chloride, inhibited by bicuculline and tert-butyl-bicyclophosphorothionate, and potentiated by diazepam, pentobarbital sodium, and alfaxalone. The EC50 for GABA was 15 microM.

Inhibition of P-type and N-type calcium channels by dopamine receptor antagonists.

P-type Ca2+ channels in cerebellar Purkinje neurons and N-type Ca2+ channels in sympathetic neurons were found to be inhibited by D2 dopamine receptor antagonists with diverse structures, including phenothiazines (chlorpromazine and thioridazine), diphenylbutylpiperidines (fluspirilene and pimozide), butyrophenones (haloperidol and spiperone), and a piperazine (fluphenazine). Dopamine and quinpirole had no effect on P-type Ca2+ channels. In all cases, inhibition was characterized by slow onset and offset. The effects of P-type and N-type channels were very similar. Fluspirilene was the most potent of the drugs, with EC50 values of 6 microM for P-type current and 2 microM for N-type current. Block of P-type channels by fluspirilene was voltage dependent, being enhanced by depolarized holding potentials, and use dependent, being enhanced by higher stimulation frequencies. The effect of fluspirilene on the P-type Ca2+ channel current was not prevented by simultaneous exposure to the peptide toxin omega-agatoxin IVA, indicating that fluspirilene binds to a distinct site on the channel. The results suggest that N-type and P-type Ca2+ channels possess similar binding sites for dopamine receptor antagonists and that block of N-type and P-type channels is relatively weak, compared with that of some T-type and L-type Ca2+ channels.

Ca2+ channels in rat central and peripheral neurons: high-threshold current resistant to dihydropyridine blockers and omega-conotoxin.

Block of Ca2+ channel current by dihydropyridines and by omega-conotoxin (omega-CgTx) was studied in a variety of freshly dissociated rat neurons. In most neurons, including those from dorsal root ganglia, sympathetic ganglia, spinal cord, cerebral cortex, and hippocampus, nitrendipine and omega-CgTx each blocked a fraction of the high-threshold current, but a substantial fraction of current remained even when the two blockers were applied together at saturating concentrations. An extreme case was cerebellar Purkinje neurons, in which very little current was blocked by either nitrendipine or omega-CgTx. These results demonstrate the existence in mammalian neurons of high-threshold channels that are resistant to both omega-CgTx and dihydropyridine blockers. Such channels might underlie instances of synaptic transmission and other processes that depend on Ca2+ entry but are not sensitive to these blockers.

Different biological activities in conditioned media control the expression of a variety of neuropeptides in cultured sympathetic neurons.

An intriguing question regarding neuronal development is how neurons choose which neurotransmitter and/or peptide to express among over 40 candidates. We find that heart cell conditioned medium (CM) induces a number of neuropeptides and/or their precursor mRNAs, as well as acetylcholine, in cultured rat sympathetic neurons: substance P, somatostatin, vasoactive intestinal polypeptide, enkephalin derivatives, and cholecystokinin, but not neuropeptide Y. Different patterns of peptide induction were observed for CMs from primary cultures of heart, gut, and skin. Acetylcholine and substance P were induced most effectively by serum-free heart cell CM; enkephalin derivatives were induced most effectively by skin cell CM; and somatostatin and vasoactive intestinal polypeptide were induced equally well by all of the CMs. These observations suggest the possibility that many distinct, diffusible factors can influence the choice of transmitter and/or peptide phenotype in developing neurons.