Fulminant type 1 diabetes in pregnancy.

We report two cases of fulminant type 1 diabetes in previously well migrants from South East Asia. This entity, which is rare outside East or South-East Asia, has a high perinatal mortality. The clinical presentation differs markedly from that of typical newly recognised type 1 diabetes in pregnancy. In both our cases, the neonates required intensive care but survived.

Differences in synaptic GABA(A) receptor number underlie variation in GABA mini amplitude.

In many neurons, responses to individual quanta of transmitter exhibit large variations in amplitude. The origin of this variability, although central to our understanding of synaptic transmission and plasticity, remains controversial. To examine the relationship between quantal amplitude and postsynaptic receptor number, we adopted a novel approach, combining patch-clamp recording of synaptic currents with quantitative immunogold localization of synaptic receptors. Here, we report that in cerebellar stellate cells, where variability in GABA miniature synaptic currents is particularly marked, the distribution of quantal amplitudes parallels that of synaptic GABA(A) receptor number. We also show that postsynaptic GABA(A) receptor density is uniform, allowing synaptic area to be used as a measure of relative receptor content. Flurazepam, which increases GABA(A) receptor affinity, prolongs the decay of all miniature currents but selectively increases the amplitude of large events. From this differential effect, we show that a quantum of GABA saturates postsynaptic receptors when <80 receptors are present but results in incomplete occupancy at larger synapses.

NMDA receptor subunits: diversity, development and disease.

N-methyl-D-aspartate receptors (NMDARs) are present at many excitatory glutamate synapses in the central nervous system and display unique properties that depend on their subunit composition. Biophysical, pharmacological and molecular methods have been used to determine the key features conferred by the various NMDAR subunits, and have helped to establish which NMDAR subtypes are present at particular synapses. Recent studies are beginning to address the functional significance of NMDAR diversity under normal and pathological conditions.

CNQX increases GABA-mediated synaptic transmission in the cerebellum by an AMPA/kainate receptor-independent mechanism.

GABA(A) receptor-mediated inhibitory synaptic transmission within the CNS is often studied in the presence of glutamate receptor antagonists. However, for nearly a decade it has been known that, in the hippocampus, one of the most commonly used alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), can increase the frequency of spontaneous GABA(A) receptor-mediated postsynaptic currents (sIPSCs). In the present study we examined the effect of CNQX and related compounds on GABA-mediated synaptic transmission in the cerebellum. At various stages of development, low concentrations of CNQX increased the frequency of sIPSCs recorded from granule cells. This effect was independent of the blocking action of CNQX on ionotropic glutamate receptors, as it was not observed with the broad-spectrum glutamate receptor antagonist kynurenate. No increase in sIPSC frequency was observed with the NMDA receptor antagonists D-AP5 or 7-ClK, the selective AMPA receptor antagonists GYKI 52466 or GYKI 53655, or the kainate receptor antagonist NS-102. In contrast, two other quinoxaline derivatives, NBQX and DNQX, were capable of increasing sIPSC frequency. These results demonstrate that the novel excitatory action of CNQX, unrelated to blockade of ionotropic glutamate receptors, is not restricted to the hippocampus and can be observed with structurally related compounds.

NMDA receptor diversity in the cerebellum: identification of subunits contributing to functional receptors.

Recent studies of N-methyl-D-aspartate (NMDA) receptors have led to the suggestion that there are two distinct classes of native NMDA receptors, identifiable from their single-channel conductance properties. 'High-conductance' openings arise from NR2A- or NR2B-containing receptors, and 'low-conductance' openings arise from NR2C- or NR2D-containing receptors. In addition, the low-conductance channels show reduced sensitivity to block by Mg2+. The readily identified cell types and simple architecture of the cerebellum make it an ideal model system in which to determine the contribution of specific subunits to functional NMDA receptors. Furthermore, mRNA for all of these four NR2 subunits are represented in this brain region. We have examined NMDA channels in Purkinje cells, deep cerebellar nuclei (DCN) neurons and Golgi cells. First we find that NR2D-containing NMDA receptors give rise to low-conductance openings in cell-attached recordings from Purkinje cells. The characteristic conductance of these events cannot, therefore, be ascribed to patch excision. Second, patches from some DCN neurons exhibit mixed populations of high- and low-conductance openings. Third, Golgi cells also exhibit a mixed population of high- and low-conductance NMDA receptor openings. The features of these low-conductance openings are consistent with the presence of NR2D-containing NMDA receptors, as suggested by in situ hybridization data. On the other hand the existence of high-conductance channels, with properties typical of NR2B-containing receptors, was not expected. Our results provide new evidence about the subunit composition of NMDA receptors in identified cerebellar cells, and suggest that examination of single-channel properties is a potentially powerful approach for determining the possible subunit composition of native NMDA receptors.

Excitatory amino acid receptor-channels in Purkinje cells in thin cerebellar slices.

Glutamate receptors of the N-methyl-D-aspartate (NMDA) and non-NMDA type serve different functions during excitatory synaptic transmission. Although many central neurons bear both types of receptor, the evidence concerning the sensitivity of cerebellar Purkinje cells to NMDA is contradictory. To investigate the receptor types present in Purkinje cells, we have used whole-cell and outside-out patch-clamp methods to record from cells in thin cerebellar slices from young rats. At a holding potential of -70 mV (in nominally Mg(2+)-free medium, with added glycine) NMDA caused a whole-cell current response which consisted of a dramatic increase in the frequency of synaptic currents. In the presence of tetrodotoxin (TTX) and the gamma-aminobutyric acidA (GABAA) receptor antagonist bicuculline, spontaneous synaptic currents and responses to NMDA were inhibited. In a proportion of cells a small polysynaptic response to NMDA persisted, which was further reduced by the non-NMDA receptor antagonist 6-cyano-2,3-dihydro-7-nitroquinoxalinedione (CNQX). The non-NMDA glutamate receptor agonists kainate (KA), quisqualate (QA) and s-alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (s-AMPA), evoked large inward currents due to the direct activation of receptors in Purkinje cells. NMDA applied to excised membrane patches failed to evoke any single-channel currents, whereas s-AMPA and QA caused small inward currents accompanied by marked increases in current noise. Spectral analysis of the s-AMPA noise in patches gave an estimated mean channel conductance of approximately 4 pS.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuronal activity, amino acid concentration and amino acid release in the substantia nigra of the rat after sodium valproate.

The effects of sodium valproate on extracellularly recorded spontaneous neuronal activity and striatal-evoked inhibition in the substantia nigra zona reticulata of the rat were compared with its effects on the tissue concentration of endogenous amino acids and their spontaneous release into perfusates of this region obtained with a push-pull cannula. Valproate (200 mg/kg i.p.) produced a rapid and sustained reduction in the firing rate of all reticulata neurones tested and a concomitant increase in the duration of striatal-evoked inhibition. No change in the spontaneous release of any amino acid was observed. A significant elevation of nigral gamma-aminobutyric acid concentration was seen in both anaesthetized and non-anaesthetized animals, but this occurred only after 60 minutes. Valproate produced a rapid decline in nigral aspartate in non-anaesthetized but not in anaesthetized animals. The results of this study suggest that the acute depressant effect of valproate is unrelated to its ability to alter the concentration of GABA or aspartate in brain and is most likely due to a postsynaptic action.

Compartmental distribution of endogenous amino acids in the substantia nigra of the rat.

Using a push-pull cannula we have monitored the spontaneous efflux of 8 endogenous amino acids into perfusates of the substantia nigra of the rat. The extracellular concentrations of the amino acids were estimated and compared to their respective tissue levels. High intra-/extracellular concentration ratios were found for gamma-aminobutyric acid (GABA), aspartate, glutamate and taurine. Much lower values were found for glycine, alanine, serine and glutamine. These results provide evidence for possible neurotransmitter roles for aspartate, glutamate and taurine in the substantia nigra in addition to that, long recognized, for GABA.

Work-related population health indicators.

OBJECTIVE: To provide evidence-based recommendations for work-related population health indicators. METHODS: Drawing on a framework of work-related experiences, we systematically reviewed studies that assess the association between these experiences and health and reviewed related measures at the population level that could be used as indicators. RESULTS: We recommend (and grade the strength of evidence supporting our recommendation for) the following indicators for which data are already routinely collected: unemployment rate (strong), long-term unemployment rate (limited), and permanent lay-off rate (limited). As well, we recommend and grade our support for the following new indicators: insecurity associated with pending job loss (limited), with possible major organizational change (limited), and with actual major organizational change (limited); and job strain (medium). CONCLUSION: These evidence-based indicators can be used to monitor work-related determinants of health and thus to inform the conceptualization, development, and evaluation of policies and programs related to these determinants.

"Run-down" of gamma-aminobutyric acidA receptor function during whole-cell recording: a possible role for phosphorylation.

When using whole-cell recording methods and a minimal intracellular medium containing only inorganic ions, ethyleneglycolbis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, we have observed a time-dependent decrease in the responsiveness of cultured chick spinal cord neurons to gamma-aminobutyric acid (GABA). The current evoked by 30 microM GABA progressively declined to approximately 30% of its initial value after five applications at 10-min intervals. This was accompanied by an equivalent decline in the GABA-evoked membrane conductance. "Run-down" of the response was reduced when Mg2+-ATP was present in the pipet solution. Inclusion of ATP-gamma-S, an analog that donates a thiophosphate group resistant to hydrolysis, also reduced run-down. The nonhydrolyzable analog beta, gamma-imidoadenosine-5'-triphosphate was without effect. These results suggest that an ATP-dependent process, possibly phosphorylation, is involved in the maintenance of GABAA receptor function.

A direct comparison of the single-channel properties of synaptic and extrasynaptic NMDA receptors.

The assumption that synaptic and extrasynaptic glutamate receptors are similar underpins many studies that have sought to relate the behavior of channels in excised patches to the macroscopic properties of the EPSC. We have examined this issue for NMDA receptors in cerebellar granule cells, the small size of which allows the opening of individual synaptic NMDA channels to be resolved directly. We have used whole-cell patch-clamp recordings to determine the conductance and open time of NMDA channels activated during the EPSC and used cell-attached and outside-out recordings to examine NMDA receptors in somatic membrane. Conductance and open time of synaptic channels were indistinguishable from those of extrasynaptic channels in cell-attached patches. However, the channel conductance in outside-out patches was 20% lower than in cell-attached recordings. This change was partially reduced by dantrolene and phalloidin, suggesting that it may involve depolymerization of actin following Ca2+ release from intracellular stores. Our results demonstrate that synaptic and extrasynaptic NMDA receptors have similar microscopic properties. However, NMDA channel conductance is reduced following the formation of an outside-out patch.

Whole-cell and single-channel currents activated by GABA and glycine in granule cells of the rat cerebellum.

1. Patch-clamp methods have been used to characterize GABA-and glycine-activated channels and spontaneous synaptic currents in granule cells in thin cerebellar slices from 7- to 20-day-old rats. 2. All granule cells responded to 10 microM GABA, while approximately 60% responded to 100 microM glycine. With repeated against application, whole-cell responses to GABA, but not those to glycine, declined over a period of minutes unless the pipette solution contained Mg-ATP. 3. Whole-cell concentration-response curves gave EC50 values at 45.2 and 99.6 microM and Hill slopes of 0.94 and 2.6 for GABA and glycine, respectively. At saturating concentrations, currents evoked by GABA were fivefold larger than those evoked by glycine. 4. Whole-cell current-voltage (I-V) relationships of GABA- and glycine-activated currents reversed close to the predicted Cl- equilibrium potential. Partial replacement of intracellular Cl- with F- shifted the GABA reversal potential to a more negative value. 'Instantaneous' I-V relationships produced by ionophoretic application of GABA were linear, while 'steady-state' I-V relationships produced by ramp changes in potential showed outward rectification. For glycine, 'steady-state' I-V plots were linear. 5. Responses to GABA were blocked by the GABAA receptor antagonists bicuculline (15 microM), SR-95531 (10 microM) and picrotoxinin (100 microM) while responses to glycine were selectively blocked by strychnine (200 nM), indicating the presence of two separate receptor types. 6. In outside-out membrane patches, GABA opened channels with conductances of 16 and 28 pS. The proportion of openings to each of the conductances varied between patches, possibly indicating the activation of two distinct channel types. Glycine-activated single-channel currents had conductances of 32, 55 and 104 pS. Single-channel I-V relationships were linear. 7. Spontaneous synaptic currents with a rapid rise time and biexponential decay were present in more than half of the cells examined. These currents were eliminated by bicuculline (15 microM) or SR-95331 (10 microM) and were greatly reduced in frequency by tetrodotoxin (TTX; 300 nM), suggesting that they were mediated by GABA and arose from spontaneous activity in Golgi interneurones. In granule cells where this spontaneous synaptic activity was apparent, glycine and low concentrations of GABA increased the frequency of the synaptic currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors.

1. To investigate the origin and functional significance of a recently described tonic GABAA receptor-mediated conductance in cerebellar granule cells we have made recordings from cells in cerebellar slices from rats of different ages (postnatal days P4 to P28). 2. During development there was a dramatic change in the properties of GABA-mediated synaptic transmission. The contribution to GABAA receptor-mediated charge transfer from the tonic conductance (GGABA), relative to that resulting from discrete spontaneous postsynaptic currents (sPSCs), was increased from 5% at P7 to 99% at P21. GGABA was reduced by bicuculline, tetrodotoxin and by lowering extracellular Ca2+, and was initially present only in those cells which exhibited sPSCs. 3. At P7 sPSCs were depolarizing, occasionally triggering a single action potential. By P18 the GABA reversal potential was shifted close to the resting potential and GGABA produced a shunting inhibition. Removal of GGABA by bicuculline increased granule cell excitability in response to current injection. 4. This novel tonic inhibition is present despite the low number of Golgi cell synapses on individual granule cells and appears to result from 'overspill' of synaptically released GABA leading to activation of synaptic and extrasynaptic GABAA receptors.

Barriers to employment-related healthy public policy in Canada.

The Ottawa Charter for Health Promotion calls for building healthy public policy, that is for '[putting] health on the agenda of policy makers in all sectors and at all levels, directing them to be aware of the health consequences of their decisions and to accept their responsibilities for health'. The objective of this study was to assess the past and potential future influence of information about the health consequences of unemployment and job insecurity on policy making and to identify the barriers to the use of such information in policy making. We conducted telephone interviews with 38 policy makers in the health and employment sectors of all three levels of Canadian government, as well as the executive directors of 10 Canadian non-governmental organizations that are active on employment issues. The interviews included both numerical ratings of the influence of this information and semi-structured questions about how this information could be used in policy making. Using an interpretive approach grounded in the political science literature, we identified barriers to using this information in their responses to these questions. Respondents rated the potential future influence of this information (mean 4.2 and median 5 on a seven-point Likert scale) higher than its past influence (mean 3.5 and median 3 on a seven-point Likert scale). Barriers related to the information itself or more commonly to the values of those who could respond to the information (i.e. idea-related barriers) were cited more frequently than either barriers related to how decisions are made (i.e. institution-related barriers) or barriers related to who would win and who would lose if the information were acted upon (i.e. interest-related barriers). We concluded that to build employment-related healthy public policy, these barriers would have to be overcome. Policy makers in health departments could, for example, frame information about health consequences in language that fits more easily with the values of other departments and advocate for institutional innovations that establish cross-departmental or cross-governmental accountability for health.

Single-channel properties of synaptic and extrasynaptic GABAA receptors suggest differential targeting of receptor subtypes.

Many neurons express a multiplicity of GABAA receptor subunit isoforms. Despite having only a single source of inhibitory input, the cerebellar granule cell displays, at various stages of development, more than 10 different GABAA subunit types. This subunit diversity would be expected to result in significant receptor heterogeneity, yet the functional consequences of such heterogeneity remain poorly understood. Here we have used single-channel properties to characterize GABAA receptor types in the synaptic and extrasynaptic membrane of granule cells. In the presence of high concentrations of GABA, which induced receptor desensitization, extrasynaptic receptors in outside-out patches from the soma entered long-lived closed states interrupted by infrequent clusters of openings. Each cluster of openings, which is assumed to result from the repeated activation of a single channel, was to one of three main conductance states (28, 17, or 12 pS), the relative frequency of which differed between patches. Such behavior indicates the presence of at least three different receptor types. This heterogeneity was not replicated by individual recombinant receptors (alpha1beta2gamma2S or alpha1beta3gamma2S), which gave rise to clusters of a single type only. By contrast, the conductance of synaptic receptors, determined by fluctuation analysis of the synaptic current or direct resolution of channel events, was remarkably uniform and similar to the highest conductance value seen in extrasynaptic patches. These results suggest that granule cells express multiple GABAA receptor types, but only those with a high conductance, most likely containing a gamma subunit, are activated at the synapse.

Identification of subunits contributing to synaptic and extrasynaptic NMDA receptors in Golgi cells of the rat cerebellum.

1. To investigate the properties of N-methyl-D-aspartate receptors (NMDARs) in cerebellar Golgi cells, patch-clamp recordings were made in cerebellar slices from postnatal day 14 (P14) rats. To verify cell identity, cells were filled with Neurobiotin and examined using confocal microscopy. 2. The NR2B subunit-selective NMDAR antagonist ifenprodil (10 microM) reduced whole-cell NMDA-evoked currents by approximately 80 %. The NMDA-evoked currents were unaffected by the Zn2+ chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN; 1 microM) suggesting the absence of NMDARs containing NR2A subunits. 3. Outside-out patches from Golgi cells exhibited a population of 'high-conductance' 50 pS NMDAR openings. These were inhibited by ifenprodil, with an IC50 of 19 nM. 4. Patches from these cells also contained 'low-conductance' NMDAR channels, with features characteristic of NR2D subunit-containing receptors. These exhibited a main conductance of 39 pS, with a sub-conductance level of 19 pS, with clear asymmetry of transitions between the two levels. As expected of NR2D-containing receptors, these events were not affected by ifenprodil. 5. The NMDAR-mediated component of EPSCs, evoked by parallel fibre stimulation or occurring spontaneously, was not affected by 1 microM TPEN. However, it was reduced (by approximately 60 %) in the presence of 10 microM ifenprodil, to leave a residual NMDAR-mediated current that exhibited fast decay kinetics. This is, therefore, unlikely to have arisen from receptors composed of NR1/NR2D subunits. 6. We conclude that in cerebellar Golgi cells, the high- and low-conductance NMDAR channels arise from NR2B- and NR2D-containing receptors, respectively. We found no evidence for NR2A-containing receptors in these cells. While NR2B-containing receptors are present in both the synaptic and extrasynaptic membrane, our results indicate that NR1/NR2D receptors do not contribute to the EPSC and appear to be restricted to the extrasynaptic membrane.

Lymphomas of the cervix and upper vagina: a report of five cases and a review of the literature.

Five cases of non-Hodgkin's lymphoma of the cervix or upper vagina presenting over the last 20 years are described. The international literature has been reviewed for similar cases and a further 72 found. In 37 of these cases the pathology had been described according to one of the modern lymphoma classifications and details of clinical presentation, staging, treatment, and outcome were adequately described. The management and outcome of these patients have been critically reviewed and recommendations for the management of patients presenting with this disease have been made.

NMDA-receptor channel diversity in the developing cerebellum.

In the cerebellum, NMDA (N-methyl-D-aspartate) receptors play an important role in neuronal differentiation and excitatory synaptic transmission. During early cerebellar development, marked changes occur in the distribution of messenger RNAs encoding various NMDA-receptor subunits. To determine whether these changes result in the appearance of functionally distinct NMDA receptors, we have recorded single-channel currents in rat cerebellar granule cells during the period of their migration from the external germinal layer to the inner granular layer. Here we show that before synapse formation, pre-migratory and migrating granule cells express NMDA receptors possessing single-channel properties similar to those previously described for many central neurons. In contrast, mature post-migratory cells also express an atypical form of NMDA receptor that has a lower single-channel conductance and distinct kinetic behaviour. The properties of these 'low-conductance' channels correspond to those described for recombinant NMDA receptors formed by coexpression of NR1 and NR2C subunits. The NR2C subunit appears postnatally and is found predominantly in the adult cerebellum. Our data demonstrate developmental changes in NMDA-receptor properties at the single-channel level, and suggest that in the cerebellum the expression of a specific subunit protein results in a distinct form of native receptor.

Chronic agonist exposure induces down-regulation and allosteric uncoupling of the gamma-aminobutyric acid/benzodiazepine receptor complex.

Using [3H]flunitrazepam as a probe for the benzodiazepine-sensitive modulator site located on the gamma-aminobutyric acid (GABA)A receptor complex, we have investigated the cellular regulation of the GABAA receptor in neuronal cultures derived from embryonic chick brain. Treatment of cultures with 1 mM GABA for 48 hr causes a reversible 35% decrease in the number of [3H]flunitrazepam binding sites with no change in affinity. The EC50 for chronic GABA-induced down-regulation is 94 microM and the half-time is 25 hr. The effect of GABA is blocked by SR-95531, a GABAA receptor antagonist, and mimicked by muscimol but not baclofen. Consistent with the decrease in [3H]flunitrazepam binding, chronic GABA exposure causes a 43% decrease in the binding of [35S]t-butylbicyclophosphorothionate, a ligand for the receptor-associated chloride ionophore. In addition to chronic GABA-induced down-regulation, allosteric interactions between GABA and benzodiazepine recognition sites are uncoupled by 34%. The half-time and pharmacology for chronic GABA-induced uncoupling is indistinguishable from that for GABA-induced down-regulation, consistent with the hypothesis that the action of GABA at a common site induces both down-regulation and uncoupling.

Improving biliary-enteric drainage in primary sclerosing cholangitis: experience with endoscopic methods.

Six jaundiced patients with primary sclerosing cholangitis and a dominant biliary stricture were managed by endoscopic placement of endoprostheses. Five showed considerable improvement within weeks of stenting: their serum bilirubin concentration fell from mean (range) 266 mumol/l (63-681) to 65 mumol/l (10-280) after one month. One patient required a liver transplant at five months because of continued deterioration in hepatic function. Follow up of 12-49 months in the remaining five patients shows sustained biochemical improvement, with repeat cholangiograms indicating doubling of the minimum calibre of the extrahepatic bile duct in four patients and considerable shortening of stricture length in three. Three patients developed sepsis at the time of the initial endoprosthesis insertion: surgical drainage was necessary in one. Endoscopic methods of improving biliary-enteric drainage in jaundiced patients with primary sclerosing cholangitis may be preferable to surgical and percutaneous methods, which may complicate subsequent liver transplantation.