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M Farrant

Publications and source records attributed to M Farrant.

At least 19 recordsLinked to original sources
PubMed2003

Cerebellar granule cell Cre recombinase expression.

The cerebellum maintains balance and orientation, refines motor action, stores motor memories, and contributes to the timing aspects of cognition. We generated two mouse lines for making Cre recombinase-mediated gene disruptions largely confined to adult cerebellar granule cells. For this purpose we chose the GABA(A) receptor alpha6 subunit gene, whose expression marks this cell type. Here we describe mouse lines expressing Cre recombinase generated by 1) Cre knocked into the native alpha6 subunit gene by homologous recombination in embryonic stem cells; and 2) Cre recombined into an alpha6 subunit gene carried on a bacterial artificial chromosome (BAC) genomic clone. The fidelity of Cre expression was tested by crossing the mouse lines with the ROSA26 reporter mice. The particular alpha6BAC clone we identified will be valuable for delivering other gene products to cerebellar granule cells.

Animals↗
PubMed2001

Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance.

Many neurons receive a continuous, or 'tonic', synaptic input, which increases their membrane conductance, and so modifies the spatial and temporal integration of excitatory signals. In cerebellar granule cells, although the frequency of inhibitory synaptic currents is relatively low, the spillover of synaptically released GABA (gamma-aminobutyric acid) gives rise to a persistent conductance mediated by the GABA A receptor that also modifies the excitability of granule cells. Here we show that this tonic conductance is absent in granule cells that lack the alpha6 and delta-subunits of the GABAA receptor. The response of these granule cells to excitatory synaptic input remains unaltered, owing to an increase in a 'leak' conductance, which is present at rest, with properties characteristic of the two-pore-domain K+ channel TASK-1 (refs 9,10,11,12). Our results highlight the importance of tonic inhibition mediated by GABAA receptors, loss of which triggers a form of homeostatic plasticity leading to a change in the magnitude of a voltage-independent K + conductance that maintains normal neuronal behaviour.

Adaptation, Physiological↗
PubMed2001

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.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2001

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.

Alternative Splicing↗
PubMed2000

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.

2-Amino-5-phosphonovalerate↗
PubMed1999

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

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.

Aging↗
PubMed1995

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)

Animals↗
PubMed1994

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.

Animals↗
PubMed1992

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.

Adult↗
PubMed1991

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)

Animals↗
PubMed1991

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.

Adult↗
PubMed1990

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.

Allosteric Regulation↗