Quantifying noise-induced stability of a cortical fast-spiking cell model with Kv3-channel-like current.

Population oscillations in neural activity in the gamma (>30 Hz) and higher frequency ranges are found over wide areas of the mammalian cortex. Recently, in the somatosensory cortex, the details of neural connections formed by several types of GABAergic interneurons have become apparent, and they are believed to play a significant role in generating these oscillations through synaptic and gap-junctional interactions. However, little is known about the mechanism of how such oscillations are maintained stably by particular interneurons and by their local networks, in a noisy environment with abundant synaptic inputs. To obtain more insight into this, we studied a fast-spiking (FS)-cell model including Kv3-channel-like current, which is a distinctive feature of these cells, from the viewpoint of nonlinear dynamical systems. To examine the specific role of the Kv3-channel in determining oscillation properties, we analyzed basic properties of the FS-cell model, such as the bifurcation structure and phase resetting curves (PRCs). Furthermore, to quantitatively characterize the oscillation stability under noisy fluctuations mimicking small fast synaptic inputs, we applied a recently developed method from random dynamical system theory to estimate Lyapunov exponents, both for the original four-dimensional dynamics and for a reduced one-dimensional phase-equation on the circle. The results indicated that the presence of the Kv3-channel-like current helps to regulate the stability of noisy neural oscillations and a transient-period length to stochastic attractors.

Spatio-temporal cholinergic modulation in cultured networks of rat cortical neurons: spontaneous activity.

Activation of the cholinergic innervation of the cortex has been implicated in sensory processing, learning, and memory. At the cellular level, acetylcholine both increases excitability and depresses synaptic transmission, and its effects on network firing are hard to predict. We studied the effects of carbachol, a cholinergic agonist, on network firing in cultures of rat cortical neurons, using electrode arrays to monitor the activity of large numbers of neurons simultaneously. These cultures show stable spontaneous synchronized burst firing which propagates through dense synaptic connections. Carbachol (10-50 microM), acting through muscarinic receptors, was found to induce a switch to asynchronous single-spike firing and to result in a loss of regularity and fragmentation of the burst structure. To obtain a quantitative measure of cholinergic actions on cortical networks, we applied a cluster Poisson-process model to sets of paralleled spike-trains in the presence and absence of carbachol. This revealed that the time series can be well-characterized by such a simple model, consistent with the observed 1/f(b)-like spectra (0.04

Spatio-temporal cholinergic modulation in cultured networks of rat cortical neurons: evoked activity.

We studied the effects of carbachol, a cholinergic agonist, on extracellularly evoked firing of networks in mature cultures of rat cortical neurons, using multi-electrode arrays to monitor the activity of large numbers of neurons simultaneously. These cultures show evoked burst firing which propagates through dense synaptic connections. When a brief voltage pulse was applied to one extracellular electrode, spiking electrical responses were evoked in neurons throughout the network. The response had two components: an early phase, terminating within 30-80 ms, and a late phase which could last several hundreds of milliseconds. Action potentials evoked during the early phase were precisely timed, with only small jitter. In contrast, the late phase characteristically showed clusters of electrical activity with significant spatio-temporal fluctuations. The late phase was suppressed by applying a relatively small amount of carbachol (5 microM) in the external solution, even though the spontaneous firing rate was not significantly changed. Carbachol increased both the spike-timing precision and the speed of propagation of population spikes, and selectively increased the firing coincidence in a subset of neuron pairs in the network, while suppressing late variable firing in responses. Hence, the results give quantitative support for the idea that cholinergic activation in the cortex has a general role of focusing or enhancing significant associative firing of neurons.

Disturbance of membrane function preceding ischemic delayed neuronal death in the gerbil hippocampus.

Slice preparations were made from the hippocampus of gerbils after 5 min of ischemia by carotid artery occlusion and the membrane properties of pyramidal neurons were examined. A majority of CA1 neurons lost the capacity for long-term potentiation following tetanic stimulation of the input fibers. CA3 pyramidal neurons, in contrast, preserved responses similar to those in the normal gerbil. Following ischemia, CA1 pyramidal neurons showed increased spontaneous firing that was highly voltage dependent and was blocked by intracellular injection of the Ca2+ chelator, EGTA. Thirty-five percent of CA1 neurons showed an abnormal slow oscillation of the membrane potential after 24 h following ischemia. Intracellular injection of GTP gamma S or IP3 produced facilitation of the oscillations followed by irreversible depolarization. Our results indicate that ischemia-damaged CA1 neurons suffer from abnormal Ca2+ homeostasis, involving IP3-induced liberation of Ca2+ from internal stores.

Block of long-term potentiation by intracellular application of anti-phosphatidylinositol 4,5-bisphosphate antibody in hippocampal pyramidal neurons.

We examined the effects of black widow spider toxin and anti-phosphatidylinositol 4,5-bisphosphate antibody on the changes in excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents accompanying long-term potentiation using whole-cell recording from hippocampal CA1 pyramidal neurons of rodents. In the presence of black widow spider toxin, tetanic stimulation of input fibers produced a short-lived potentiation followed by a gradual decline of the excitatory postsynaptic current amplitude. With an anti-phosphatidylinositol 4,5-bisphosphate antibody containing pipette, tetanus elicited only decremental potentiation of excitatory postsynaptic currents with a reduced frequency of spontaneous excitatory postsynaptic currents, suggesting inhibition of retrograde reinforcement from the antibody-injected neuron. With both black widow spider toxin and anti-phosphatidylinositol 4,5-bisphosphate antibody, neurons showed a rapid depression of excitatory postsynaptic currents after tetanus. The results indicate that time-dependent interactions between presynaptic terminals and the postsynaptic spikes take place during long-term potentiation.

Inositol 1,3,4,5-tetrakisphosphate as a mediator of neuronal death in ischemic hippocampus.

Selective death of CA1 pyramidal neurons after transient forebrain ischemia has attracted interest for its possible relation to the pathogenesis of memory deficits and dementia. Using whole cell patch-clamp recording from CA1 pyramidal neurons in hippocampal slices of gerbils after ischemia we studied the intracellular signaling mechanisms related to the phosphoinositide cycle. Intracellular application of an antibody against phosphatidylinositol 4,5-bisphosphate rescued ischemic neurons from stimulus-induced irreversible depolarization. Furthermore, application of inositol 1,3,4,5-tetrakisphosphate in normal cells caused an irreversible depolarization in response to synaptic input, which mimicked the deterioration of ischemic neurons. Depolarization of both ischemic and normal neurons in the presence of inositol 1,3,4,5-tetrakisphosphate was prevented by the addition of the Ca2+ chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetate. Application of antibody against inositol 1,4,5-triphosphate 3-kinase, which blocks formation of inositol 1,3,4,5-tetrakisphosphate, also protected against cell deterioration. Our results suggest that the vulnerability of hippocampal pyramidal neurons following ischemia is caused by a disturbed phosphoinositide cascade, with one metabolite, inositol 1,3,4,5-tetrakisphosphate, playing a key role in the induction of Ca2+ accumulation, which leads to neuronal death.

Abnormal Ca2+ homeostasis before cell death revealed by whole cell recording of ischemic CA1 hippocampal neurons.

Slices were made from the hippocampus of gerbils following transient ischemia achieved by clamping the carotid arteries for 5 min, and changes in the electrophysiology of CA1 pyramidal neurons were studied by whole cell patch-clamp recording as well as conventional intracellular recording. The great majority of CA1 neurons in slices made 2.5-3 days after ischemia showed reduced resting potentials and were easily depolarized by prolonged low-frequency stimulation or by tetanic stimulation of the Schaffer collateral/commissural input. This stimulus-induced depolarization was accelerated by intracellular injection of D-myo-inositol 1,4,5-triphosphate, which depolarized membrane potentials towards 0 mV without synaptic input stimulation. Intracellular application of BAPTA, a Ca2+ chelator, effectively blocked the stimulus-induced depolarization. When recording from ischemic neurons with patch pipettes containing both D-myo-inositol 1,4,5-triphosphate and BAPTA, excitatory postsynaptic currents were transiently potentiated by stimulation, but the membrane potential did not show stimulus-induced depolarization and remained steady for long periods. These results lend support to the view that the intracellular Ca2+ regulation system is severely disturbed following ischemia, and that input fiber stimulation leads to abnormal Ca2+ accumulation in ischemic neurons resulted in neuronal death. The reduction of free Ca2+ inside the ischemic neuron by BAPTA apparently saves neurons which are otherwise destined to delayed neuronal death.

Single channel properties at the synaptic site.

The properties of single channels activated during spontaneous postsynaptic currents in small cultured rat hippocampal neurons were investigated in low-noise whole cell recordings. The technique of nonstationary fluctuation analysis, which has previously been applied to sodium currents, was modified so that fluctuations were measured around the least-squares scaled fit of the ensemble average to individual synaptic currents. This had the effect of separating channel gating fluctuations from the quantal fluctuations of scale from event to event. Single channel amplitude was estimated from the variance--current distribution, and the kinetics of channel gating fluctuations were studied. Channels involved in the non-N-methyl-D-aspartate (non-NMDA) phase of the excitatory glutamatergic postsynaptic current showed a single channel amplitude of 1.5 pS, while those in the NMDA phase had a conductance of 42 pS. The single channel conductance estimated for the inhibitory chloride synaptic current was 14 pS. In addition, NMDA phase channel openings could be resolved directly in the whole cell current against the low noise level afforded by the small cells. Single channel lifetime and amplitude distributions of the channels activated during the postsynaptic current were measured, and confirmed the accuracy of the fluctuation method.

Purification of AMPA type glutamate receptor by a spider toxin.

A glutamate receptor was purified from Triton X-100-solubilized bovine cerebellum membranes. The purification was carried out in two steps: affinity chromatography using a spider toxin (Joro spider toxin; JSTX) immobilized on a lysine-agarose column, and a Mono Q anion exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified active fraction showed a single band with Coomassie Blue staining, which migrated with a M(r) = 130,000. The specific [3H]amino-3-hydroxy-5-methyl-isoxazole propionate ([3H]AMPA) binding activity of the affinity-purified fraction was 2095-fold higher than that of the crude soluble fraction. Lineweaver-Burk plot analysis showed a Kd of 12.7 nM [3H]AMPA in the purified fraction. The purified fraction was examined with patch-clamp recording methods in reconstituted liposomes. A glutamate-activated channel was observed and was inhibited with JSTX. The rank order of potency of agonists inducing channel currents was AMPA = glutamate greater than quisqualate much greater than kainate greater than NMDA. Thus, there is strong evidence that the 130 kDa protein is a purified component of the native AMPA type glutamate channel of bovine cerebellum.

Single glutamate channels in CA1 pyramidal neurones after transient ischaemia.

Patch clamp recordings were made from CA1 pyramidal neurones to study changes in the glutamate receptor subtypes in the gerbil hippocampus after transient ischaemia. In whole-cell recordings, the maximum chord conductances of AMPA currents in ischaemic neurones were increased over those of control neurones but NMDA-induced currents in the ischaemic neurones were smaller than the control. In AMPA-activated single channel currents, an open time histogram of the control neurones was well fitted by a single exponential function whereas in the ischaemic patches it was fitted by a double exponential function, indicating that currents consisted of at least two kinetically different types. These functional changes of the glutamate receptor channels may contribute to the abnormalities of the excitatory synaptic currents recorded in post-ischaemic CA1 neurones.

Ultrasound timing of human chorionic gonadotropin administration in clomiphene-stimulated cycle.

Seventeen patients treated with clomiphene citrate were examined using real-time ultrasound timing 28 ovulatory cycles to determine the pattern of follicular development under clomiphene stimulation. The rate of growth was faster, but the follicular diameter range at ovulation was similar to that during spontaneous cycles. This information was used to time midcycle human chorionic gonadotropin (hCG) administration in over 97 cycles in 21 patients responsive to clomiphene who had not conceived. When hCG was given when the mean follicular diameter reached 18 mm, 92% of these cycles were ovulatory. Fourteen patients (67%) conceived within 6 ovulatory treatment cycles. Five of 7 patients (71%) who did not conceive were found to have endometriosis at laparoscopy. Midcycle cervical scores were significantly lower in clomiphene-treated as compared with spontaneous ovulatory cycles, and additional treatment with ethinyl estradiol did not effect a significant improvement. Timing of midcycle hCG using ultrasound is an effective method of inducing ovulation in patients in whom an estrogenic follicular response without ovulation is obtained with clomiphene treatment.

Use of ultrasound in monitoring ovulation induction with human pituitary gonadotropins.

Serial follicular growth was studied using real-time ultrasound scanning in 26 anovulatory patients in 55 cycles stimulated with exogenous human pituitary gonadotropin (hPG) and human chorionic gonadotropin (hCG). Dosage was monitored with urinary total estrogen estimations. The ultrasound findings were not made known to the attending physician. Forty-four cycles (80%) were ovulatory, and conception occurred in 13. The mean follicular diameter of 17 to 25 mm at ovulation corresponded with the range observed in spontaneous cycles, although the rate of follicular growth was faster with hPG. On the day the ovulatory dose of hCG was given, as determined by the first urinary estrogen value greater than 50 microgram/24 hours, estrogen excretion was similar whether single or multiple follicles of potential ovulatory size were present, but mean follicular diameters were significantly smaller when there was more than 1 follicle. During the last 4 days before ovulation, the mean diameters in conceptual cycles increased from 14.2 mm to 19.7 mm, values similar to those observed in spontaneous cycles, but different growth patterns occurred in some nonconceptual cycles. Ovarian hyperstimulation was identified by ultrasound scan in 4 of 5 cycles before urinary estrogen excretion exceeded the normal range. Although ultrasound scan does not supplant estrogen monitoring, it can assist in the early detection of hyperstimulation, in the more accurate timing of the ovulatory dose of hCG, and in the withholding of hCG when 3 or more ovulations are possible. It is recommended that ultrasound examination be performed in all patients before the ovulation-inducing dose of hCG is given to provide information on the number of follicles that are likely to ovulate and thus avoid conception of more than twins.

Diagnostic ultrasound: early detection of fetal neural tube defects.

In a series of 366 patients identified as at risk for a fetal neural tube defect (NTD) before the 24th week of pregnancy, 64 had an abnormal fetus. The abnormalities included anencephaly (39), open spinal defect (17), closed spinal defect (2), encephalocele (1), and a miscellany of other abnormalities (5). An ultrasound examinaton prior to diagnostic anmiocentesis positively identified all anencephalic fetuses, the fetus with the encephalocele, and 15 of the 19 fetuses with spina bifida. The spinal defects in 3 of the remaining 4 fetuses were demonstrated at a second examination. Since both amniotic fluid alpha-fetoprotein (AFP) assays and ultrasound examination have been shown to give false results in the diagnosis of NTDs, the importance of using 2 independent diagnostic techniques is stressed. In patients with elevated levels of maternal serum AFP, a careful ultrasound examination, in addition to identifying the majority of cases associated with an abnormal fetus, provided a good explanation for the elevation in over half of the remainder. In this series more than half the patients (40/69) who underwent amniocentesis because of raised maternal serum AFP levels were shown to have an abnormal fetus.

Ultrasound-guided fetal blood transfusion for severe rhesus isoimmunization.

Four fetuses with severe rhesus isoimmunization were transfused with packed red blood cells directly into the umbilical vein. The outcome was successful in three. In one infant, this ultrasound-guided technique resulted in resolution of severe fetal hydrops at 27 weeks, allowing delivery of a healthy nonhydropic infant at 33 weeks, and in the other three infants in prolongation of the pregnancy. The last four transfusions were performed after fetal neuromuscular blockade with curare. The procedure would appear to be associated with a low risk of complication and to provide an excellent chance of a successful outcome of a fetus with severe rhesus isoimmunization even when fetal hydrops is present.

A voltage-clamp study of the effects of Joro spider toxin and zinc on excitatory synaptic transmission in CA1 pyramidal cells of the guinea pig hippocampal slice.

Using the single-electrode voltage-clamp technique, we have examined the effects of a non-N-methyl-D-aspartate (NMDA) antagonist. Joro spider toxin (JSTX), and of an NMDA antagonist, zinc, on excitatory postsynaptic currents (EPSCs) evoked by stimulation of stratum radiatum in CA1 pyramidal cells of the guinea-pig hippocampal slice. Pressure application of a synthesized JSTX (JSTX-3) at 10-200 microM greatly reduced the EPSCs (14/19 cells). The block by JSTX-3 was observed in pyramidal cells where the EPSCs showed linear peak current-voltage (I-V) relations in the control. EPSCs remaining after JSTX-3 application showed non-linear peak I-V relationships (10/14 cells), and were blocked by puff application of the selective NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (APV) at 200 microM (6/10 cells). In the presence of JSTX-3, the decay time constant of the EPSC was increased and was less affected by membrane potential. JSTX-3 had no detectable effects on EPSCs apparently mediated solely by NMDA receptor. These observations suggest that JSTX-3 blocks excitatory synaptic transmission mainly by suppressing non-NMDA-receptor-mediated EPSCs, and that the JSTX-3-insensitive component is mediated at least in part by NMDA receptors in the hippocampal slice. Zinc (100-200 microM) reversibly attenuated EPSCs (6/9 cells) and appeared to block a slower component of the EPSCs, suggesting that mainly NMDA receptor-mediated currents were affected.

Injection of digitally synthesized synaptic conductance transients to measure the integrative properties of neurons.

A novel technique was developed for injecting a time-varying conductance into a neuron, to allow quantitative measurement of the processing of synaptic inputs. In current-clamp recording mode, the membrane potential was sampled continuously and used to calculate and update the level of injected current within 60 microseconds, using a real-time computer, so as to mimic the electrical effect of a given conductance transient. Cellular responses to synthetic conductance transients modelled on the fast (non-N-methyl-D-aspartate) phase of the glutamatergic postsynaptic potential were measured in cultured rat hippocampal neurons.

Ultrasound in an in vitro fertilization program.

Ultrasound examinations of the preovulatory follicle were performed on 39 patients in 58 consecutive spontaneous cycles in which ovum aspiration for in vitro fertilization was planned. Examinations during the follicular phase helped to indicate when patients should be admitted for intensive monitoring of urinary luteinizing hormone (LH) levels and as a means of lateralizing the side of follicular development in those patients in whom one ovary was known to be inaccessible to laparoscopic aspiration. The technique was also of value in determining whether ovulation had occurred in those patients in whom the anticipated midcycle LH surge was not detected and as a routine measure prior to laparoscopy to ensure the continuing presence of the follicle.

Ultrasound control of clomiphene/human chorionic gonadotropin stimulated cycles for oocyte recovery and in vitro fertilization.

Three pregnancies have been achieved through in vitro fertilization (IVF) following clomiphene/human chorionic gonadotropin (hCG) stimulation monitored only with ultrasound. When the results of 120 stimulated cycles were compared with those from 213 spontaneous cycles during a one-year period, the clomiphene-ultrasound-hCG method led to a significantly higher laparoscopy rate as well as significantly better yields of mature oocytes and embryos for intrauterine transfer. The luteal phase was normal in the stimulated group. This ultrasound-monitored technique was simpler to manage and less costly and appears to be the current method of choice for obtaining oocytes for IVF.

Spontaneous periodic synchronized bursting during formation of mature patterns of connections in cortical cultures.

Long-term recording of spontaneous activity in cultured cortical neuronal networks was carried out using substrates containing multi-electrode arrays. Spontaneous uncorrelated firing appeared within the first 3 days and transformed progressively into synchronized bursting within a week. By 30 days from the establishment of the culture, the network exhibited a complicated non-periodic, synchronized activity pattern which showed no changes for more than 2 months and thus represented the mature state of the network. Pharmacological inhibition of activity only during the period when regular synchronized bursting was observed was capable of producing a different mature activity pattern from the control. These results suggest that periodic synchronized bursting plays a critical role in the development of synaptic connections.

Screening for ovarian cancer using serum CA125 and vaginal examination: report on 2550 females.

This study was undertaken to assess the effectiveness of using serum CA125 and vaginal examination as a screening test for ovarian cancer in apparently healthy females. Two thousand five hundred and fifty healthy females aged 40 and over were recruited to participate in a screening study involving a questionnaire, serum CA125 measurement and vaginal examination. Females with either an elevated CA125 level or abnormal vaginal examination had a pelvic ultrasound performed as a secondary procedure. The positive predictive values of an elevated serum CA125 level, and a combination of CA125 level measurement and vaginal examination for ovarian cancer, were 1/100 and 1/3, respectively. The specificities of serum CA125 levels, vaginal examination and both in combination were 96.1%, 98.5% and 99.9%, respectively. In postmenopausal females the positive predictive values were improved with CA125 measurement alone, giving a positive predictive value of 1/24. Seventeen females underwent operative procedure as a result of the screening-only one of these was for an ovarian cancer. The combination of serum CA125 measurement and vaginal examination is not an effective screening test in the general population, although in postmenopausal females it does achieve acceptable specificities and positive predictive values.