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PubMed1996

Model clamp and its application to synchronization of rabbit sinoatrial node cells.

A method for coupling an isolated cardiac cell to a simulated cardiac cell, i.e., the real-time solution of a mathematical model of such cell, has been developed. With this "model clamp" technique, the real cell and the model cell are coupled by any desired value of intercellular coupling conductance, producing the effect of mutual interaction by electrical coupling through gap junctional channels. We implemented the model clamp technique with our previously published model of an isolated rabbit sinoatrial node cell. We used this model clamp system to study synchronization of sinoatrial node cells with regard to the critical value of intercellular coupling conductance required for frequency entrainment and the common interbeat interval during frequency entrainment. This common interbeat interval lay between the intrinsic intervals of the real cell and the model cell, but was closer to that of the intrinsically faster beating cell. Critical coupling conductance increased with increasing difference in intrinsic interbeat interval of the real cell and the model cell and ranged between 50 and 300 pS in 11 hybrid cell pairs.

Action Potentials↗

PubMed2001

Sequential hyperglycemic-euglycemic clamp to assess beta-cell and peripheral tissue: studies in female athletes.

Insulin secretion and rate of utilization (R(d)) of glucose were tested during a newly developed sequential clamp in 42 highly trained female athletes (A; 18-69 yr old) and 14 sedentary control women (C; 18--50 yr old; body mass index <25 kg/m(2)). The A women were categorized into four age groups: 18--29, 30--39, 40--49, and 50--69 yr old. The C women were also grouped by age (18--29 and 40--50 yr old). During the three-step clamp (hyperglycemia, return to euglycemia, and hyperinsulinemia), glucose turnover was assessed with [3-(3)H]glucose. Among the A, the youngest group had the largest first- and second-phase insulin response, which was significantly different from the oldest A (P < 0.05). Among the two C groups, first-phase response of both groups and second-phase response of the older group was higher than respective age-matched A (P < 0.05). During the hyperglycemic period, glucose R(d) was similar among A groups and between A and C. Despite similar levels of insulin between groups during the hyperinsulinemic period (approximately 400 pmol/l), A utilized 36% more glucose than C (P < 0.001). Glucose R(d) was not different across the age groups of A. This newly developed sequential clamp procedure allows assessment of both beta-cell sensitivity to glucose and peripheral tissue sensitivity to insulin in a single session. We have shown that physical activity improves beta-cell efficiency across the age span in women and ameliorates the effect of age on the decline of peripheral tissue sensitivity to insulin.

Adipose Tissue↗

PubMed2005

A comparative voltage and current-clamp analysis of feedback and feedforward synaptic transmission in the striatal microcircuit in vitro.

Striatal spiny projection (SP) neurons control movement initiation by integrating cortical inputs and inhibiting basal ganglia outputs. Central to this control lies a "microcircuit" that consists of a feedback pathway formed by axon collaterals between GABAergic SP neurons and a feedforward pathway from fast spiking (FS) GABAergic interneurons to SP neurons. Here, somatically evoked postsynaptic potentials (PSPs) and currents (PSCs) were compared for both pathways with dual whole cell patch recording in voltage- and current-clamp mode using cortex-striatum-substantia nigra organotypic cultures. On average, feedforward inputs were 1 ms earlier, more reliable, and about twice as large in amplitude compared with most feedback inputs. On the other hand, both pathways exhibited widely varying, partially overlapping amplitude distributions. This variability was already established for single FS neurons targeting many SP neurons. In response to precisely timed action potential bursts, feedforward and feedback inputs consistently showed short-term depression < or =50-70% in voltage-clamp, although feedback inputs also displayed strong augmentation in current-clamp in line with previous reports. The augmentation of feedback inputs was absent in gramicidin D perforated-patch recording, which also showed the natural reversal potential for both inputs to be near firing threshold. Preceding depolarizing feedback inputs during the down state did not consistently change subsequent postsynaptic action potentials. We conclude that feedback and feedforward inputs have their dominant effect during the up-state. The reversal potential close to the up-state potential, which supports shunting operation with millisecond precision and the strong synaptic depression, should enable both pathways to carry time-critical information.

Action Potentials↗

PubMed2004

Angiotensin II excites paraventricular nucleus neurons that innervate the rostral ventrolateral medulla: an in vitro patch-clamp study in brain slices.

Neurons of the hypothalamic paraventricular nucleus (PVN) are key controllers of sympathetic nerve activity and receive input from angiotensin II (ANG II)-containing neurons in the forebrain. This study determined the effect of ANG II on PVN neurons that innervate in the rostral ventrolateral medulla (RVLM)-a brain stem site critical for maintaining sympathetic outflow and arterial pressure. Using an in vitro brain slice preparation, whole cell patch-clamp recordings were made from PVN neurons retrogradely labeled from the ipsilateral RVLM of rats. Of 71 neurons tested, 62 (87%) responded to ANG II. In current-clamp mode, bath-applied ANG II (2 muM) significantly (P < 0.05) depolarized membrane potential from -58.5 +/- 2.5 to -54.5 +/- 2.0 mV and increased the frequency of action potential discharge from 0.7 +/- 0.3 to 2.8 +/- 0.8 Hz (n = 4). Local application of ANG II by low-pressure ejection from a glass pipette (2 pmol, 0.4 nl, 5 s) also elicited rapid and reproducible excitation in 17 of 20 cells. In this group, membrane potential depolarization averaged 21.5 +/- 4.1 mV, and spike activity increased from 0.7 +/- 0.4 to 21.3 +/- 3.3 Hz. In voltage-clamp mode, 41 of 47 neurons responded to pressure-ejected ANG II with a dose-dependent inward current that averaged -54.7 +/- 3.9 pA at a maximally effective dose of 2.0 pmol. Blockade of ANG II AT1 receptors significantly reduced discharge (P < 0.001, n = 5), depolarization (P < 0.05, n = 3), and inward current (P < 0.01, n = 11) responses to locally applied ANG II. In six of six cells tested, membrane input conductance increased (P < 0.001) during local application of ANG II (2 pmol), suggesting influx of cations. The ANG II current reversed polarity at +2.2 +/- 2.2 mV (n = 9) and was blocked (P < 0.01) by bath perfusion with gadolinium (Gd(3+), 100 muM, n = 8), suggesting that ANG II activates membrane channels that are nonselectively permeable to cations. These findings indicate that ANG II excites PVN neurons that innervate the ipsilateral RVLM by a mechanism that depends on activation of AT1 receptors and gating of one or more classes of ion channels that result in a mixed cation current.

Anesthetics, Local↗

PubMed1999

Current clamp and modeling studies of low-threshold calcium spikes in cells of the cat's lateral geniculate nucleus.

Current clamp and modeling studies of low-threshold calcium spikes in cells of the cat's lateral geniculate nucleus. All thalamic relay cells display a voltage-dependent low-threshold Ca2+ spike that plays an important role in relay of information to cortex. We investigated activation properties of this spike in relay cells of the cat's lateral geniculate nucleus using the combined approach of current-clamp intracellular recording from thalamic slices and simulations with a reduced model based on voltage-clamp data. Our experimental data from 42 relay cells showed that the actual Ca2+ spike activates in a nearly all-or-none manner and in this regard is similar to the conventional Na+/K+ action potential except that its voltage dependency is more hyperpolarized and its kinetics are slower. When the cell's membrane potential was hyperpolarized sufficiently to deinactivate much of the low-threshold Ca2+ current (IT) underlying the Ca2+ spike, depolarizing current injections typically produced a purely ohmic response when subthreshold and a full-blown Ca2+ spike of nearly invariant amplitude when suprathreshold. The transition between the ohmic response and activated Ca2+ spikes was abrupt and reflected a difference in depolarizing inputs of <1 mV. However, activation of a full-blown Ca2+ spike was preceded by a slower period of depolarization that was graded with the amplitude of current injection, and the full-blown Ca2+ spike activated when this slower depolarization reached a sufficient membrane potential, a quasithreshold. As a result, the latency of the evoked Ca2+ spike became less with stronger activating inputs because a stronger input produced a stronger depolarization that reached the critical membrane potential earlier. Although Ca2+ spikes were activated in a nearly all-or-none manner from a given holding potential, their actual amplitudes were related to these holding potentials, which, in turn, determined the level of IT deinactivation. Our simulations could reproduce all of the main experimental observations. They further suggest that the voltage-dependent K+ conductance underlying IA, which is known to delay firing in many cells, does not seem to contribute to the variable latency seen in activation of Ca2+ spikes. Instead the simulations indicate that the activation of IT starts initially with a slow and graded depolarization until enough of the underling transient (or T) Ca2+ channels are recruited to produce a fast, "autocatalytic" depolarization seen as the Ca2+ spike. This can produce variable latency dependent on the strength of the initial activation of T channels. The nearly all-or-none nature of Ca2+ spike activation suggests that when a burst of action potentials normally is evoked as a result of a Ca2+ spike and transmitted to cortex, this signal is largely invariant with the amplitude of the input activating the relay cell.

Action Potentials↗

PubMed2006

Clamp techniques in paediatrics: what have we learned?

The marked increase in conditions associated with insulin resistance in youth, including obesity, polycystic ovary syndrome, type 2 diabetes mellitus etc., has prompted the need to assess insulin sensitivity in this age group. Even though insulin resistance plays an important role in disorders of glucose metabolism and other pathological conditions, both insulin sensitivity and insulin secretion should be determined for a comprehensive evaluation of glucose homeostasis disorders. Insulin sensitivity and secretion are intricately coupled with a delicate feedback mechanism governing their relationship. This article will delineate our paediatric experience with the clamp technique, the hyperinsulinaemic-euglycaemic clamp in assessing in vivo insulin sensitivity, and the hyperglycaemic clamp in assessing insulin secretion.

Adolescent↗

PubMedDate not supplied

Glycemia influences on glucose metabolism in sepsis during hyperinsulinemic clamp.

BACKGROUND: We investigated glucose metabolism in septic patients during hyperglycemic clamps and compared the different levels of insulinemia and glycemia. METHODS: In 10 non-diabetic stable septic patients on mechanical ventilation with baseline glycemia >6 mmol/L and continuous insulin infusion, 3 steps of hyperinsulinemic clamp were performed after 8 hours without caloric intake. In step 1, the targets were insulinemia of 250 mIU/L and glycemia of 5 mmol/L; in step 2, insulinemia of 250 mIU/L and glycemia of 10 mmol/L; in step 3, insulinemia of 1250 mIU/L and glycemia of 5 mmol/L. Glucose uptake was calculated as the amount of glucose per time needed to maintain the target level of glycemia. Glucose oxidation was calculated from indirect calorimetry and urinary nitrogen losses. Values are provided as means +/- SD. A two-way analysis of variance and Scheffe's method were used for statistical analysis and p < .05 was considered significant. RESULTS: At step 1, glucose uptake was lower than at step 2 (3.8 +/- 2.48 mg/kg/min and 7.9 +/- 3.45 mg/kg/min, respectively; p < .001). Glucose oxidation was also lower at step 1 (2.6 +/- 0.98 and 4.2 +/- 1.85 mg/kg/min, respectively; p < .01). Glucose storage was low at step 1 (0.7 +/- 1.39) and increased at step 2 (3.5 +/- 2.18; p < .05). In step 3, glucose uptake was 7.0 +/- 2.1, oxidation was 3.6 +/- 1.37, and storage was 2.9 +/- 2.79. There was no significant difference in all these parameters between steps 2 and 3. Energy expenditure between steps 1, 2 and 3 did not change (2294 + 307.42, 2334 + 341.53, and 2342 + 426.67 kcal/day, respectively). Alanine in plasma dropped significantly (p < .05): 10 mmol/L (311 +/- 55.88 mmol/L) at glycemia compared with 5 mmol/L (390 +/- 76 micromol/L) at insulinemia 250 mIU/L. It did not differ significantly from the values obtained at glycemia 5 mmol/L and insulinemia 1250 mIU/L (348 +/- 70.68 mmol/L). Even if the level of cytokines in sepsis was higher, there was no correlation between the insulin level in plasma (250 and 1250 mIU/L), glycemia (5 and 10 mmol/L) and cytokine level (IL-1beta, IL-2, IL-6, IL-8 and TNFalpha). CONCLUSION: At insulinemia 250 mIU/L, a glucose level of 10 mmol/L seems to increase glucose uptake, oxidation, and storage compared with glycemia 5 mmol/L. This glucose uptake and oxidation at glycemia 10 mmol/L is comparable with the effect of extremely high insulinemia (1250 mIU/L) clamped at glycemia 5 mmol/L. A higher level of blood glucose or a high level of insulinemia significantly increases glucose uptake but not energy expenditure.

Analysis of Variance↗

PubMed2005

A mathematical model of the euglycemic hyperinsulinemic clamp.

BACKGROUND: The Euglycemic Hyperinsulinemic Clamp (EHC) is the most widely used experimental procedure for the determination of insulin sensitivity, and in its usual form the patient is followed under insulinization for two hours. In the present study, sixteen subjects with BMI between 18.5 and 63.6 kg/m(2) were studied by long-duration (five hours) EHC. RESULTS: From the results of this series and from similar reports in the literature it is clear that, in obese subjects, glucose uptake rates continue to increase if the clamp procedure is prolonged beyond the customary 2 hours. A mathematical model of the EHC, incorporating delays, was fitted to the recorded data, and the insulin resistance behaviour of obese subjects was assessed analytically. Obese subjects had significantly less effective suppression of hepatic glucose output and higher pancreatic insulin secretion than lean subjects. Tissue insulin resistance appeared to be higher in the obese group, but this difference did not reach statistical significance. CONCLUSION: The use of a mathematical model allows a greater amount of information to be recovered from clamp data, making it easier to understand the components of insulin resistance in obese vs. normal subjects.

Blood Glucose↗

PubMed1990

Glyburide increases insulin sensitivity and responsiveness in peripheral tissues of the rat as determined by the glucose clamp technique.

The effect of chronic glyburide treatment on insulin sensitivity and responsiveness in vivo in unanesthetized male Sprague-Dawley rats was determined by the hyperinsulinemic-euglycemic clamp technique. Normal animals were surgically prepared for the clamp procedure and then gavaged with glyburide, 2 mg/kg/day, or with normal saline for 6-18 days. Basal plasma glucose concentrations were significantly lower in glyburide-treated animals compared to controls, but basal plasma insulin concentrations were the same. Rates of glucose disposal, calculated before and during insulin infusions of 2 to 40 mU/kg.min with plasma glucose concentration clamped at 125 mg/dl, were significantly greater in the glyburide-treated rats compared to controls. Insulin dose-response curves demonstrate that glyburide treatment increased both insulin sensitivity and responsiveness. Basal hepatic glucose production, estimated by D-[3-3H]Glucose infusion, was significantly greater with glyburide treatment; however the sensitivity of the liver to suppression by insulin infusions of 2 and 4 mU/kg.min was unchanged. These data suggest that the decreased basal plasma glucose concentration observed in rats chronically treated with glyburide is the result of increased glucose disposal in peripheral tissues and not associated with an increase in plasma insulin concentrations or a decrease in hepatic glucose production.

Animals↗

PubMed2004

Comparison of the insulin action parameters from hyperinsulinemic clamps with homeostasis model assessment and QUICKI indexes in subjects with different endocrine disorders.

The aim of this study was to compare insulin sensitivity expressed by the hyperinsulinemic clamp and by the homeostasis model assessment (HOMA) and QUICKI indexes in subjects with various disorders influencing insulin action. We examined 41 type 2 diabetic patients, 20 insulinoma patients, 32 women with polycystic ovary syndrome, 16 patients with primary hyperaldosteronism, 12 patients with essential high renin hypertension, and 47 healthy subjects. The metabolic clearance rate of glucose and the insulin sensitivity index calculated from the clamps were compared with both the HOMA and QUICKI indexes. The relationship of insulin action to body mass index, serum cholesterol, and triglycerides as well as to systolic and diastolic blood pressures was also evaluated. Body mass index was very strongly associated with the insulin sensitivity index (r = -0.70; P < 0.0001) in the entire cohort of 168 subjects. Cholesterol, triglycerides, and blood pressure influenced insulin action in the healthy subjects and type 2 diabetic patients. A significant relationship was observed between the insulin sensitivity index and the HOMA or QUICKI indexes in healthy subjects (r = -0.66; P < 0.0001), type 2 diabetic patients (r = -0.68; P < 0.0001), and women with polycystic ovary syndrome (r = -0.65; P < 0.0001). We did not find any relationship between the above variables in the patients with insulinoma or primary hyperaldosteronism. The HOMA and QUICKI indexes do not offer the same information as glucose clamps in the rare cases with differently impaired peripheral or hepatic insulin action.

Adolescent↗

PubMed1990

Brain glucose metabolism in noninsulin-dependent diabetes mellitus: a study in Pima Indians using positron emission tomography during hyperinsulinemia with euglycemic glucose clamp.

To determine whether insulin or noninsulin-dependent diabetes mellitus affects brain glucose metabolism, brain glucose utilization was studied in the basal state and during hyperinsulinemic euglycemic glucose clamps in nondiabetic and diabetic Pima Indians by positron emission tomography with 2-[18F]fluoro-2-deoxy-D-glucose (18FDG). Glucose utilization in 75 brain areas was determined by analysis of single scans and by least squares estimation of the rate parameters for the FDG model; these data were compared to results in normal caucasian volunteers. No effect of ethnicity or diabetic status on brain glucose utilization was observed. During the hyperinsulinemic clamps (mean insulin, 11,708 +/- 3,026 pmol/L), clearance of 18FDG from blood was accelerated, and accumulation of brain radioactivity was reduced. However, glucose utilization by the brain was identical to results during sham glucose clamps (mean insulin, 204 +/- 56 pmol/L) performed in the same patients. During the studies with hyperinsulinemia, k4 (representing loss of tissue radioactivity) was increased in most brain areas (mean increase, 0.0031 +/- 0.0018 min-1; P less than 0.02). The possible mechanisms for this effect are multiple, and the physiological significance, if any, is unknown. Further studies of the effects of insulin on brain glucose metabolism are needed.

Adult↗

PubMed1996

Electrophysiological and pharmacological characteristics of ionotropic glutamate receptors in medial vestibular nucleus neurons: a whole cell patch clamp study in acutely dissociated neurons.

A patch clamp study was performed to determine which subtype of ionotropic glutamate receptors is involved in the glutamate-induced excitation of the medial vestibular nucleus (MVN) neurons. Whole cell recording was performed on MVN neurons that were acutely dissociated by enzymatic and mechanical treatments. Application of glutamate at a concentration of 100 microM produced a current with a reversal potential of approximately 0 mV. The glutamate-induced current was completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM), a non-N-methyl-D-aspartate (NMDA)-receptor antagonist. Application of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) and kainic acid (KA), non-NMDA-receptor agonists, at concentrations of 30 and 100 microM produced a concentration-dependent depolarization concomitantly with an increase in firing rates during current clamp recording. During voltage clamp recording, glutamate, AMPA and KA elicited a concentration-dependent current with an equilibrium potential of approximately 0 mV. To clarify whether NMDA receptors are present in MVN neurons, the effects of glycine on the glutamate- and NMDA-induced current were examined. Two types of NMDA receptor-mediated current (types 1 and 2) were obtained in terms of the difference in sensitivity to both magnesium ion and MK-801, which act on the NMDA-receptor channel. In the type 1 neurons, the NMDA-induced current was not apparently blocked by magnesium ion or MK-801, although a larger current was obtained in the absence of magnesium ion. In the type 2 neurons, marked blockade of the NMDA-induced current was seen in the presence of magnesium ion and MK-801, as previously reported in other neurons of the central nervous system. These findings indicate the presence of both non-NMDA and NMDA receptors, which are involved in primary afferent transmission, in the MVN neuron, and two distinct types of NMDA receptors.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2003

Adapting the Quesant Nomad atomic force microscope for biology and patch-clamp atomic force microscopy.

The Quesant Nomad atomic force microscope (AFM) was modified to produce a reliable patch-clamp AFM for demanding biologic applications. The AFM's laser optics forms the basis of a condenser that allows simultaneous Köhler illumination and AFM imaging on an inverted optical microscope. The original AFM scan head was replaced with plastic and glass to make it biologically inert. A bevel cut in the new scan head permits clearance for patch clamp pipets. Cantilevers are attached to the scan head with a quick setting silicone rubber that is readily removable. Software was developed to (a) automate a gentle approach and set a specific feedback force, (b) provide a mouse-driven control of the X-Y position of the probe tip and recall of saved locations, and (c) measure force-distance curves over user defined paths. Additional modifications were made to minimize mechanical noise. The patch-clamp AFM achieves 600 fA (3 kHz bandwidth) and 1 A RMS noise levels (10 kHz bandwidth). The correlation of electrical and mechanical information allows signal averaging and measures sub-Angstrom, sub-millisecond electromotile responses from cells.

Cell Membrane↗

PubMed2005

Evaluation of insulin sensitivity in patients with Klinefelter's syndrome: a hyperinsulinemic euglycemic clamp study.

Patients with Klinefelter's syndrome have a higher incidence of diabetes mellitus and the percentage of insulin resistance was reported to be high in these patients. However, little is known about the insulin sensitivity assessed by the hyperinsulinemic euglycemic clamp in these patients. In the present study, subjects included 13 newly diagnosed patients with Klinefelter's syndrome, and 9 age- and body mass index-matched healthy males. The hyperinsulinemic euglycemic clamp was performed in all patients and controls. Insulin resistance was present in five (38.5%) patients with Klinefelter's syndrome. Compared with control subjects, patients with Klinefelter's syndrome had elevated plasma concentrations of fasting insulin, follicle-stimulating hormone, luteinizing hormone, estradiol, and sex hormone-binding globulin, whereas they had reduced plasma free testosterone and total testosterone concentrations. The multivariate linear regression analysis showed that fasting glucose, fasting insulin, free testosterone, and total testosterone were independently associated with M-value. In conclusion, the present study by using hyperinsulinemic euglycemic clamp indicates the high prevalence of insulin resistance in Klinefelter's syndrome patients. However, these patients did not have reduced mean M-values compared with the controls, although their plasma insulin levels were significantly elevated. It is possible that hyperinsulinemia may be the primary metabolic abnormality rather than insulin resistance.

Adult↗

PubMed2001

Prostaglandylinositol cyclic phosphate synthase activity in the liver of insulin-resistant rhesus monkeys before and after a euglycemic hyperinsulinemic clamp.

Prostaglandylinositol cyclic phosphate (cPIP), functionally a cAMP antagonist, is a novel, low-molecular weight mediator of insulin action. Both essential hypertension and type 2 diabetes may be associated with a reduction of cPIP synthesis. In intact cells and in plasma membranes, cPIP synthesis is stimulated by insulin, which activates cPIP synthase by tyrosine phosphorylation. We measured the activities of cPIP synthase in the homogenates of freeze-clamped and then lyophilized liver samples from five insulin-resistant, adult rhesus monkeys, obtained under basal fasting conditions and again under maximal insulin stimulation during a euglycemic hyperinsulinemic clamp. The mean cPIP synthase activity in basal samples (0.33 +/- 0.09 pmol/min/mg protein) was not significantly different at the end of the clamp (0.24 +/- 0.11 pmol/min/mg protein). Basal cPIP synthase activityVoL 12, No. 1, 2001 was directly related to both basal cAMP content and basal fractional activity of cAMP-dependent protein kinase (PKA): r=0.85, p<0.05 and r=0.86, p<0.05, respectively. In turn, insulin-stimulated cPIP synthase activity was inversely related to both the insulin-stimulated fractional activity of PKA (r=0.89, p<0.02) and the insulin-stimulated total PKA activity: r=0.94, p<0.005. The findings suggest that in the liver of insulin-resistant rhesus monkeys, cPIP synthase activity, which leads to the synthesis of the low-molecular weight mediator cPIP, may oppose cAMP synthesis and PKA activity.

Animals↗

PubMed2006

Direct GABAergic and glycinergic inhibition of the substantia gelatinosa from the rostral ventromedial medulla revealed by in vivo patch-clamp analysis in rats.

Stimulation of the rostral ventromedial medulla (RVM) is believed to exert analgesic effects through the activation of the serotonergic system descending to the spinal dorsal horn; however, how nociceptive transmission is modulated by the descending system has not been fully clarified. To investigate the inhibitory mechanisms affected by the RVM, an in vivo patch-clamp technique was used to record IPSCs from the substantia gelatinosa (SG) of the spinal cord evoked by chemical (glutamate injection) and electrical stimulation (ES) of the RVM in adult rats. In the voltage-clamp mode, the RVM glutamate injection and RVM-ES produced an increase in both the frequency and amplitude of IPSCs in SG neurons that was not blocked by glutamate receptor antagonists. Serotonin receptor antagonists were unexpectedly without effect, but a GABAA receptor antagonist, bicuculline, or a glycine receptor antagonist, strychnine, completely suppressed the RVM stimulation-induced increase in IPSCs. The RVM-ES-evoked IPSCs showed fixed latency and no failure at 20 Hz stimuli with a conduction velocity of >3 m/s (3.1-20.7 m/s), suggesting descending monosynaptic GABAergic and/or glycinergic inputs from the RVM to the SG through myelinated fibers. In the current-clamp mode, action potentials elicited by noxious mechanical stimuli applied to the receptive field of the ipsilateral hindlimb were suppressed by the RVM-ES in more than half of the neurons tested (63%; 10 of 16). These findings suggest that the RVM-mediated antinociceptive effects on noxious inputs to the SG may be exerted preferentially by the direct GABAergic and glycinergic pathways to the SG.

Animals↗

PubMedDate not supplied

Patch-clamp recording of human retinal photoreceptors and bipolar cells.

Photoreceptors and retinal bipolar cells are considered as nonspiking neurons; however, we recently showed that human rod photoreceptors can generate sodium action potentials in response to membrane depolarization from membrane potentials of -60 or -70 mV (Kawai et al., Neuron 30 [2001] 451). We performed patch-clamp recording of human cone photoreceptors and retinal bipolar cells to examine whether functional voltage-gated sodium channels are expressed in these cells as well as rod photoreceptors. Under current-clamp conditions, the injection of depolarizing current steps into a cone photoreceptor-induced marked action potentials. These action potentials were blocked by 1 microM tetrodotoxin, a voltage-gated sodium channel blocker. Under voltage-clamp conditions, depolarizing voltage steps-induced a fast transient inward current in several bipolar cells (n = 4/78). This current was activated from -70 to + 20 mV (maximal at -10 mV) and inactivated within 5 ms. The 10-90% rise time of this current was shorter than another inward current (less than one-hundredth). These results indicate that human cones and bipolar cells express voltage-gated sodium channels as rod photoreceptors. Sodium channels may serve to amplify the release of a neurotransmitter and to accelerate the light-dark change in photosignals.

Adult↗

PubMed2000

IGF-I treatment in adults with type 1 diabetes: effects on glucose and protein metabolism in the fasting state and during a hyperinsulinemic-euglycemic amino acid clamp.

Type 1 diabetes is associated with abnormalities of the growth hormone (GH)-IGF-I axis. Such abnormalities include decreased circulating levels of IGF-I. We studied the effects of IGF-I therapy (40 microg x kg(-1) x day(-1)) on protein and glucose metabolism in adults with type 1 diabetes in a randomized placebo-controlled trial. A total of 12 subjects participated, and each subject was studied at baseline and after 7 days of treatment, both in the fasting state and during a hyperinsulinemic-euglycemic amino acid clamp. Protein and glucose metabolism were assessed using infusions of [1-13C]leucine and [6-6-2H2]glucose. IGF-I administration resulted in a 51% rise in circulating IGF-I levels (P < 0.005) and a 56% decrease in the mean overnight GH concentration (P < 0.05). After IGF-I treatment, a decrease in the overnight insulin requirement (0.26+/-0.07 vs. 0.17+/-0.06 U/kg, P < 0.05) and an increase in the glucose infusion requirement were observed during the hyperinsulinemic clamp (approximately 67%, P < 0.05). Basal glucose kinetics were unchanged, but an increase in insulin-stimulated peripheral glucose disposal was observed after IGF-I therapy (37+/-6 vs. 52+/-10 micromol x kg(-1) x min(-1), P < 0.05). IGF-I administration increased the basal metabolic clearance rate for leucine (approximately 28%, P < 0.05) and resulted in a net increase in leucine balance, both in the basal state and during the hyperinsulinemic amino acid clamp (-0.17+/-0.03 vs. -0.10+/-0.02, P < 0.01, and 0.25+/-0.08 vs. 0.40+/-0.06, P < 0.05, respectively). No changes in these variables were recorded in the subjects after administration of placebo. These findings demonstrated that IGF-I replacement resulted in significant alterations in glucose and protein metabolism in the basal and insulin-stimulated states. These effects were associated with increased insulin sensitivity, and they underline the major role of IGF-I in protein and glucose metabolism in type 1 diabetes.

Adult↗