Efficacy and safety of initial combination treatment with sitagliptin and pioglitazone--a factorial study.

AIM: To evaluate the efficacy and safety of initial combination therapy of sitagliptin 100 mg/day coadministered with all marketed doses of pioglitazone in patients with type 2 diabetes. METHODS: Patients with A1c ≥7.5 and ≤11.0% were randomized among seven arms that received, once daily, 100 mg sitagliptin alone; 15, 30 or 45 mg pioglitazone alone, or 100 mg sitagliptin plus 15, 30 or 45 mg pioglitazone for 54 weeks. The primary endpoint was change from baseline in A1c at week 24. Protocol-specified analyses compared combination therapies with monotherapies at respective dose-strengths and combination of sitagliptin plus pioglitazone 30 mg with pioglitazone 45 mg monotherapy. Post-hoc analyses compared sitagliptin plus pioglitazone 15 mg with pioglitazone monotherapy at the two higher doses. RESULTS: Initial combination therapy with sitagliptin and pioglitazone provided significantly greater reductions in A1c (0.4-0.7% differences) and other glycaemic endpoints than either monotherapy at the same doses. Combining sitagliptin with low-dose pioglitazone generally produced greater glycaemic improvements than higher doses of pioglitazone monotherapy (0.3-0.4% differences in A1c). Combination therapy was generally well tolerated; adverse events (AEs) of hypoglycaemia were reported with similar incidence (7.8-11.1%) in all treatment groups over the 54 weeks of study; oedema was reported in 0.5% of patients in the sitagliptin monotherapy group and 2.7-5.3% among pioglitazone-treated groups. Significant weight gain was observed in all combination-treated groups compared with the sitagliptin monotherapy group. CONCLUSIONS: Initial combination therapy with sitagliptin and pioglitazone provided better glycaemic control than either monotherapy and was generally well tolerated.

Bone loss in the oestrogen-depleted rat is not exacerbated by sitagliptin, either alone or in combination with a thiazolidinedione.

Antihyperglycaemic therapy on bone was evaluated in the ovariectomized (OVX), non-diabetic adult rat. Animals were treated daily for 12 weeks with various doses of sitagliptin, pioglitazone, rosiglitazone, combinations of sitagliptin with pioglitazone or vehicle alone. Sitagliptin target engagement was confirmed by assessing inhibition of plasma dipeptidyl peptidase-4 (DPP-4) and oral glucose tolerance. Parameters related to bone health were evaluated in femur and vertebrae by dual-energy X-ray absorptiometry and histomorphometry. Bone mineral density (BMD) generally did not differ significantly between OVX-sitagliptin-treated animals and OVX-vehicle controls. In lumbar vertebrae, however, there was significantly less BMD loss with increasing sitagliptin dose. Thiazolidinedione (TZD) treatment generally resulted in lower BMD; OVX-TZD-treated (but not OVX-sitagliptin-treated) animals also had lessened cortical thickness in central femur and profoundly greater bone marrow adiposity in lumbar vertebrae. These findings support prior findings with TZDs and suggest a neutral or beneficial impact of DPP-4 inhibition on bone health.

Chronic treatment with a glucokinase activator delays the onset of hyperglycaemia and preserves beta cell mass in the Zucker diabetic fatty rat.

AIMS/HYPOTHESIS: Glucokinase activators (GKAs) are currently being developed as new therapies for type 2 diabetes and have been shown to enhance beta cell survival and proliferation in vitro. Here, we report the effects of chronic GKA treatment on the development of hyperglycaemia and beta cell loss in the male Zucker diabetic fatty (ZDF) rat, a model of type 2 diabetes with severe obesity. METHODS: Cell protection by GKA was studied in MIN6 and INS-1 cells exposed to hydrogen peroxide. Glucose homeostasis and beta cell mass were evaluated in ZDF rats dosed for 41 days with Cpd-C (a GKA) or glipizide (a sulfonylurea) as food admixtures at doses of approximately 3 and 10 mg kg(-1) day(-1). RESULTS: Incubation of MIN6 and INS-1 832/3 insulinoma cell cultures with GKA significantly reduced cell death and impairment of intracellular NADH production caused by exposure to hydrogen peroxide. Progression from prediabetes (normoglycaemia and hyperinsulinaemia) to overt diabetes (hyperglycaemia and hypoinsulinaemia) was significantly delayed in male ZDF rats by in-feed treatment with Cpd-C, but not glipizide. Glucose tolerance, tested in the fifth week of treatment, was also significantly improved by Cpd-C, as was pancreatic insulin content and beta cell area. In a limited immunohistochemical analysis, Cpd-C modestly and significantly enhanced the rate of beta cell proliferation, but not rates of beta cell apoptosis relative to untreated ZDF rats. CONCLUSIONS/INTERPRETATION: These findings suggest that chronic activation of glucokinase preserves beta cell mass and delays disease in the ZDF rat, a model of insulin resistance and progressive beta cell failure.

Synaptically driven spikes and long-term potentiation in neocortical layer 2/3.

Recently, variation upon a well-established hippocampal model has given rise to a new paradigm in which the strength of synaptic inputs to neocortical layer 2/3 is estimated in vitro by recording synaptically driven extracellular potentials elicited there by electrical stimulation applied to underlying layer 4. The analysis of these potentials is commonly based upon an assumption that postsynaptic spiking has played no significant role in their generation. Here, we have tested this assumption by quantifying in rats (using data obtained by cell-attached recording) the rate at which unit spikes are elicited in layer 2/3 under commonly used conditions of stimulation and recording. We found that spike responses were regularly elicited at the same latencies as field potential peaks and the rising phases of intracellularly recorded synaptic currents, and the incidence of such spiking (the fractional rate of cells spiking versus cells sampled) was sufficient to give this higher-order activity a major role in determining response amplitudes. We then analyzed layer 2/3 waveform characteristics before and after inducing long-term potentiation (LTP) by theta-burst stimulation (TBS) and found that the induction of LTP succeeded only when the initial response included a strong spike component. We further observed that LTP expression was always accompanied by a pronounced enhancement of such components. Our data suggest that, unlike in hippocampal CA1, LTP elicited by TBS in this neocortical paradigm depends upon modification of synaptically driven spike activity, through either enhanced synchronization of unitary responses, the recruitment of additional responding units, or both. This potentiation of the spike response could arise (as previously proposed) through an increase in the efficacy of synapses mediating projection from layer 4 to 2/3, but other mechanisms may also contribute, such as modification of short-range recurrent connections within layer 2/3, which are likely to play an important role in defining local-network cell ensembles.

Deficient neurogenesis in forebrain-specific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces.

To examine the in vivo function of presenilin-1 (PS1), we selectively deleted the PS1 gene in excitatory neurons of the adult mouse forebrain. These conditional knockout mice were viable and grew normally, but they exhibited a pronounced deficiency in enrichment-induced neurogenesis in the dentate gyrus. This reduction in neurogenesis did not result in appreciable learning deficits, indicating that addition of new neurons is not required for memory formation. However, our postlearning enrichment experiments lead us to postulate that adult dentate neurogenesis may play a role in the periodic clearance of outdated hippocampal memory traces after cortical memory consolidation, thereby ensuring that the hippocampus is continuously available to process new memories. A chronic, abnormal clearance process in the hippocampus may conceivably lead to memory disorders in the mammalian brain.

Short-term plasticity of extrinsic excitatory inputs to neocortical layer 1.

Previous studies have shown that different pyramidal cell inputs vary in the short-term plasticity expressed when they are subjected to repetition of use. Here, we describe short-term plasticity at synapses that mediate long-range input to neocortical layer 1 and compare it with that which normally occurs in the hippocampal Schaffer collateral pathway, which also involves projection by remote inputs onto apical dendrites. We isolated tangential inputs to layer 1 in neocortical slices, stimulated these with brief 40-Hz trains, and examined postsynaptic responses by recording extracellularly from layer 1 in somatosensory, prefrontal, and visual neocortex, and intracellularly from visually identified pyramidal cell somata in layer 2/3 in somatosensory and prefrontal neocortex. Train response amplitudes were characterized by calculating paired-pulse ratios, fifth-versus-first amplitude ratios (5th/1st ratios), and a center-of-mass index "M". As expected, the hippocampal train responses facilitated strongly. In contrast, layer-1 responses displayed strong synaptic depression in all regions examined. This depression was reflected in 5th/lst ratios and M scores, but not paired-pulse ratios because it did not consistently begin until the third responses in trains. It persisted unchanged in the presence of partially blocking levels of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but was converted to strong facilitation when slices were bathed in low-Ca++ media. Intracellularly, we observed response-train depression very similar to that recorded extracellularly. These findings show that long-range inputs to neocortical layer 1 display short-term plasticity markedly different from that which normally occurs at hippocampal Schaffer collateral synapses, but similar to that which has been described previously for excitatory inputs to pyramidal cells in deeper neocortical layers.

Allosteric modulation of AMPA-type glutamate receptors increases activity of the promoter for the neural cell adhesion molecule, N-CAM.

To study regulation in vivo of the promoter for the neural cell adhesion molecule, N-CAM, we have used homologous recombination to insert the bacterial lacZ gene between the transcription and translation initiation sites of the N-CAM gene. This insertion disrupts the gene and places the expression of beta-galactosidase under the control of the N-CAM promoter. Animals homozygous for the disrupted allele did not express N-CAM mRNA or protein, but the pattern of beta-galactosidase expression in heterozygous and homozygous embryos was similar to that of N-CAM mRNA in wild-type animals. The homozygotes exhibited many of the morphological abnormalities observed in previously reported N-CAM knockout mice, with the exception that hippocampal long-term potentiation in the Schaffer collaterals was identical in homozygous, heterozygous, and wild-type animals. Heterozygous mice were used to examine the regulation of the N-CAM promoter in response to enhanced synaptic transmission. Treatment of the mice with an ampakine, an allosteric modulator of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that enhances normal glutamate-mediated synaptic transmission, increased the expression of beta-galactosidase in vivo as well as in tissue slices in vitro. Similar treatments also increased the expression of N-CAM mRNA in the heterozygotes. The effects of ampakine in slices were strongly reduced in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA receptor antagonist. Taken together, these results indicate that facilitation of AMPA receptor-mediated transmission leads to activation of the N-CAM promoter and provide support for the hypothesis that N-CAM synthesis is regulated in part by synaptic activity.

Posttetanic potentiation and presynaptically induced long-term potentiation at the mossy fiber synapse in rat hippocampus.

A form of long-term potentiation (LTP) is induced at the mossy fiber (MF) synapse in the hippocampus by high-frequency presynaptic stimulation (HFS). It is generally accepted that induction of this form of LTP (MF LTP) does not depend on postsynaptic Ca2+ current gated by N-methyl-D-aspartate receptors, but it has remained controversial whether induction depends on postsynaptic depolarization and voltage-gated entry of Ca2+. There are also contradictory data on the time course of both LTP and post-tetanic potentiation (PTP), a shorter duration form of potentiation observed at MF synapses immediately following HFS. It has been proposed that some of these differences in results may have arisen because of difficulties in isolating monosynaptic responses to MF input. In the present study, whole cell recording was used to observe excitatory postsynaptic currents (EPSCs) elicited in CA3 pyramidal cells by input from MFs. Postsynaptic cells were dialyzed with 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) and F- to inhibit postsynaptic mechanisms that required Ca2+, cells were under voltage clamp during HFS, and conditions were selected to minimize the likelihood of polysynaptic contamination. Under these conditions, HFS nevertheless induced robust LTP (mean magnitude, 62%). The possibility that EPSCs were contaminated by polysynaptic components was investigated by exposing the slices to a suppressing medium (one that partially blocked neurotransmission). EPSC waveforms did not change shape during suppression, indicating that contamination was absent. The LTP observed always was accompanied by prominent PTP that lasted through the first 5 to 15 min following HFS (mean decay time constant, 3.2 min). Induction of this LTP was not cooperative; there was no relationship between the size of responses and the magnitude of the LTP induced. LTP magnitude also was unrelated to the extent to which postsynaptic cells depolarized during HFS. These results show that high rates of presynaptic MF activity elicit robust LTP whether or not there is accompanying postsynaptic depolarization or increase in the concentration of postsynaptic Ca2+. High-frequency MF activity also results in a PTP that is unusually large and long.

Asynchrony of mossy fibre inputs and excitatory postsynaptic currents in rat hippocampus.

1. Excitatory postsynaptic currents (EPSCs) were studied by whole-cell voltage-clamp recording (WCR) from pyramidal cells in the CA3 field of rat hippocampal slices. Input from mossy fibres was evoked by stimuli applied to stratum granulosum ('dentate gyrus stimulation'). This often resulted in complex, multi-component EPSCs with rise times as long as 5.0 ms (mean = 2.5 ms). In contrast, individual EPSC components typically had rise times between 0.3 and 1.0 ms. 2. To isolate monosynaptic, mossy fibre-driven EPSC components, slices were exposed to 'suppressing' media that reduced response amplitudes by 64-88%. In five out of six cases, long EPSC rising phases (> 3 ms) retained the same shape during suppression. This implied that EPSCs were driven by asynchronously active mossy fibre inputs. 3. From latencies of antidromically driven granule cell population spikes (GCPSs) a mean conduction velocity of 0.67 m/s was inferred. Conduction distance had practically no correlation with GCPS duration, implying that velocity dispersion was small and did not desynchronize mossy fibre impulses. EPSC components exhibited 'surplus' latency; they occurred 0.9-4.8 ms after latencies expected on the basis of direct conduction distances. 4. Mossy fibre volleys (MFVs) were evoked by dentate gyrus stimulation and studied with neurotransmission disabled. MFV negative phases lasted from 2.5 to 4.5 ms and had multiple components. By comparison, negative phases of Schaffer collateral fibre volleys (SCFVs) were always simple in shape and lasted 1.5 ms or less. MFV components had surplus latencies similar to those of EPSC components. Late MFV components did not require high stimulus intensities. 5. Widespread activation of granule cells occurred when stimuli were applied to single loci in the stratum granulosum. This implies that such stimuli elicit antidromic impulses in hilar collaterals of mossy fibres, which could result in activation of orthodromic impulses in mossy fibre trunks that had not been stimulated directly. After anti-, then orthodromic conduction, impulses would arrive in the CA3 subfield with 'surplus' latency. 6. When cuts were made in the hilus to prevent anti-/orthodromic conduction, MFV durations were reduced, but only to a small extent. This implies that surplus latency and asynchrony arise in part by anti-/orthodromic conduction, and partly by a mechanism that is intrinsic to mossy fibres or their 'giant' boutons. 7. Because of desynchronization of mossy fibre inputs, there probably are significant differences between kinetic properties of averaged, compound mossy fibre EPSCs and those of unitary mossy fibre EPSCs (i.e. currents driven by input from single presynaptic axons).(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of selective glutamate receptor antagonists on synchronized firing bursts in layer III of rat visual cortex.

In the rat visual cortex in vitro, single-shock stimulations applied to the border between layer VI and the white matter evoke synchronized burst-firing by units in layer III. We have examined the effects of glutamate receptor antagonists on this activity, with antagonists applied via the bath to allow correlation of effects with concentrations. All synaptically driven components (recorded extracellularly as field potential 'S2' spikes, dipoles 'W1' and 'W2', and coinciding single-unit spikes) were inhibited by greater than 90% in 1.0 mM kynurenic acid and in 3 or 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, which selectively blocks AMPA/kainate receptors). S2 spike amplitudes were reduced by half in 0.7 microM CNQX. 2-Amino-5-phosphonovalerate (APV), a specific blocker of NMDA receptors, did not prevent S2 spike burst or horizontal spread of bursting within layer III. However, APV reduced the duration of synchronized bursts and the slower potentials which followed. In Mg(2+)-free medium, new components appeared which were APV-sensitive: (1) low amplitude spikes, distributed spatially like S2 spike, but recurring more slowly, and (2) slow potentials, distributed spatially like W1 and W2 potentials, but lasting for hundreds of milliseconds. The amplitudes of these spikes were reduced by half in 3 microM D-APV. Our data imply that: (1) glutamate receptors play a major role in mediating local, excitatory neurotransmission in the supragranular layers of neocortex, with NMDA and AMPA/kainate subtypes each subserving somewhat different functions; (2) AMPA/kainate receptors mediate rapid excitatory transmission between layer III neurons, responsible for driving the first 15 ms of synchronized bursts; (3) currents gated by NMDA receptors determine the duration of coherent firing bursts, and drive asynchronous neuronal firing following bursts; and (4) under conditions which circumvent block by extracellular Mg2+, activation of NMDA receptors greatly enhances and prolongs the response to single-shock stimulations. In vivo, activation of layer III neurons is likely to depend significantly upon currents gated by NMDA receptors whenever repetitively firing excitatory inputs summed over several tens of milliseconds provide enough depolarization to lift block by extracellular Mg2+.

Transient retinal axon collaterals to visual and somatosensory thalamus in neonatal hamsters.

We have studied the postnatal development of individual axons in the optic tract and thalamus of the Syrian hamster, concentrating attention on retinal ganglion cell axons that make a transient projection to the main somatosensory nucleus, the ventrobasal complex. We bulk-filled axons with horseradish peroxidase in hemithalami maintained en bloc, in vitro. After processing and reaction with diaminobenzidine, we reconstructed individual axons from serial sections. In hamsters and other rodents, the optic tract is composed of superficial and internal components, either or both being possible sources of the retino-ventrobasal projection. Both project to the midbrain, but in normal adults only the superficial optic tract maintains collaterals in the thalamus. We found that the axons of the internal component bear numerous transient thalamic collaterals on postnatal days 0, 1, and 2, and some of these extend into the ventrobasal complex. Axons in the superficial optic tract also bear collaterals on days 0 to 2, but these are confined to the superficial half of the dorsal lateral geniculate nucleus. Thus the transient retino-ventrobasal projection comprises solely transient collaterals originating from axon trunks in the internal optic tract. On days 1 and 2, some collaterals from the superficial optic tract appear to have begun to arborize in the lateral geniculate nucleus. In contrast, collaterals from internal optic tract axons to the ventrobasal complex branch little if at all as they traverse the lateral geniculate nucleus, and at no time prior to their elimination do they develop an appreciable terminal arbor. These long collaterals often terminate in growth cones that include lamellopodia. Our HRP-impregnation method also revealed some transient non-retinofugal axons that pass medially from the ventral lateral geniculate nucleus to the ventrobasal complex but then return without terminating or branching. By day 4, they are absent, as are collaterals from the internal optic tract to the ventrobasal complex.

Disruption of retinogeniculate afferent segregation by antagonists to NMDA receptors.

Afferent activity has an important role in the formation of connections in the developing mammalian visual system. But the extent to which the activity of target neurons shapes patterns of afferent termination and synaptic contact is not known. In the ferret's visual pathway, retinal ganglion cell axons from each eye segregate early in development into eye-specific laminae in the lateral geniculate nucleus (LGN). The dorsal laminae (termed laminae A and A1) then segregate further into inner and outer sublaminae that retain input from on-centre and off-centre retinal axons, respectively. Thus, individual retinogeniculate axons form terminal arbors within laminae A and A1 that are restricted to one inner or outer sublamina. We report here that blockade of N-methyl-D-aspartate (NMDA) receptors on LGN cells with specific antagonists during the period of sublamina formation prevents retinal afferents from segregating into 'On' and 'Off' sublaminae. Retinogeniculate axons have arbors that are not restricted appropriately, or are restricted in size but inappropriately positioned within the eye-specific laminae. NMDA receptor antagonists may specifically disrupt a mechanism by which LGN neurons detect correlated afferent and target activity, and have been shown to reduce retinogeniculate transmission more generally, causing LGN cells to have markedly reduced levels of activity. These results therefore indicate that the activity of postsynaptic cells can significantly influence the patterning of inputs and the structure of presynaptic afferents during development.

Components of field potentials evoked by white matter stimulation in isolated slices of primary visual cortex: spatial distributions and synaptic order.

1. We have recorded profiles of the spatial distributions of extracellular field potentials in transverse slices of rat primary visual cortex. Responses were evoked by electrical stimulation near the white matter/layer VI border and sampled from layers I to V along the radial axis orthogonal to the laminae and intersecting the stimulation site ("on-beam" recording). To assess the activity of "horizontal" connections, we also recorded profiles along axes parallel to the cortical lamination ("off-beam" recording), usually in layer III. Overall, our goal was to extend understanding of the physiology and organization of neocortical circuitry and to provide a basis for comparisons of data from different experiments and experimenters when neocortical field potentials are used in studies of plasticity and pharmacology. 2. Responses were highly specific with respect to the cortical layers. We distinguish four major components: two kinds of population spike ("S1" and "S2") and two slower waveforms ("W1" and "W2"). The latter appear to represent flow of current in apical dendrites of the supragranular layers. Component W1, the earliest slow component, is a synaptically driven field potential dipole that is positive in layer I and negative in layer II. Based on estimates of current source densities (CSDs), we attribute this to entry of depolarizing current into dendrites and/or cell somata in layer II, ascending intradendritic current, and passive depolarization of inactive dendritic membrane in layer I. Component W1 rises during the 20 ms after stimulation and falls during the 50-100 ms thereafter. Component W2 is also positive in layer I but maximally negative in layer III. It rises for approximately 100 ms after stimulation and decays during the following 400-800 ms. 3. Component S1 does not depend on synaptic transmission because it persists during the application of glutamate receptor antagonists or medium that is low in Ca2+. This component is largest in layer III, radial to the site of stimulation. There, it is a negative deflection, typically 1-2 mV in amplitude and lasting roughly 2 ms, with a latency to peak between 2 and 4.5 ms. Component S1 is most likely a population spike due to synchronized firing of cell somata activated antidromically via unmyelinated efferent axons. 4. Component S2 is a short (less than 20 ms) burst of population spikes specifically in layer III. Individual S2 spikes closely resemble S1 spikes, and we propose that the same neuronal population generates both. However, S2 spikes require glutamatergic synaptic transmission.(ABSTRACT TRUNCATED AT 400 WORDS)

Goldfish retinotectal transmission in vitro: component current sink-source pairs isolated by varying calcium and magnesium levels.

Field potentials and radial current source-densities (CSDs) evoked by optic tract stimulation were observed in goldfish tectum in vitro. The effects of different concentrations of calcium and magnesium were studied to improve understanding of component events in retinotectal transmission and interpretations of effects of pharmacological agents. Responses were of 3 forms: 'non-synaptic', 'subthreshold', and 'complex.' Subthreshold responses occurred when amplitudes were less than 40% of their maximal levels. They consisted of a sink-source pair with the sink in the superficial optic neuropil and the source 100-150 micron deeper. They were monophasic, rising in 1-2 ms and decaying as simple exponentials with time constants between 3.3 and 4.5 ms. Several other conditions which reduce amplitudes (besides low [Ca2+] and/or high [Mg2+]) produce responses of the subthreshold form. Complex responses, observed when amplitudes were 40-100% of maximal, were characterized by more rapid rise and decay and included, in the most extreme cases, a late, long, low-amplitude sink-source pair of opposite polarity. We propose that the time course of decay of subthreshold responses is determined by the passive cable properties of bi- or multistratified neurons with one dendritic arbor in the optic neuropil and a second arbor 100-250 micron deeper. Complex responses probably include recurrent inhibition to depolarized dendrites of the optic neuropil, latent by 4 ms to the monosynaptic excitation. Pharmacological assessments of retinotectal transmission may be made more precise by using low [Ca2+]/high [Mg2+] media to isolate monosynaptic activity.

Pharmacology of retinotectal transmission in the goldfish: effects of nicotinic ligands, strychnine, and kynurenic acid.

The goal of this study was to evaluate different neurotransmitters and their receptors that might be involved in retinotectal transmission in the goldfish. Sections of tectum were isolated and maintained in vitro while pharmacological agents were administered via the tissue bath. Field potentials were elicited by electrical stimulation of the optic nerve and recorded at 50 micron depth intervals, and profiles of current source densities (CSDs) were computed from the second spatial derivatives of these potentials. The preparations were treated with low [Ca2+]/high [Mg2+] media, various cholinergic agonists and antagonists, eserine, strychnine, or kynurenic acid, via the tissue bath. Prior to treatment, depth profiles of these in vitro field potentials and CSDs closely resembled those previously reported in vivo, including 2 prominent sink-source pairs with their sinks in the superficial optic neuropil, followed by a smaller and more prolonged sink-source pair of opposite polarity. These were rapidly and reversibly eliminated by low [Ca2+]/high [Mg2+] bathing media, and substantially reduced by 0.5 or 1.0 mM kynurenic acid. By contrast, d-tubocurarine (d-TC; up to 0.16 mM) reduced peak response amplitudes by less than 40%, eliminated the third sink-source pair, and more than doubled the duration of decay of sink-source pairs 1 and 2 in a concentration-dependent manner. Strychnine had a similar action to d-TC but was slightly more potent. The time course and amplitudes of responses were not much affected by the following nicotinic agonists or antagonists (concentrations in microM): mecamylamine, 50; dihydro-beta-erythroidine, 50; nicotine, 200; tetramethylammonium, 500; ACh (protected by eserine, 20), 200; alpha-bungarotoxin, 2 microM for 2.5 hr, and 0.4 microM for up to 10.5 hr; and lophotoxin, 32 microM for up to 94 min. Eserine (20 microM) and carbachol (200 microM) increased peak response amplitudes by up to 80% within 5-10 min, and amplitudes remained elevated during 20-33 min of continued treatment. The onset of the effects of d-TC, strychnine, and kynurenic acid began in 5-10 min and was completed in 30 min or less, indicating that test substances could adequately penetrate into the interior of the isolated sections of tectum. The failure of these cholinergic ligands to prevent postsynaptic responses indicates that excitatory retinotectal transmission does not depend on an intact nicotinic (or other cholinergic) system, as previously proposed. The action by kynurenic acid suggests the involvement of an excitatory amino acid neurotransmitter in retinotectal transmission.(ABSTRACT TRUNCATED AT 400 WORDS)

Antagonists of glutaminergic neurotransmission block retinotectal transmission in goldfish.

The hypothesis that excitatory retinotectal transmission is mediated primarily by a glutamate or glutamate-related transmitter-receptor system was examined by recording extracellular field potentials in isolated sections of goldfish tectum while stimulating the optic tract and applying antagonists of excitatory amino acid (EAA) neurotransmission via the tissue bath. Three antagonists of EAA receptors produced greater than 90% reduction in the postsynaptic components of these evoked potentials. In order of potency, these were (with the concentrations that produced 50% block): kynurenic acid (0.15 mM), gamma-D-glutamylglycine (0.33 mM), and cis-2,3-piperidine dicarboxylic acid (0.47 mM). All 3 log concentration-effect curves were parallel, symmetrically sigmoidal, and somewhat steeper than non-cooperative single-site binding isotherms. All antagonist actions stabilized within 15 min and were completely reversible. An EAA antagonist potent and selective for the N-methyl-D-aspartate (NMDA) subtype of receptor, 2-amino-5-phosphonovalerate, had little or no effect in either normal, low [Ca2+]/high [Mg2+], or Mg2+-free media. These data indicate that an excitatory amino acid receptor not of the NMDA subtype plays an essential role in fast excitatory retinotectal transmission, and would be most consistent with the mediation of most or all excitatory retinotectal transmission by a single class and subtype of glutamate receptor.

Irreversible autonomic actions by lophotoxin suggest utility as a probe for both C6 and C10 nicotinic receptors.

The marine natural product lophotoxin has produced a non-reversible antagonism of parasympathetic and sympathetic functions that are known to be mediated by C6 sub-type nicotinic receptors. Transmission through anuran paravertebral ganglia was eliminated in 20-40 min by 10-30-min treatments with 16-32 microM lophotoxin, in a time course resembling the onset of block of C10 sub-type nicotinic receptors at the neuromuscular junction and in cultured BC3H-1 cells. The action persisted through 16 h of washout. Nerve conduction was unaffected. Somewhat longer treatments (80 min) of in vitro ileal sections resulted in loss of sensitivity to nicotine, but not to acetylcholine, for at least 5 h. These data indicate that lophotoxin can serve as a more universal nicotinic receptor probe than the alpha-neurotoxins, which may bind to both C6 and C10 sub-types, but block only the C10.

Software that detects and analyzes bioelectrical events by assessing the fit of modeled subintervals.

A software system is described which has been used to identify, average, and otherwise analyse different kinds of electrophysiological events. Spontaneous miniature end-plate potentials were identified by assessing the fit of subintervals within suspected event-containing intervals to flexibly defined model subevents. It was thus practical to apply complex, explicit, and stable criteria in the estimation of the frequency and mean time course of these spontaneous events. This software also mediated the objective rejection of noise-contaminated elements from samples of either evoked or spontaneous potentials. Average event time courses were comprised of a series of estimates of the standard deviations as well as the means. Each analysis generated thousands of individual event amplitudes which were used to improve estimates of quantal release and response parameters. This software allowed more complete analysis of tape-recorded experiments which conserved test compounds and animals, and enabled the observation of simultaneous and equal degrees of action upon spontaneous and evoked potentials, which suggested that a common mechanism was involved.

Quantal analysis indicates an alpha-toxin-like block by lophotoxin, a non-ionic marine natural product.

The effects of a structurally novel paralytic substance (lophotoxin) on quantal transmission parameters and the time course of synaptic potentials have been examined. This substance completely abolished potentials by reducing quantal size without affecting the release of quanta. Nerve conduction, membrane potential, and the passive electrical properties of the muscle end-plate remained unaffected. Lophotoxin appears to act directly on the acetylcholine receptor-channel complex, although perhaps not the cholinoreceptive site itself, as suggested by the unusual chemistry and onset kinetics of this toxin.