Application Of Glycine Research Articles

Mechanisms of Interaction between GABA- and Glycine-Medicated Responses of Frog Spinal Cord Neurons

Whole-cell patch-clamp recordings were made from isolated frog (R. temporaria) spinal cord neurons to study the interaction between processes evoked by application of glycine and GABA. When GABA and glycine were applied simultaneously, neuron responses were always smaller than the sum of the responses to separate application of these neurotransmitters. The mechanisms underlying this occlusion phenomenon were identified. Firstly, GABA could activate glycine receptors (EC50 = 1.2 mM) in frog spinal cord neurons relatively effectively. Secondly, studies of the effects of application of glycine on the course of “rundown” of GABA-mediated responses showed that activation of glycine receptors facilitated inhibition of GABAA receptors. Both of these mechanisms of interaction of neuron responses are presumptively mediated via simultaneous release of the neurotransmitters glycine and GABA in synapses.

P.1.011 Identification of a significant role for the ventral hippocampus in neuropeptide S-elicited anxiolysis

The negative symptoms of schizophrenia are reverted by treatment with glycine or other agonists of the glycine-B site which facilitate NMDA receptor function. On the other hand, there are experimental observations showing that exogenous application of glycine (0.5–10 mM) results in a long-lasting potentiation of glutamatergic synaptic transmission (LTP-GLY). The characterization of the mechanisms underlying LTP-GLY could be useful to develop new therapies for schizophrenia. The main goal of this work is to deepen the understanding of this potentiation phenomenon. The present study demonstrates in rat hippocampal slices that superfusion of glycine 1 mM during 30 min produces a potentiation of excitatory postsynaptic potentials in CA3-CA1 pathway lasting at least 1 h. Glycine application does not modify neither presynaptic fiber volley nor paired-pulse facilitation of synaptic potentials. This LTP-GLY is independent of both strychnine-sensitive glycine receptors and nifedipine-sensitive calcium channels. Interestingly, LTP-GLY is not inhibited but strengthened by NMDA receptors antagonists such as AP-5 or MK-801. In contrast, LTP-GLY is partially or totally blocked with the antagonists of glycine transporter GLYT1, sarcosine or ALX-5407, respectively. These results indicate that LTP-GLY requires the activation of GLYT1, a glycine transporter co-localized and associated to NMDA receptors. In addition, the fact that NMDA receptor inhibition increases LTP-GLY magnitude, opens the possibility that these receptors could have a negative control on GLYT1 activity.

Presynaptic Glycine Receptors Increase GABAergic Neurotransmission in Rat Periaqueductal Gray Neurons

The periaqueductal gray (PAG) is involved in the central regulation of nociceptive transmission by affecting the descending inhibitory pathway. In the present study, we have addressed the functional role of presynaptic glycine receptors in spontaneous glutamatergic transmission. Spontaneous EPSCs (sEPSCs) were recorded in mechanically dissociated rat PAG neurons using a conventional whole-cell patch recording technique under voltage-clamp conditions. The application of glycine (100 µM) significantly increased the frequency of sEPSCs, without affecting the amplitude of sEPSCs. The glycine-induced increase in sEPSC frequency was blocked by 1 µM strychnine, a specific glycine receptor antagonist. The results suggest that glycine acts on presynaptic glycine receptors to increase the probability of glutamate release from excitatory nerve terminals. The glycine-induced increase in sEPSC frequency completely disappeared either in the presence of tetrodotoxin or Cd2+, voltage-gated Na+, or Ca2+ channel blockers, suggesting that the activation of presynaptic glycine receptors might depolarize excitatory nerve terminals. The present results suggest that presynaptic glycine receptors can regulate the excitability of PAG neurons by enhancing glutamatergic transmission and therefore play an important role in the regulation of various physiological functions mediated by the PAG.

Glycine Gating of NR1/NR2A NMDA Receptors

N-Methyl-D-Aspartate receptors (NMDARs) are ligand-gated ion channels that mediate excitatory neurotransmission in the mammalian central nervous system. They are required for normal neuronal function and are a factor in several neuropathies including Alzheimer's disease and schizophrenia. Classical NMDARs require both glycine (NR1) and glutamate (NR2) bound for receptor activation. Reaction mechanisms for NMDA receptors have been developed for several isoforms, however these models assume saturation of glycine sites and a quantitative understanding of glycine association and dissociation kinetics is currently inadequate. We measured current responses with fast application of glycine (in 1 mM glutamate) onto outside-out patches containing several NR1/NR2A receptors and observed relatively slow kinetics, with a rise time of 4.9 ± 0.6ms and deactivation of 105 ± 8ms. Anticipating a slow dissociation rate for glycine, we first developed models for two low-affinity glycine-site full-agonists: L-serine (95% efficacy; EC50=0.21mM) and 3,3,3-trifluoro-DL-alanine (132% efficacy; EC50=2.2mM). We used these data to select best fitting multi-state kinetic models of several arrangements and further validated these with cycles of simulations and experimental measurements which included macroscopic responses to several stimulation patterns. These models will be used to estimate glycine association and dissociation rate constants. Work is in progress to validate these results with macroscopic measurements. Knowledge about how glycine gates NMDA receptors will offer insight to glycine-dependent NR1/NR2A kinetic mechanisms and contribute to a more comprehensive understanding of the activation of these physiologically important receptors.

Regulation of acetylcholinesterase activity by nitric oxide in rat neuromuscular junction via N‐methyl‐d‐aspartate receptor activation

Acetylcholinesterase (AChE) is an enzyme that hydrolyses the neurotransmitter acetylcholine, thereby limiting spillover and duration of action. This study demonstrates the existence of an endogenous mechanism for the regulation of synaptic AChE activity. At the rat extensor digitorum longus neuromuscular junction, activation of N-methyl-d-aspartate (NMDA) receptors by combined application of glutamate and glycine led to enhancement of nitric oxide (NO) production, resulting in partial AChE inhibition. Partial AChE inhibition was measured using increases in miniature endplate current amplitude. AChE inhibition by paraoxon, inactivation of NO synthase by N(x)-nitro-L-arginine methyl ester, and NMDA receptor blockade by DL-2-amino-5-phosphopentanoic acid prevented the increase in miniature endplate current amplitude caused by amino acids. High-frequency (10 Hz) motor nerve stimulation in a glycine-containing bathing solution also resulted in an increase in the amplitude of miniature endplate currents recorded during the interstimulus intervals. Pretreatment with an NO synthase inhibitor and NMDA receptor blockade fully eliminated this effect. This suggests that endogenous glutamate, released into the synaptic cleft as a co-mediator of acetylcholine, is capable of triggering the NMDA receptor/NO synthase-mediated pathway that modulates synaptic AChE activity. Therefore, in addition to well-established modes of synaptic plasticity (e.g. changes in the effectiveness of neurotransmitter release and/or the sensitivity of the postsynaptic membrane), another mechanism exists based on the prompt regulation of AChE activity.

Long term depression of MNTB-LSO synapses is expressed postsynaptically in developing circling mice

Early onset long term depression (LTD) during the first postnatal week has rarely been demonstrated at the medial nucleus of trapezoid body (MNTB) – lateral superior olive (LSO) synapses in spite of many favorable conditions, such as depolarizing synapses and glutamate co-release from MNTB terminals. Thus, we tested the early expression of LTD at MNTB-LSO synapses during the first postnatal week using circling mice, whose main transmitter is glutamate at MNTB-LSO synapses. Tetanic stimulation on MNTB elicited LTD of postsynaptic currents recorded at LSO neurons in P0–P3 homozygous (cir/cir) mice (45.8±0.3% of the control, n=7) and heterozygous (+/cir) mice (43.3±0.4% of the control, n=7). The magnitude of LTD decreased in P8–P12 heterozygous (+/cir) mice (84.5±0.3% of the control, n=7), but was maintained in P8–P12 homozygous (cir/cir) mice (38.2±0.3% of the control, n=9). Glutamatergic LTD observed in homozygous (cir/cir) mice and glycinergic LTD observed heterozygous (+/cir) mice showed similar pattern of change. As currents induced by the pressure application of glycine on LSO neurons were reduced by tetanic stimulation in P0–P3 heterozygous (+/cir) mice, LTD was thought to occur at postsynaptic sites. Our results suggest that LTD might occur in vivo and participate in the synaptic silencing and strengthening of MNTB-LSO synapses, which is most active during the first postnatal week.

The Importance of TM3-4 Loop Subdomains for Functional Reconstitution of Glycine Receptors by Independent Domains

Truncated glycine receptors that have been found in human patients suffering from the neuromotor disorder hyperekplexia or in spontaneous mouse models resulted in non-functional ion channels. Rescue of function experiments with the lacking protein portion expressed as a separate independent domain demonstrated restoration of glycine receptor functionality in vitro. This construct harbored most of the TM3-4 loop, TM4, and the C terminus and was required for concomitant transport of the truncated α1 and the complementation domain from the endoplasmic reticulum toward the cell surface, thereby enabling complex formation of functional glycine receptors. Here, the complementation domain was stepwise truncated from its N terminus in the TM3-4 loop. Truncation of more than 49 amino acids led again to loss of functionality in the receptor complex expressed from two independent domain constructs. We identified residues 357-418 in the intracellular TM3-4 loop as being required for reconstitution of functional glycine-gated channels. All complementation constructs showed cell surface protein expression and correct orientation according to glycine receptor topology. Moreover, we demonstrated that the truncations did not result in a decreased protein-protein interaction between both glycine receptor domains. Rather, deletions of more than 49 amino acids abolished conformational changes necessary for ion channel opening. When the TM3-4 loop subdomain harboring residues 357-418 was expressed as a third independent construct together with the truncated N-terminal and C-terminal glycine receptor domains, functionality of the glycine receptor was again restored. Thus, residues 357-418 represent an important determinant in the process of conformational rearrangements following ligand binding resulting in channel opening.

Interaction between taurine and GABAA/glycine receptors in neurons of the rat anteroventral cochlear nucleus

Taurine, one of the most abundant endogenous amino acids in the mammalian central nervous system (CNS), is involved in neural development and many physiological functions. In this study, the interaction between taurine and GABAA/glycine receptors was investigated in young rat (P13–P15) anteroventral cochlear nucleus (AVCN) neurons using the whole-cell patch-clamp method. We found that taurine at low (0.1mM) and high (1mM) concentrations activated both GABAA and glycine receptors, but not AMPA and NMDA receptors. The reversal potentials of taurine-, GABA- or glycine-evoked currents were close to the expected chloride equilibrium potential, indicating that receptors activated by these agonists were mediating chloride conductance. Moreover, our results showed that the currents activated by co-application of GABA and glycine were cross-inhibitive. Sequential application of GABA and glycine or vice versa also reduced the glycine or GABA evoked currents. There was no cross-inhibition when taurine and GABA or taurine and glycine were applied simultaneously, but the response was larger than that evoked by GABA or glycine alone. These results suggest that taurine can serve as a neuromodulator to strengthen GABAergic and glycinergic neurotransmission in the rat AVCN.

Searching for Presynaptic NMDA Receptors in the Nucleus Accumbens

The nucleus accumbens shell (NAc) is a key brain region mediating emotional and motivational learning. In rodent models, dynamic alterations have been observed in synaptic NMDA receptors (NMDARs) within the NAc following incentive stimuli, and some of these alterations are critical for acquiring new emotional/motivational states. NMDARs are prominent molecular devices for controlling neural plasticity and memory formation. Although synaptic NMDARs are predominately located postsynaptically, recent evidence suggests that they may also exist at presynaptic terminals and reshape excitatory synaptic transmission by regulating presynaptic glutamate release. However, it remains unknown whether presynaptic NMDARs exist in the NAc and contribute to emotional and motivational learning. In an attempt to identify presynaptically located NMDARs in the NAc, the present study uses slice electrophysiology combined with pharmacological and genetic tools to examine the physiological role of the putative presynaptic NMDARs in rats. Our results show that application of glycine, the glycine-site agonist of NMDARs, potentiated presynaptic release of glutamate at excitatory synapses on NAc neurons, whereas application of 5,7-dichlorokynurenic acid or 7-chlorokynurenic acid, the glycine-site antagonists of NMDARs, produced the opposite effect. However, these seemingly presynaptic NMDAR-mediated effects could not be prevented by application of d-APV, the glutamate-site NMDAR antagonist, and were still present in the mice in which NMDAR NR1 or NR3 subunits were genetically deleted. Thus, rather than suggesting the existence of presynaptic NMDARs, our results support the idea that an unidentified type of glycine-activated substrate may account for the presynaptic effects appearing to be mediated by NMDARs.

Pharmacokinetics and cerebral distribution of glycine administered to rats

High doses of glycine have been reported to improve negative schizophrenic symptoms, suggesting that ingested glycine activates glutamatergic transmission via N-methyl-D-aspartate (NMDA) receptors. However, the pharmacokinetics of administered glycine in the brain has not been evaluated. In the present study, the time- and dose-dependent distributions of administered glycine were investigated from a pharmacokinetic viewpoint. Whole-body autoradiography of radiolabeled glycine was performed, and time-concentration curves for glycine and serine in plasma, cerebrospinal fluid (CSF), and brain tissues were obtained. Furthermore, pharmacokinetic parameters were calculated. For a more detailed analysis, the amount of glycine uptake in the brain was evaluated using the brain uptake index method. Radiolabeled glycine was distributed among periventricular organs in the brain. Oral administration of 2 g/kg of glycine significantly elevated the CSF glycine concentration above the ED50 value for NMDA receptors. The glycine levels in CSF were 100 times lower than those in plasma. Glycine levels were elevated in brain tissue, but with a slower time-course than in CSF. Serine, a major metabolite of glycine, was elevated in plasma, CSF, and brain tissue. Glycine uptake in brain tissue increased in a dose-dependent manner. Time-concentration curves revealed that glycine was most likely transported via the blood-CSF barrier and activated NMDA receptors adjacent to the ventricles. The pharmacokinetic analysis and the brain uptake index for glycine suggested that glycine was transported into brain tissue by passive diffusion. These results provide further insight into the potential therapeutic applications of glycine.

Role of Glycine Receptors in Glycine-Induced LTD in Hippocampal CA1 Pyramidal Neurons

Glycine in the hippocampus can exert its effect on both synaptic NMDA receptors (NMDARs) and extrasynaptic functional glycine receptors (GlyRs) via distinct binding sites. Previous studies have reported that glycine induces long-term potentiation (LTP) through the activation of synaptic NMDARs. However, little is known about the potential role of the activated GlyRs that are largely located in extrasynaptic regions. We report here that relatively high levels of glycine achieved either by exogenous glycine application or by the elevation of endogenous glycine accumulation with an antagonist of the glycine transporter induced long-term depression (LTD) of excitatory postsynaptic currents (EPSCs) in hippocampal CA1 pyramidal neurons. The co-application of glycine with the selective GlyR antagonist strychnine changed glycine-induced LTD (Gly-LTD) to LTP. Blocking the postsynaptic GlyR-gated net chloride flux by manipulating intracellular chloride concentrations failed to elicit any changes in EPSCs. These results suggest that GlyRs are involved in Gly-LTD. Furthermore, this new form of chemical LTD was accompanied by the internalization of postsynaptic AMPA receptors and required the activation of NMDARs. Therefore, our present findings reveal an important function of GlyR activation and modulation in gating the direction of synaptic plasticity.

Probing glycine receptor stoichiometry in superficial dorsal horn neurones using the spasmodic mouse

Inhibitory glycine receptors (GlyRs) are pentameric ligand gated ion channels composed of α and β subunits assembled in a 2:3 stoichiometry. The α1/βheteromer is considered the dominant GlyR isoform at 'native' adult synapses in the spinal cord and brainstem. However, the α3 GlyR subunit is concentrated in the superficial dorsal horn (SDH: laminae I-II), a spinal cord region important for processing nociceptive signals from skin, muscle and viscera. Here we use the spasmodic mouse, which has a naturally occurring mutation (A52S) in the α1 subunit of the GlyR, to examine the effect of the mutation on inhibitory synaptic transmission and homeostatic plasticity, and to probe for the presence of various GlyR subunits in the SDH.We usedwhole cell recording (at 22-24◦C) in lumbar spinal cord slices obtained from ketamine-anaesthetized (100 mg kg⁻¹, I.P.) spasmodic and wild-type mice (mean age P27 and P29, respectively, both sexes). The amplitude and decay time constants of GlyR mediated mIPSCs in spasmodic micewere reduced by 25% and 50%, respectively (42.0 ± 3.6 pA vs. 31.0 ± 1.8 pA, P <0.05 and 7.4 ± 0.5 ms vs. 5.0 ± 0.4 ms, P <0.05; means ± SEM, n =34 and 31, respectively). Examination of mIPSC amplitude versus rise time and decay time relationships showed these differences were not due to electrotonic effects. Analysis of GABAAergic mIPSCs and A-type potassium currents revealed altered GlyR mediated neurotransmission was not accompanied by the synaptic or intrinsic homeostatic plasticity previously demonstrated in another GlyR mutant, spastic. Application of glycine to excised outside-out patches from SDH neurones showed glycine sensitivity was reduced more than twofold in spasmodic GlyRs (EC50 =130 ± 20 μM vs. 64 ± 11 μM, respectively; n =8 and 15, respectively). Differential agonist sensitivity and mIPSC decay times were subsequently used to probe for the presence of α1-containing GlyRs in SDHneurones.Glycine sensitivity, based on the response to 1-3 μM glycine, was reduced in>75% of neurones tested and decay times were faster in the spasmodic sample. Together, our data suggest most GlyRs and glycinergic synapses in the SDH contain α1 subunits and few are composed exclusively of α3 subunits. Therefore, future efforts to design therapies that target the α3 subunit must consider the potential interaction between α1 and α3 subunits in the GlyR.

Activation of glycine receptor phase‐shifts the circadian rhythm in neuronal activity in the mouse suprachiasmatic nucleus

In mammals, the master clock in the suprachiasmatic nucleus (SCN) of the hypothalamus is composed of numerous synchronized oscillating cells that drive daily behavioural and physiological processes. Several entrainment pathways, afferent inputs to the SCN with their neurotransmitter and neuromodulator systems, can reset the circadian system regularly and also modulate neuronal activity within the SCN. In the present study, we investigated the function of the inhibitory neurotransmitter glycine on neuronal activity in the mouse SCN and on resetting of the circadian clock. The effects of glycine on the electrical activity of SCN cells from C57Bl/6 mice were studied either by patch-clamp recordings from acute brain slices or by long-term recordings from organotypic brain slices using multi-microelectrode arrays(MEA). Voltage-clamp recordings confirmed the existence of glycine-induced, chloride-selective currents in SCN neurons. These currents were reversibly suppressed by strychnine, phenylbenzeneω-phosphono-α-amino acid (PMBA) or ginkgolide B, selective blockers of glycine receptors(GlyRs). Long-term recordings of the spontaneous activity of SCN neurons revealed that glycine application induces a phase advance during the subjective day and a phase delay during the early subjective night. Both effects were suppressed by strychnine or by PMBA. These results suggest that glycine is able to modulate circadian activity by acting directly on its specific receptors in SCN neurons.

Zinc modulation of glycine receptors

Glycine receptors are widely expressed in the mammalian central nervous system, and previous studies have demonstrated that glycine receptors are modulated by endogenous zinc. Zinc is concentrated in synaptic vesicles in several brain regions but is particularly abundant in the hippocampus and olfactory bulb. In the present study, we used patch-clamp electrophysiology of rat hippocampal and olfactory bulb neurons in primary culture to examine the effects of zinc on glycine receptors. Although glycine has been reported to reach millimolar concentrations during synaptic transmission, most previous studies on the effects of zinc on glycine receptors have used relatively low concentrations of glycine. High concentrations of glycine cause receptor desensitization. Our current results extend our previous demonstration that the modulatory actions of zinc are largely prevented when co-applied with desensitizing concentrations of glycine (300 μM), suggesting that the effects of zinc are dependent on the state of the receptor. In contrast, pre-application of 300 μM zinc, prior to glycine (300 μM) application, causes a slowly developing inhibition with a slow rate of recovery, suggesting that the timing of zinc and glycine release also influences the effects of zinc. Furthermore, previous evidence suggests that synaptically released zinc can gain intracellular access, and we provide the first demonstration that low concentrations of intracellular zinc can potentiate glycine receptors. These results support the notion that zinc has complex effects on glycine receptors and multiple factors may interact to influence the efficacy of glycinergic transmission.

The long activations of α2 glycine channels can be described by a mechanism with reaction intermediates (“flip”)

The α2 glycine receptor (GlyR) subunit, abundant in embryonic neurons, is replaced by α1 in the adult nervous system. The single-channel activity of homomeric α2 channels differs from that of α1-containing GlyRs, as even at the lowest glycine concentration (20 µM), openings occurred in long (>300-ms) groups with high open probability (Popen; 0.96; cell-attached recordings, HEK-expressed channels). Shut-time intervals within groups of openings were dominated by short shuttings of 5–10 µs. The lack of concentration dependence in the groups of openings suggests that they represent single activations, separated by very long shut times at low concentrations. Several putative mechanisms were fitted by maximizing the likelihood of the entire sequence of open and shut times, with exact missed-events allowance (program hjcfit). Records obtained at several glycine concentrations were fitted simultaneously. The adequacy of the different schemes was judged by the accuracy with which they predicted not only single-channel data but also the time course and concentration dependence of macroscopic responses elicited by rapid glycine applications to outside-out patches. The data were adequately described only with schemes incorporating a reaction intermediate in the activation, and the best was a flip mechanism with two binding sites and one open state. Fits with this mechanism showed that for α2 channels, the opening rate constant is very fast, ∼130,000 s−1, much as for α1β GlyRs (the receptor in mature synapses), but the estimated true mean open time is 20 times longer (around 3 ms). The efficacy for the flipping step and the binding affinity were lower for α2 than for α1β channels, but the overall efficacies were similar. As we previously showed for α1 homomeric receptors, in α2 glycine channels, maximum Popen is achieved when fewer than all five of the putative binding sites in the pentamer are occupied by glycine.

Direct and indirect control of orexin/hypocretin neurons by glycine receptors.

Hypothalamic hypocretin/orexin (hcrt/orx) neurons promote arousal and reward seeking, while reduction in their activity has been linked to narcolepsy, obesity and depression. However, the mechanisms influencing the activity of hcrt/orx networks in situ are not fully understood. Here we show that glycine, a neurotransmitter best known for its actions in the brainstem and spinal cord, elicits dose dependent postsynaptic Cl⁻ currents in hcrt/orx cells in acute mouse brain slices. The effect was blocked by the glycine receptor (GLyR) antagonist strychnine and mimicked by the GlyR agonist alanine. Postsynaptic GlyRs on hcrt/orx cells remained functional during both early postnatal and adult periods, and gramicidin-perforated patch-clamp recordings revealed that they progressively switch from excitatory to inhibitory during the first two postnatal weeks. The pharmacological profile of the glycine response suggested that developed hcrt/orx neurons contain α/β-heteromeric GlyRs that lack α2-subunits, whereas α2-subunits, whereas α2-subunits are present in early postnatal hcrt/orx neurons. All postsynaptic currents (PSCs) in developed hcrt/orx cells were blocked by inhibitors of GABA and glutamate receptors, with no evidence of GlyR-mediated PSCs. However, the frequency but not amplitude of miniature PSCs was reduced by strychnine and increased by glycine in ~50% of hcrt/orx neurons. Together, these results provide the first evidence for functional GlyRs in identified hcrt/orx circuits and suggest that the activity of developed hcrt/orx cells is regulated by two GlyR pools: inhibitory extrasynaptic GlyRs located on all hcrt/orx cells and excitatory GlyRs located on presynaptic terminals contacting some hcrt/orx cells.

Application of Silver Nitrate Induces Functional Bisexual Flowers in Gynoecious Cucumber Plants (Cucumis sativus L.)

The most common cucumber plants (Cucumis sativus L.) show monoecious sex expression and produce both male and female flowers on the same plant, whereas plants of the gynoecious type produce only female flowers under natural conditions. Ethylene is a plant hormone that affects sex differentiation in cucumber plants. In the gynoecious cucumber, application of the ethylene biosynthesis inhibitor aminoethoxyvinyl glycine (AVG) induced only male flowers, whereas application of the ethylene action inhibitor silver nitrate (AgNO3) induced male and morphologically bisexual flowers. To investigate whether the morphologically bisexual flowers that were induced by AgNO3 in gynoecious cucumber plants have functional sexual organs, we analyzed the function of pistils and stamens. Seeds were produced by cross-fertilization and the pollen tubes were elongated on sucrose medium in eight of the twenty-five morphologically bisexual flowers. These results show that the application of AgNO3 to gynoecious cucumber plants produces functional bisexual flowers. In functional bisexual flowers, the length of pistils was similar to that of the female flowers of gynoecious cucumber plants and the length of stamens was similar to that of male and bisexual flowers of andromonoecious cucumber plants. In contrast, stamen length was inversely proportional to pistil length in the group of morphologically bisexual flowers that were induced by AgNO3. These results suggest that the morphology of the sexual organs is different in morphologically bisexual flowers and in functional bisexual flowers. It is possible that the functional bisexual flowers that were induced by AgNO3 in gynoecious cucumber plants are the result of exclusive blockade of ethylene signals that inhibit stamen development.

The possible additional role of the cystic fibrosis transmembrane regulator to motoneuron inhibition produced by glycine effects

In the present work we study the contribution of the chloride channel of the Cystic Fibrosis Transmembrane Regulator (CFTR) in the postsynaptic inhibition of somatic motoneurons during rapid-eye-movement (REM) sleep atonia. Postsynaptic inhibition of motoneurons is partially responsible for the atonia that occurs during REM sleep. Disfacilitation is an additional mechanism that lowers motoneuron excitability in this state. Postsynaptic inhibition is mediated by the release of glycine from synaptic terminals on motoneurons, and by GABA that plays a complementary role to that of glycine. In this work we look in brain stem motoneurons of neonatal rats at a mechanism unrelated to the actions of glycine, GABA or to disfacilitation which depends on the chloride channel of the CFTR. We studied the presence of CFTR by immunocytochemistry. In electrophysiological experiments utilizing whole cell recordings in in vitro slices we examined the consequences of blocking this chloride channel. The effects on motoneurons of the application of glycine, of the application of glibenclamide (a CFTR blocker) and again of glycine during the effects of glibenclamide were studied. Glycine produced an hyperpolarization, a decrease in motoneuron excitability and a decrease in input resistance, all characteristic changes of the postsynaptic inhibition produced by this neurotransmitter. Glibenclamide produced an increase in input resistance and in motoneurons' repetitive discharge as well as a shift in the equilibrium potential for chloride ions as indicated by the displacement of the reversal potential for glycinergic actions. In motoneurons treated with glibenclamide, glycine produced postsynaptic inhibition but this effect was smaller when compared to that elicited by glycine in control conditions. The fact that blocking of the CFTR-chloride channel in brain stem motoneurons influences glycinergic inhibition suggests that this channel may play a complementary role in the glycinergic inhibition that occurs during REM sleep.

Transmission of phase-coupling accuracy from the auditory nerve to spherical bushy cells in the Mongolian gerbil

The phase of low-frequency sinusoids is encoded in phase-coupled discharges of spherical bushy cells (SBCs) of the anteroventral cochlear nucleus and transmitted to the medial superior olive, where binaural input-coincidence is used for processing of sound source localization. SBCs are innervated by auditory nerve fibers through large, excitatory synapses (endbulbs of Held) and by inhibitory inputs, which effectively reduce SBC discharge rates. Here we monitor presynaptic potentials of endbulb-terminals and postsynaptic spikes of SBCs in extracellular single unit recordings in vivo. We compare postsynaptic phase-coupling of SBCs and their presynaptic immediate auditory nerve input. In all but one SBC discharge rates at the characteristic frequency were reduced pre-to-postsynaptically and phase-coupling accuracy was increased in one-third of them. We investigated the contribution of systemic inhibition on spike timing in SBCs by iontophoretic application of glycine- and GABA-receptor antagonists (strychnine, bicuculline). Discharge rate increased in one-third of the units during antagonist application, which was accompanied by a deterioration of phase-coupling accuracy in half of those units. These results suggest that the phase-coupling accuracy is improved in a subpopulation of SBCs during transmission from the auditory nerve to the SBCs by reduction of spike rates.

Plasma membrane ferric reductase activity of iron-limited algal cells is inhibited by ferric chelators

Iron-limited cells of the green alga Chlorella kesslerii use a reductive mechanism to acquire Fe(III) from the extracellular environment, in which a plasma membrane ferric reductase reduces Fe(III)-chelates to Fe(II), which is subsequently taken up by the cell. Previous work has demonstrated that synthetic chelators both support ferric reductase activity (when supplied as Fe(III)-chelates) and inhibit ferric reductase. In the present set of experiments we extend these observations to naturally-occurring chelators and their analogues (desferrioxamine B mesylate, schizokinen, two forms of dihydroxybenzoic acid) and also two formulations of the commonly-used herbicide N-(phoshonomethyl)glycine (glyphosate). The ferric forms of the larger siderophores (desferrioxamine B mesylate, schizokinen) and Fe(III)-N-(phoshonomethyl)glycine (as the isopropylamine salt) all supported rapid rates of ferric reductase activity, while the iron-free forms inhibited reductase activity. The smaller siderophores/siderophore precursors, 2,3- and 3,4-dihydroxybenzoic acids, did not support high rates of reductase in the ferric form but did inhibit reductase activity in the iron-free form. Bioassays indicated that Fe(III)-chelates that supported high rates of ferric reductase activity also supported a large stimulation in the growth of iron-limited cells, and that an excess of iron-free chelator decreased the growth rate. With respect to N-(phosphonomethyl)glycine, there were differences between the pure compound (free acid form) and the most common commercial formulation (which also contains isopropylamine) in terms of supporting and inhibiting ferric reductase activity and growth. Overall, these results suggest that photosynthetic organisms that use a reductive strategy for iron acquisition both require, and are potentially simultaneously inhibited by, ferric chelators. Furthermore, these results also may provide an explanation for the frequently contradictory results of N-(phosphonomethyl)glycine application to crops: we suggest that low concentrations of this molecule likely solubilize Fe(III), making it available for plant growth, but that higher (but sub-lethal) concentrations decrease iron acquisition by inhibiting ferric reductase activity.