Glycinergic feedback enhances synaptic gain in the distal retina.

Glycine input originates with interplexiform cells, a group of neurons situated within the inner retina that transmit signals centrifugally to the distal retina. The effect on visual function of this novel mechanism is largely unknown. Using gramicidin-perforated patch whole cell recordings, intracellular recordings and specific antibody labelling techniques, we examined the effects of the synaptic connections between glycinergic interplexiform cells, photoreceptors and bipolar cells. To confirm that interplexiform cells make centrifugal feedback on bipolar cell dendrites, we recorded the postsynaptic glycine currents from axon-detached bipolar cells while stimulating presynaptic interplexiform cells. The results show that glycinergic interplexiform cells activate bipolar cell dendrites that express the α3 subunit of the glycine receptor, as well as a subclass of unidentified receptors on photoreceptors. By virtue of their synaptic contacts, glycine centrifugal feedback increases glutamate release from photoreceptors and suppresses the uptake of glutamate by the type 2A excitatory amino acid transporter on photoreceptors. The net effect is a significant increase in synaptic gain between photoreceptors and their second-order neurons.

Cytoprotection by endogenous zinc in the vertebrate retina.

Our recent studies have shown that endogenous zinc, co-released with glutamate from the synaptic terminals of vertebrate retinal photoreceptors, provides a feedback mechanism that reduces calcium entry and the concomitant vesicular release of glutamate. We hypothesized that zinc feedback may serve to protect the retina from glutamate excitotoxicity, and conducted in vivo experiments on the retina of the skate (Raja erinacea) to determine the effects of removing endogenous zinc by chelation. These studies showed that removal of zinc by injecting the zinc chelator histidine results in inner retinal damage similar to that induced by the glutamate receptor agonist kainic acid. In contrast, when an equimolar quantity of zinc followed the injection of histidine, the retinal cells were unaffected. Our results are a good indication that zinc, co-released with glutamate by photoreceptors, provides an auto-feedback system that plays an important cytoprotective role in the retina.

Review: Zinc's functional significance in the vertebrate retina.

This review covers a broad range of topics related to the actions of zinc on the cells of the vertebrate retina. Much of this review relies on studies in which zinc was applied exogenously, and therefore the results, albeit highly suggestive, lack physiologic significance. This view stems from the fact that the concentrations of zinc used in these studies may not be encountered under the normal circumstances of life. This caveat is due to the lack of a zinc-specific probe with which to measure the concentrations of Zn(2+) that may be released from neurons or act upon them. However, a great deal of relevant information has been garnered from studies in which Zn(2+) was chelated, and the effects of its removal compared with findings obtained in its presence. For a more complete discussion of the consequences of depletion or excess in the body's trace elements, the reader is referred to a recent review by Ugarte et al. in which they provide a detailed account of the interactions, toxicity, and metabolic activity of the essential trace elements iron, zinc, and copper in retinal physiology and disease. In addition, Smart et al. have published a splendid review on the modulation by zinc of inhibitory and excitatory amino acid receptor ion channels.

Taurine activates delayed rectifier Kv channels via a metabotropic pathway in retinal neurons.

Taurine is one of the most abundant amino acids in the retina, throughout the CNS, and in heart and muscle cells. In keeping with its broad tissue distribution, taurine serves as a modulator of numerous basic processes, such as enzyme activity, cell development, myocardial function and cytoprotection. Despite this multitude of functional roles, the precise mechanism underlying taurine's actions has not yet been identified. In this study we report findings that indicate a novel role for taurine in the regulation of voltage-gated delayed rectifier potassium (K(V)) channels in retinal neurons by means of a metabotropic receptor pathway. The metabotropic taurine response was insensitive to the Cl(-) channel blockers, picrotoxin and strychnine, but it was inhibited by a specific serotonin 5-HT(2A) receptor antagonist, MDL11939. Moreover, we found that taurine enhanced K(V) channels via intracellular protein kinase C-mediated pathways. When 5-HT(2A) receptors were expressed in human embryonic kidney cells, taurine and AL34662, a non-specific 5-HT(2) receptor activator, produced a similar regulation of K(IR) channels. In sum, this study provides new evidence that taurine activates a serotonin system, apparently via 5-HT(2A) receptors and related intracellular pathways.

Regulation of synaptic transmission at the photoreceptor terminal: a novel role for the cation-chloride co-transporter NKCC1.

The Na(+)-K(+)-2Cl(-) co-transporter type 1 (NKCC1) is localized primarily throughout the outer plexiform layer (OPL) of the distal retina, a synaptic lamina that is comprised of the axon terminals of photoreceptors and the dendrites of horizontal and bipolar cells. Although known to play a key role in development, signal transmission and the gating of sensory signals in other regions of the retina and in the CNS, the contribution of NKCC1 to synaptic transmission within the OPL is largely unknown. In the present study, we investigated the function of NKCC1 at the photoreceptor-horizontal cell synapse by recording the electrical responses of photoreceptors and horizontal cells before and after blocking the activity of the transporter with bumetanide (BMN). Because NKCC1 co-transports 1 Na(+), 1 K(+) and 2 Cl(-), it is electroneutral and its activation had little effect on membrane conductance. However, recordings from postsynaptic horizontal cells revealed that inhibiting NKCC1 with BMN greatly increased glutamate release from both rod and cone terminals. In addition, we found that NKCC1 directly regulates Ca(2+)-dependent exocytosis at the photoreceptor synapse, raising the possibility that NKCC1 serves to suppress bulk release of glutamate vesicles from photoreceptor terminals in the dark and at light offset. Interestingly, NKCC1 gene and protein expressions were upregulated by light, which we attribute to the light-induced release of dopamine acting on D1-like receptors. In sum, our study reveals a new role for NKCC1 in the regulation of synaptic transmission in photoreceptors.

Zinc modulation of calcium activity at the photoreceptor terminal: a calcium imaging study.

There is abundant experimental evidence that zinc ions (Zn(2+)) are present in the synaptic vesicles of vertebrate photoreceptors, and that they are co-released with glutamate. Here we show that increasing the concentration of extracellular zinc (2 µM-2 mM) suppresses the entry of calcium into the synaptic terminals of isolated salamander double cones. The resultant dose-dependent curve was fit by an inverse Hill equation having an IC50 of 38 µM, and Hill coefficient of 1.1. Because there is currently no reliable way to measure the concentration of extracellular zinc, it is not known whether the zinc released under normal circumstances is of physiological significance. In an attempt to circumvent this problem we used zinc chelators to reduce the available pool of endogenous zinc. This enabled us to determine how the absence of zinc affected calcium entry. We found that when intra- or extra-cellular zinc was chelated by 250 µM of membrane-permeable TPEN or 500 µM of membrane-impermeable histidine, there was a significant rise in the depolarization-induced intracellular calcium level within photoreceptor terminals. This increase in internal [Ca(2+)] will undoubtedly lead to a concomitant increase in glutamate release. In addition, we found that blocking the L-type calcium channels that are expressed on the synaptic terminals of photoreceptors with 50 µM nicardipine or 100 µM verapamil abolished the effects of zinc chelation. These findings are a good indication that, when released in vivo, the zinc concentration is sufficient to suppress voltage-gated calcium channels, and reduce the rate of glutamate release from photoreceptor terminals.

The modulatory role of taurine in retinal ganglion cells.

Taurine (2-aminoethylsuphonic acid) is present in nearly all animal tissues, and is the most abundant free amino acid in muscle, heart, CNS, and retina. Although it is known to be a major cytoprotectant and essential for normal retinal development, its role in retinal neurotransmission and modulation is not well understood. We investigated the response of taurine in retinal ganglion cells, and its effect on synaptic transmission between ganglion cells and their presynaptic neurons. We find that taurine-elicited currents in ganglion cells could be fully blocked by both strychnine and SR95531, glycine and GABA(A) receptor antagonists, respectively. This suggests that taurine-activated receptors might share the antagonists with GABA and glycine receptors. The effect of taurine at micromolar concentrations can effectively suppress spontaneous vesicle release from the presynaptic neurons, but had limited effects on light-evoked synaptic signals in ganglion cells. We also describe a metabotropic effect of taurine in the suppression of light-evoked response in ganglion cells. Clearly, taurine acts in multiple ways to modulate synaptic signals in retinal output neurons, ganglion cells.

Taurine regulation of voltage-gated channels in retinal neurons.

Taurine activates not only Cl(-)-permeable ionotropic receptors but also receptors that mediate metabotropic responses. The metabotropic property of taurine was revealed in electrophysiological recordings obtained after fully blocking Cl(-)-permeable receptors with an inhibitory "cocktail" consisting of picrotoxin, SR95531, and strychnine. We found that taurine's metabotropic effects regulate voltage-gated channels in retinal neurons. After applying the inhibitory cocktail, taurine enhanced delayed outward rectifier K(+) channels preferentially in Off-bipolar cells, and the effect was completely blocked by the specific PKC inhibitor, GF109203X. Additionally, taurine also acted through a metabotropic pathway to suppress both L- and N-type Ca(2+) channels in retinal neurons, which were insensitive to the potent GABA(B) receptor inhibitor, CGP55845. This study reinforces our previous finding that taurine in physiological concentrations produces a multiplicity of metabotropic effects that precisely govern the integration of signals being transmitted from the retina to the brain.

Review: taurine: a "very essential" amino acid.

Taurine is an organic osmolyte involved in cell volume regulation, and provides a substrate for the formation of bile salts. It plays a role in the modulation of intracellular free calcium concentration, and although it is one of the few amino acids not incorporated into proteins, taurine is one of the most abundant amino acids in the brain, retina, muscle tissue, and organs throughout the body. Taurine serves a wide variety of functions in the central nervous system, from development to cytoprotection, and taurine deficiency is associated with cardiomyopathy, renal dysfunction, developmental abnormalities, and severe damage to retinal neurons. All ocular tissues contain taurine, and quantitative analysis of ocular tissue extracts of the rat eye revealed that taurine was the most abundant amino acid in the retina, vitreous, lens, cornea, iris, and ciliary body. In the retina, taurine is critical for photoreceptor development and acts as a cytoprotectant against stress-related neuronal damage and other pathological conditions. Despite its many functional properties, however, the cellular and biochemical mechanisms mediating the actions of taurine are not fully known. Nevertheless, considering its broad distribution, its many cytoprotective attributes, and its functional significance in cell development, nutrition, and survival, taurine is undoubtedly one of the most essential substances in the body. Interestingly, taurine satisfies many of the criteria considered essential for inclusion in the inventory of neurotransmitters, but evidence of a taurine-specific receptor has yet to be identified in the vertebrate nervous system. In this report, we present a broad overview of the functional properties of taurine, some of the consequences of taurine deficiency, and the results of studies in animal models suggesting that taurine may play a therapeutic role in the management of epilepsy and diabetes.

Fast glutamate uptake via EAAT2 shapes the cone-mediated light offset response in bipolar cells.

Excitatory amino acid transporters (EAATs) are responsible for extracellular glutamate uptake within the retina, and are expressed by retinal neurons and Müller cells. Their role within glutamatergic synapses is not completely understood. In the salamander retina, five distinct EAAT-encoding genes have been cloned, making the amphibian retina an excellent system to study EAAT function. This study focused on sEAAT2, which is expressed in photoreceptor terminals and Off-bipolar cells in two isoforms, sEAAT2A and sEAAT2B. Using whole-cell patch-clamp recording, florescence imaging and antibody labelling methods, we systematically studied the functions of these two isoforms at the synapse between photoreceptors and bipolar cells, both in dark and with photic stimulation. Both sEAAT2A and sEAAT2B were sensitive to dihydrokainic acid (DHKA), a known EAAT2-specific inhibitor. Each isoform of sEAAT2 was found to play a role in tonic glutamate uptake at the cone synapse in darkness. Furthermore, presynaptic sEAAT2A strongly suppressed the rapid, transient glutamate signal from cones following light-offset. This was achieved by quickly binding exocytosed glutamate, which subsequently limited glutamate spillover to adjacent receptors at postsynaptic sites. Since the intensity and duration of photic stimulation determine the magnitude of these cone transient signals, we postulate that presynaptic cone EAATs contribute to the encoding of contrast sensitivity in cone vision.

Functional analysis of hemichannels and gap-junctional channels formed by connexins 43 and 46.

PURPOSE: The gap junctions (GJs) mediating direct cell-cell interaction are formed by clusters of membrane-spanning proteins known as connexins (Cxs). These channels play a key role in signal transmission, and their permeability, time-, and voltage-dependence are governed by the properties of the specific Cxs forming the gap junctions. Retinal pigment epithelium (RPE) cells express Cx43 and Cx46. Here, we employed a heterologous expression system to explore the functional properties of the hemichannels and GJs that could be formed by different combinations of these Cxs. Specifically, we examined the response kinetics of GJs formed by pairing cells expressing Cx43 or Cx46, or those expressing both, i.e., designated as Cx43*Cx46. METHODS: The Xenopus oocyte expression system and a two-electrode voltage clamp technique were used to study the properties of hemichannels and GJs formed in oocytes transfected with Cx43 and/or Cx46 mRNA. RESULTS: Depolarizing voltages activated hemicurrents of similar amplitude from single oocytes transfected with Cx46 or Cx43*Cx46, but not in oocytes expressing Cx43 alone. Incorporating Cx43 with Cx46 altered the gating charge, but not the voltage sensitivity of the hemichannels. In addition, Cx43*Cx46 hemichannel currents exhibited faster activation kinetics than homomeric Cx46 hemichannels. Both homotypic GJs formed by Cx43 and Cx46, and heteromeric Cx43*Cx46 GJs exhibited large junctional conductances with amplitudes of 6.5+/-3.0 microS (Cx43), 8.9+/-3.4 microS (Cx46), and 8.5+/-1.8 microS (Cx43*46); a significantly lower conductance (1.8+/-0.7 microS) was observed for heterotypic GJs formed by Cx43 and Cx46. There were also differences in their gating kinetics. Whereas the kinetics of homotypic Cx46 could be described by a single exponential function (tau=0.91 s), double exponential functions were required for homotypic Cx43 (tau(1)=0.24, tau(2)=3.4 s), heterotypic Cx43/Cx46 (tau(1)=0.29, tau(2)=3.6 s), and heteromeric Cx43*Cx46/Cx43*Cx46 (tau(1)=1.2, tau(2)=8.1 s) junctions. CONCLUSIONS: The failure of oocytes expressing Cx43 to exhibit hemichannel activity is an intrinsic membrane property of this Cx, and cannot be attributed to a lack of expression; western blot analysis showed clearly that Cx43 was expressed in oocytes in which it was injected. Our results provide further evidence that Cx43 and Cx46 form both heterotypic and heteromeric channels when co-expressed, an indication that various combinations of Cxs may participate in gap-junctional communication between RPE cells.

Harmonic analysis of the cone flicker ERG of rabbit.

Harmonic analysis was used to characterize the rabbit flicker ERG elicited by sinusoidally modulated full-field stimuli under light-adapted conditions. The frequency-response function for fundamental amplitude, derived from Fourier analysis of the ERG waveforms, exhibited two limbs, with an amplitude minimum at approximately 30Hz, and a high-frequency region peaking at around 45Hz and extending to more than 100Hz at higher adapting levels. At low frequencies (<20Hz), the fundamental response amplitude was independent of mean luminance (Weber law behavior), whereas the response amplitude at high stimulus frequencies varied nonlinearly with mean luminance. At low frequencies, intravitreal administration of L-AP4, which blocks ON-pathway activity, reduced the fundamental response amplitude and produced a phase shift. On the other hand, PDA, which reduces OFF-pathway activity, had a minimal effect on both the response amplitude and phase at low frequencies. At high frequencies, L-AP4 increased the fundamental response amplitude at low mean luminances, whereas PDA had only a small effect on amplitude and phase. Both pharmacologic agents removed the minimum in the amplitude-frequency function as well as the abrupt change in phase at stimulus frequencies near 30Hz. The results suggest that there is a nonlinear interaction between ON- and OFF-pathway activity over the entire stimulus frequency range examined in this study. These findings provide a basis for formulating protocols to evaluate the effect of pharmacologic agents and/or disease on the cone flicker ERG of rabbit.

Characteristics of period doubling in the rat cone flicker ERG.

When the eye is stimulated by a flickering light, the electroretinogram (ERG) and other electrophysiological responses in the visual pathway often exhibit period doubling. This phenomenon is manifested as an alternation in the shape of the response waveform from cycle to cycle, and also as spectral components at the half-fundamental frequency (F/2) and its odd multiples. Although period doubling has been described in humans as well as in other animals, its features in the rodent flicker ERG have not been characterized. We investigated the properties of period doubling in the rat cone flicker ERG elicited with full field, sinusoidal photic stimuli. Period doubling was observed when the temporal frequency of the stimulus was in the range of 20-30 Hz. The F/2 component of the Fourier spectrum of the ERG was more pronounced than its odd harmonics. The magnitude of the cycle-to-cycle variation in amplitude differed depending on whether measurements were based on peak-to-trough or trough-to-peak amplitudes, owing to the relative phase relationship between F/2 and F as a function of stimulus frequency. The frequency-response characteristics of period doubling varied with stimulus contrast, such that reducing the contrast shifted the peak F/2 amplitude to a lower stimulus frequency. Period doubling was evident in rat eyes in which PDA was administered intravitreally, indicating that the phenomenon can occur independently of OFF pathway activity in the rat retina. The period doubling properties we observed in the flicker ERG response of the rat cone system provide constraints on the nature of the nonlinear feedback mechanism presumed to underlie the period doubling phenomenon.

Restoration of electroretinogram activity in exenterated swine eyes following ophthalmic artery anastomosis.

PURPOSE: To determine the feasibility of restoring electroretinogram (ERG) activity of exenterated swine eyes following in vivo arterial anastomosis. METHODS: The carotid artery was exposed and cannulated. The eye was exenterated along with the extraocular muscles and surrounding connective tissue. Prior to eye transplantation, the ophthalmic artery was identified and anastomosed to the carotid artery. Perfusion was confirmed by injecting FITC-conjugated tomato lectin into the anastomotic tubing and performing confocal microscopy of retinal flat-mounts. Dark-adapted ERG and optic nerve responses were analyzed to assess retinal function, and dilated eye examination and retinal imaging were performed. RESULTS: Arterial anastomosis resulted in perfusion of blood from the carotid artery through the anastomosis and into the ophthalmic artery. Arterial perfusion was confirmed by the presence of tomato lectin-stained retinal vessels. Immediately following the anastomosis, ERG and optic nerve activities were minimal. However, an "a" wave (representing photoreceptor activity), "b" wave (representing bipolar cell activity), and optic nerve responses (representing RGC activity) were detected 30 min after reperfusion. CONCLUSIONS: Electroretinographic function is partially recovered following re-anastomosis of exenterated swine eyes. This model would be useful for further studies on eye transplantation.

Cyclothiazide: a subunit-specific inhibitor of GABAC receptors.

We tested the effects of cyclothiazide (CTZ), an agent used to block desensitization of AMPA-type glutamate receptors, on heterologously expressed GABA(C) receptors formed by homomeric rho subunits. CTZ inhibition of GABA(C) receptors was subunit specific; it produced a dose-dependent reduction of the GABA-elicited current on homomeric rho2 receptors with an IC(50) of about 12 microm, but had no significant effect on homomeric rho1 receptors. This differential sensitivity was attributable to a single amino acid located on the second transmembrane domain of the rho subunits. Mutating the residue at this position from serine to proline on the rho2 subunit eliminated CTZ sensitivity, whereas switching proline to serine on the rho1 subunit made the receptor CTZ sensitive. The inhibitory properties of CTZ were consistent with its action as a channel blocker on the receptors formed by rho2 subunits. The effect showed a small degree of voltage dependence, and was due mainly to a non-competitive mechanism that reduced the maximum response elicited by GABA. In addition, the prominent membrane current rebound when co-application of GABA and CTZ was terminated suggests that the binding site for CTZ on the GABA(C) receptor is distinct from that for GABA, and that CTZ acts as a non-competitive antagonist on the GABA(C) receptor. CTZ inhibited the open channel of the GABA(C) receptor with a time constant of about 0.4 s, but the kinetics were approximately 10-fold slower when GABA is absent. The ability of CTZ to interact with various types of neurotransmitter receptors indicates that the drug has multiple actions in the CNS.

Taurine suppresses the spread of cell death in electrically coupled RPE cells.

PURPOSE: To determine whether taurine exerts a protective effect on retinal pigment epithelium (RPE) cells exposed to a cytotoxic agent, cytochrome C (cyC), shown previously to induce apoptosis and produce cell death in electrically coupled neighboring cells. METHODS: Monolayer cultures of confluent human RPE (ARPE-19) cells, which express gap-junctional proteins, were incubated in culture medium with or without taurine. After scrape loading cyC into the cells, we assayed these cells for caspase 3 activity and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to determine the spread of apoptosis. RESULTS: We found that cyC, too large a molecule to traverse gap junctional channels, produced apoptosis in cells injured by the scrape as well as those distant from the site of the scrape, presumably by the intercellular transmission of a toxic agent through the gap junctions that couple these cells. Incubation in taurine, or the gap-junction blocker, octanol, before application of cyC, reduced significantly the fraction of cells undergoing apoptosis. Voltage clamp recordings from electrically coupled Xenopus oocytes transfected with Cx43 showed that junctional communication was unaffected by taurine. CONCLUSIONS: Our results indicate that taurine can serve to suppress cell death in RPE cells independent of any effect on gap junctions. We have considered various avenues by which taurine can exert its protective effect, but the precise mechanism involved under these experimental conditions has yet to be identified.

Zinc-mediated feedback at the synaptic terminals of vertebrate photoreceptors.

There is mounting evidence that zinc release from glutamatergic nerve terminals serves as a neuromodulator at synaptic sites within the retina and CNS. However, it has not been possible to reliably measure the concentration of zinc co-released with glutamate in the confines of the synaptic cleft. Thus, much of the evidence supporting this view derives from electrophysiological studies showing the modulatory effects of exogenous zinc on the membrane currents of ligand- and voltage-gated channels. In the present study, we took advantage of the unique properties of the glutamatergic photoreceptor terminal to demonstrate a feedback signal mediated by endogenous zinc at the synaptic sites from which it is discharged. Through its ability to block voltage-gated calcium channels in the photoreceptor terminal, zinc suppresses the radial dark current of the visual cell, and reduces its release of glutamate. It follows that chelation of extracellular zinc, e.g., with histidine, will lead to an increase both in the dark current and in the release of glutamate, changes that result in an enhancement of the light-evoked a-wave of the ERG and can account for the b-wave enhancement observed previously after zinc chelation when inner retinal responses were not blocked by aspartate.

A single amino acid in the second transmembrane domain of GABA rho receptors regulates channel conductance.

GABAC receptors, expressed predominately in vertebrate retina, are thought to be formed mainly by GABA rho subunits, each of which exhibits distinct physiological and pharmacological properties. In this study, the receptors formed by perch GABA rho subunits were expressed in HEK cells, and their single channel conductances were determined using noise analysis techniques. The receptors formed by the perch rho1A subunit gate a channel with a conductance of 0.2 pS, whereas the receptors formed by GABA rho2 subunits exhibit much higher channel conductances, i.e., 3.2 and 3.5 pS for perch rho2A and rho2B receptors, respectively. A comparison of the amino acid sequences of the channel-forming TMII regions of the various subunits suggested that a single amino acid at position 2' was a potential site for the large differential in conductance. We found that switching the serine residue at that site in the GABA rho2 subunit to the proline residue present in the rho1 subunit reduced the channel conductance to a level similar to that of the wild type rho1 receptor. Conversely, mutating proline to serine in the amino acid sequence of the rho1 receptor significantly increased its unitary conductance. These results indicate that a single amino acid in the TMII region plays an important role in determining the single channel conductance of the GABAC receptors.

GABAB receptor attenuation of GABAA currents in neurons of the mammalian central nervous system.

Ionotropic receptors are tightly regulated by second messenger systems and are often present along with their metabotropic counterparts on a neuron's plasma membrane. This leads to the hypothesis that the two receptor subtypes can interact, and indeed this has been observed in excitatory glutamate and inhibitory GABA receptors. In both systems the metabotropic pathway augments the ionotropic receptor response. However, we have found that the metabotropic GABAB receptor can suppress the ionotropic GABAA receptor current, in both the in vitro mouse retina and in human amygdala membrane fractions. Expression of amygdala membrane microdomains in Xenopus oocytes by microtransplantation produced functional ionotropic and metabotropic GABA receptors. Most GABAA receptors had properties of α-subunit containing receptors, with ~5% having ρ-subunit properties. Only GABAA receptors with α-subunit-like properties were regulated by GABAB receptors. In mouse retinal ganglion cells, where only α-subunit-containing GABAA receptors are expressed, GABAB receptors suppressed GABAA receptor currents. This suppression was blocked by GABAB receptor antagonists, G-protein inhibitors, and GABAB receptor antibodies. Based on the kinetic differences between metabotropic and ionotropic receptors, their interaction would suppress repeated, rapid GABAergic inhibition.