DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery.

More than 800 G protein-coupled receptors (GPCRs) comprise the largest class of membrane receptors in humans. While there is ample biological understanding and many approved drugs for prototypic GPCRs, most GPCRs still lack well-defined biological ligands and drugs. Here, we report our efforts to tap the potential of understudied GPCRs by developing yeast-based technologies for high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) engineering and GPCR ligand discovery. We refer to these technologies collectively as Dynamic Cyan Induction by Functional Integrated Receptors, or DCyFIR. A major advantage of DCyFIR is that GPCRs and other assay components are CRISPR-integrated directly into the yeast genome, making it possible to decode ligand specificity by profiling mixtures of GPCR-barcoded yeast strains in a single tube. To demonstrate the capabilities of DCyFIR, we engineered a yeast strain library of 30 human GPCRs and their 300 possible GPCR-Gα coupling combinations. Profiling of these 300 strains, using parallel (DCyFIRscreen) and multiplex (DCyFIRplex) DCyFIR modes, recapitulated known GPCR agonism with 100% accuracy, and identified unexpected interactions for the receptors ADRA2B, HCAR3, MTNR1A, S1PR1, and S1PR2. To demonstrate DCyFIR scalability, we profiled a library of 320 human metabolites and discovered several GPCR-metabolite interactions. Remarkably, many of these findings pertained to understudied pharmacologically dark receptors GPR4, GPR65, GPR68, and HCAR3. Experiments on select receptors in mammalian cells confirmed our yeast-based observations, including our discovery that kynurenic acid activates HCAR3 in addition to GPR35, its known receptor. Taken together, these findings demonstrate the power of DCyFIR for identifying ligand interactions with prototypic and understudied GPCRs.

Membrane properties of identified lateral and medial perforant pathway projection neurons.

The physiological characteristics of neurons that project to the hippocampus and dentate gyrus via the medial perforant pathway (projection neurons) are well known, but the characteristics of neurons that project to these areas via the lateral perforant pathway (projection neurons) are less well known. We have used retrograde tracing and whole-cell recording in brain slices to compare the membrane and firing properties of medial perforant pathway and lateral perforant pathway projection neurons in layer II of the medial and lateral entorhinal cortex. The properties of medial perforant pathway projection neurons were identical to those reported previously for spiny stellate neurons in the medial entorhinal cortex. In contrast, lateral perforant pathway projection neurons were characterized by a higher input resistance, a lack of time-dependent inward (anomalous) rectification, and a lack of prominent depolarizing spike afterpotentials. Voltage-clamp recordings suggest that the absence of anomalous rectification in lateral perforant pathway projection neurons is due to smaller hyperpolarization activated cation currents in these cells, and the lack of depolarizing afterpotential may be due to smaller low-threshold calcium currents. Persistent sodium current was also smaller in lateral perforant pathway projection neurons, but the difference in persistent sodium current between medial perforant pathway and lateral perforant projection neurons was much less pronounced than the difference in low voltage activated currents. These results underscore the functional differences between the medial entorhinal cortex and lateral entorhinal cortex, and may help to explain the differing abilities of these cortical areas to participate in certain types of network activity.

Endogenous regulators of G protein signaling proteins regulate presynaptic inhibition at rat hippocampal synapses.

Presynaptic inhibition mediated by G protein-coupled receptors (GPCRs) can develop and decay in a few seconds. This time course is too rapid to be accounted for by the intrinsic GTPase activity of Galpha subunits alone. Here, we test the hypothesis that endogenous regulators of G protein signaling (RGS proteins) are required for rapid, brief presynaptic inhibition. Endogenous G protein alpha subunits were uncoupled from GPCRs by treating cultures with pertussis toxin (PTX). Adenoviral expression of mutant PTX-insensitive (PTX-i) Galpha(i1-3) or Galpha(o) subunits rescued adenosine-induced presynaptic inhibition in cultured hippocampal neurons. Expression of double mutant Galpha(i1) or Galpha(o) subunits that were both PTX-insensitive and unable to bind RGS proteins (PTX/RGS-i) also rescued presynaptic inhibition. Presynaptic inhibition mediated by PTX/RGS-i subunits decayed much more slowly after agonist removal than that mediated by PTX-i subunits or native G proteins. The onset of presynaptic inhibition mediated by PTX/RGS-i Galpha(o) was also slower than that mediated by PTX-i Galpha(o). In contrast, the onset of presynaptic inhibition mediated by PTX/RGS-i Galpha(i1) was similar to that mediated by PTX-i Galpha(i1). These results suggest that endogenous RGS proteins regulate the time course of G protein signaling in mammalian central nervous system presynaptic terminals.

Neuronal differentiation of cryopreserved neural progenitor cells derived from mouse embryonic stem cells.

Embryonic stem cells (ES cells) are developmentally pluripotent cells isolated from pre-implantation mammalian embryos. In cell culture ES cells can be easily differentiated to generate cultures of neural progenitors. We present a simple method for the cryopreservation of these ES-derived neural progenitors. Cryopreserved neural progenitor stocks can be thawed, expanded with FGF2, and differentiated into functional neurons. This method will facilitate studies using ES-derived neural progenitor cells as a cell culture model system for neural development and differentiation. It will also aid studies designed to test the ability of these progenitor cells to functionally engraft and repair damaged neural tissue.

GABA(B) receptors couple to potassium and calcium channels on identified lateral perforant pathway projection neurons.

Activation of presynaptic GABA(B) receptors inhibits neurotransmitter release at most cortical synapses, at least in part because of inhibition of voltage-gated calcium channels. One synapse where this is not the case is the lateral perforant pathway synapse onto dentate granule cells in the hippocampus. The current study was conducted to determine whether the neurons that make these synapses express GABA(B) receptors that can couple to ion channels. Perforant pathway projection neurons were labeled by injecting retrograde tracer into the dorsal hippocampus. The GABA(B) receptor agonist baclofen (10 microM) activated inwardly rectifying potassium channels and inhibited currents mediated by voltage-gated calcium channels in retrogradely labeled neurons in layer II of the lateral entorhinal cortex. These effects were reversed by coapplication of the selective GABA(B) receptor antagonist CGP 55845A (1 microM). Equivalent effects were produced by 100 microM adenosine, which inhibits neurotransmitter release at lateral perforant pathway synapses. The effects of baclofen and adenosine on inward currents were largely occlusive. These results suggest that the absence of GABA(B) receptor-mediated presynaptic inhibition at lateral perforant pathway synapses is not simply due to a failure to express these receptors and imply that GABA(B) receptors can either be selectively localized or regulated at terminal versus somatodendritic domains.

Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis.

Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of retrieval. Here we demonstrate that retrieval is limited to sites of prior exocytosis. We tested whether channel distribution can account for the localization of retrieval at exocytotic sites. We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis. Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

Calcium influx is required for endocytotic membrane retrieval.

Cells use endocytotic membrane retrieval to compensate for excess surface membrane after exocytosis. Retrieval is thought to be calcium-dependent, but the source of this calcium is not known. We found that, in sea urchin eggs, endocytotic membrane retrieval required extracellular calcium. Inhibitors of P-type calcium channels-cadmium, omega-conotoxin MVIIC, omega-agatoxin IVA, and omega-agatoxin TK-blocked membrane retrieval; selective inhibitors of N-type and L-type channels did not. Treatment with calcium ionophores overcame agatoxin inhibition in a calcium-dependent manner. Cadmium blocked membrane retrieval when applied during the first 5 minutes after fertilization, the period when the membrane potential is depolarized. We conclude that calcium influx through omega-agatoxin-sensitive channels plays a key role in signaling for endocytotic membrane retrieval.

Inhibition of dendritic calcium influx by activation of G-protein-coupled receptors in the hippocampus.

Gi proteins inhibit voltage-gated calcium channels and activate inwardly rectifying K+ channels in hippocampal pyramidal neurons. The effect of activation of G-protein-coupled receptors on action potential-evoked calcium influx was examined in pyramidal neuron dendrites with optical and extracellular voltage recording. We tested the hypotheses that 1) activation of these receptors would inhibit calcium channels in dendrites; 2) hyperpolarization resulting from K+ channel activation would deinactivate low-threshold, T-type calcium channels on dendrites, increasing calcium influx mediated by these channels; and 3) activation of these receptors would inhibit propagation of action potentials into dendrites, and thus indirectly decrease calcium influx. Activation of adenosine receptors, which couple to Gi proteins, inhibited calcium influx in cell bodies and proximal dendrites without inhibiting action-potential propagation into the proximal dendrites. Inhibition of dendritic calcium influx was not changed in the presence of 50 microM nickel, which preferentially blocks T-type channels, suggesting influx through these channels is not increased by activation of G-proteins. Adenosine inhibited propagation of action potentials into the distal branches of pyramidal neuron dendrites, leading to a three- to fourfold greater inhibition of calcium influx in the distal dendrites than in the soma or proximal dendrites. These results suggest that voltage-gated calcium channels are inhibited in pyramidal neuron dendrites, as they are in cell bodies and terminals and thatG-protein-mediated inhibition of action-potential propagation can contribute substantially to inhibition of dendritic calcium influx.

High-threshold Ca2+ currents in rat hippocampal interneurones and their selective inhibition by activation of GABA(B) receptors.

1. Whole-cell calcium currents were recorded from visually identified inhibitory interneurones located in stratum radiatum (near the border with stratum lacunosum-moleculare) of area. CA1 in rat hippocampal slices. Current-voltage (I-V) relationships in relatively well-clamped neurones showed that inward current activated between -50 and -40 mV (holding potential, -80 mV) and was maximal near -10 mV. Currents showed little inactivation over the course of 85 ms steps, and were completely blocked by removal of Ca2+ or addition of Cd2+. Prominent low-threshold currents were not observed under these conditions. 2. The calcium channels contributing to whole-cell currents in interneurones were examined using selective channel antagonists. The selective N-type calcium channel blocker omega-conotoxin GVIA (omega-CgTX-GVIA; 10 microM) irreversibly blocked 23.2 +/- 2.8% of whole-cell currents. The P/Q-type antagonist omega-agatoxin IVA (omega-Aga-IVA; 1-5 microM) blocked 10.4 +/- 3.3% of whole-cell currents. Block by omega-Aga-IVA was highly variable, ranging from 0 to 30%. The less selective conotoxin, omega-conotoxin MVIIC (omega-CTX-MVIIC; 5 microM) blocked 31.0 +/- 2.7% of whole-cell currents. The selective L-type channel antagonist nifedipine (20 microM) blocked 27.5 +/- 3.5% of whole-cell currents. 3. Whole-cell calcium currents were reversibly inhibited by the selective GABA(B) receptor agonists (+/-)-baclofen or CGP 27492 (1-3 microM; 18.9 +/- 1.4%). This inhibition was reversed or prevented by the selective GABAB receptor antagonist CGP 55845A (1 microM). Inhibition of inward current activated by voltage ramps was voltage dependent, being greatest near -10 mV, and less pronounced at more positive or negative potentials. Inhibition of calcium currents by GABAB receptor agonists was accompanied by an apparent change in the kinetics of whole-cell currents consistent with a slowing of the rate of activation. CGP 27492 depressed calcium currents by 16.1 +/- 1.9% before application of omega-CgTX-GVIA, and by 3.9 +/- 2.0% after application of omega-CgTX-GVIA in the same cells (P < 0.005), consistent with preferential block of N-type calcium channels. 4. Neither adenosine (200 microM) nor the selective mu-opioid receptor agonist Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO; 2 microM) inhibited calcium currents. Similarly, CGP 27492, but not adenosine or DAMGO, induced an outward current (at - 70 mV) consistent with activation of inwardly rectifying potassium channels. 5. These results indicate that hippocampal inhibitory neurones located in stratum radiatum possess multiple calcium channel subtypes, including N-type, L-type, and at least two other types of high-threshold channels. Activation of GABAB receptors (but not adenosine or mu-opioid receptors) preferentially inhibits N-type channels in these neurones. Similar inhibition occurring in the terminals of interneurones could contribute to depression of inhibitory synaptic transmission by activation of GABAB autoreceptors.

Induction and reversal of long-term potentiation by low- and high-intensity theta pattern stimulation.

Reversal of long-term potentiation by low-frequency stimulation is often referred to as depotentiation. However, it is not clear whether depotentiation induced by low-frequency stimulation and long-term depression (LTD) induced by similar stimuli are distinct phenomena. We have performed a series of experiments in area CA1 of rat hippocampal slices in which a single pattern of theta-burst (TB) stimulation (Larson, et al., 1986; Staubli and Lynch, 1987) was found to produce either LTP or reversal of LTP depending on the intensity of the stimulation. TB stimulation (10 trains consisting of 4 pulses at 100 Hz, 200 msec apart) delivered at test pulse-intensity induced LTP. However, the same stimulation delivered at a higher intensity (10 times that of the test pulse, evoking a maximal response) did not induce LTP or depression of control responses, but produced lasting depression of previously potentiated responses. This reversal of LTP (TB depotentiation) was observed when the stimulus was delivered between 0.5 and 110 min after induction of LTP. TB depotentiation was reversible, cumulative and saturable when high-intensity TB trains were delivered in succession. TB depotentiation was also blocked by antagonists at NMDA receptors. Low-frequency (1 Hz) stimulation induced LTD, indicating that responses were not already maximally depressed. In addition, high-intensity TB stimulation did not reverse LTD. These results suggest that depotentiation induced by maximal TB stimulation and LTD induced by low-frequency stimulation are distinct phenomena, yet share some characteristics common to forms synaptic plasticity mediated by NMDA receptor activation.

Temporally distinct mechanisms of use-dependent depression at inhibitory synapses in the rat hippocampus in vitro.

1. Gamma-aminobutyric acid-B (GABAB) autoreceptor-dependent and -independent components of paired-pulse depression (PPD) at inhibitory synapses in area CA3 of the rat hippocampus were studied using whole-cell recording techniques. Inhibitory fibers were activated directly in the presence of the ionotropic glutamate receptor antagonists 6,7-dinitroquinoxaline-2,3,dione (20 microM) and D-2-amino-5-phosphonovalerate (20 microM). 2. When pairs of monosynaptic inhibitory postsynaptic currents (eIPSCs) were evoked with an interstimulus interval of 200 ms, the amplitude of the second response (eIPSC2) was depressed when compared with the first (eIPSC1). The GABAB receptor agonist baclofen (10 microM) depressed both responses, but eIPSC1 was depressed more than eIPSC2, resulting in PPD that was comparatively smaller. Addition of the GABAB receptor antagonist CGP 55845A (1 microM) completely reversed depression of eIPSC1 by baclofen and increased the amplitude of eIPSC2 above the control value, such that PPD in the combination of baclofen and CGP 55845A was equivalent to that in baclofen alone. The ratio eIPSC2/eIPSC1 was 0.64 under control conditions, 0.77 in the presence of baclofen, and 0.79 in the presence of baclofen and CGP 55845A. These results demonstrate the existence of two components of PPD at inhibitory synapses, one that depends on activation of GABAB autoreceptors (GABAB receptor-dependent PPD) and one that does not (GABAB receptor-independent PPD). 3. When the number of inhibitory fibers activated was lowered by decreasing the stimulus intensity, eIPSC2/eIPSC1 was 0.76 under control conditions, 0.75 in the presence of baclofen, and 0.76 in the presence of baclofen and CGP 55845A. These results indicate that GABAB receptor-dependent PPD requires activation of several presynaptic inhibitory neurons, whereas GABAB receptor-independent PPD does not. 4. The time-courses of the GABAB-dependent and -independent components of PPD were compared by varying the interstimulus interval in the absence and presence of CGP 55845A. GABAB-dependent PPD was maximal at an interstimulus interval of 100 ms and was undetectable at 1 s. In contrast, GABAB-independent PPD was maximal at 5 ms and 1 s, was slightly less pronounced at intermediate intervals (50-200 ms), and was present at intervals as long as 5 s. 5. GABAB-independent PPD was not blocked by antagonists at opioid receptors (10 microM naloxone) or muscarinic acetylcholine receptors (10 microM atropine). GABAB-independent PPD could not be accounted for by a decrease in driving force because of Cl- redistribution.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneity in presynaptic regulation of GABA release from hippocampal inhibitory neurons.

Release of GABA from the terminals of hippocampal inhibitory neurons is inhibited by activation of GABAB autoreceptors and mu opioid receptors. However, it is not known whether these presynaptic processes affect all inhibitory synapses equally. We examined the effects of the GABAB receptor agonist baclofen and the mu opioid receptor agonist DAGO on postsynaptic currents evoked by minimal stimulation of inhibitory fibers (meIPSCs) in area CA3. Baclofen reversibly depressed approximately half of the meIPSCs evoked in the stratum pyramidale. The remaining meIPSCs were unaffected despite a coincident depression of spontaneous IPSCs. In contrast, all meIPSCs were depressed by DAGO. In addition, minimal stimulation in the stratum radiatum evoked meIPSCs that were always depressed by baclofen. These results indicate that regulation of GABA release by GABAB autoreceptors occurs at a subset of inhibitory synapses and that GABAB-resistant inhibitory synapses are located on pyramidal neuron somata. Hippocampal inhibitory neurons may be heterogeneous with respect to presynaptic receptor-mediated regulation of GABA release.

Role of HCO3- ions in depolarizing GABAA receptor-mediated responses in pyramidal cells of rat hippocampus.

1. Activation of GABAA receptors can produce both hyperpolarizing and depolarizing responses in CA1 pyramidal cells. The hyperpolarizing response is mediated by a Cl- conductance, but the ionic basis of the depolarizing response is not clear. We compared the GABAA receptor-mediated depolarizations induced by synaptically released gamma-aminobutyric acid [GABA; depolarizing inhibitory postsynaptic potentials (dIPSPs)] with those produced by exogenous GABA (depolarizing GABA responses). Short trains of high-frequency (200 Hz) stimuli were used to generate dIPSPs. We found that dIPSPs generated by trains of stimuli and depolarizing responses to exogenous GABA were accompanied by a conductance increase and had a similar reversal potential, indicating a similar ionic basis for both responses. 2. We wished to determine whether an HCO3- current contributed to the GABAA-mediated depolarizations. We found that dIPSPs and depolarizing GABA responses were sensitive to perfusion with HCO3(-)-free medium. Interpretation of these data was complicated by the mixed nature of the responses: dIPSPs were invariably accompanied by conventional, Cl(-)-mediated fast hyperpolarizing IPSPs (fIPSPs), and response to exogenous GABA usually consisted of biphasic hyperpolarizing and depolarizing responses. However, it was sometimes possible to elicit responses to GABA that appeared purely depolarizing (monophasic depolarizing GABA responses). 3. We analyzed monophasic depolarizing GABA responses and found no change in reversal potential when slices were perfused with HCO(3-)-free medium. We also made whole-cell recordings from CA1 pyramidal cells, attempting to reduce [HCO3-]i, and compared the reversal potential for monophasic depolarizing GABA responses with similar responses recorded with fine intracellular microelectrodes. We found no difference in reversal potential. We also examined effects of the carbonic anhydrase inhibitor acetazolamide (ACTZ) on depolarizing GABA responses. ACTZ reduced these responses but did not change their reversal potential. 4. Effects of HCO(3-)-free medium were not specific to GABAA receptor-mediated responses. GABAB receptor-mediated slow IPSPs (sIPSPs) were also reduced, as were excitatory postsynaptic potentials (EPSPs). Analyses of field potentials and spontaneous fIPSPs suggested a decrease in presynaptic excitability during perfusion with HCO(3-)-free medium. In addition, pyramidal cells showed decreased input resistance when perfused with HCO(3-)-free medium. 5. The sensitivity of GABAA receptor-mediated depolarizations to HCO(3-)-free medium can be explained by a decrease in presynaptic excitability and an increased resting conductance in postsynaptic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Discrimination of post- and presynaptic GABAB receptor-mediated responses by tetrahydroaminoacridine in area CA3 of the rat hippocampus.

1. The effects of the K+ channel blocker 9-amino-1,2,3,4-tetrahydroacridine (THA) on the actions of baclofen and gamma-aminobutyric acid (GABA) at post- and presynaptic GABAB receptors were studied with whole-cell voltage-clamp recording in area CA3 of rat hippocampal slices. 2. The effect of THA on postsynaptic GABAB receptor-mediated responses was studied in neurons perfused internally with potassium gluconate and guanosine triphosphate (GTP). At a holding potential of -70 mV, the GABAB receptor agonist (+/-)-baclofen (30 microM) induced an outward current and increased membrane conductance. In the presence of the excitatory amino acid receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and (+/-)-2-amino-5-phosphonovalerate (APV), stimulation in stratum pyramidale or proximal stratum radiatum evoked GABAA receptor-mediated, fast monosynaptic inhibitory postsynaptic currents (IPSCs) and GABAB receptor-mediated, late monosynaptic IPSCs. THA (0.3 mM) blocked the baclofen-induced current and conductance increase and GABAB receptor-mediated IPSCs. 3. The effect of THA on presynaptic GABAB receptor-mediated responses was studied in neurons perfused internally with Cs+ and lidocaine N-ethyl bromide (QX-314), which blocked post-synaptic GABAB receptor-mediated responses. Stimulation in the presence of DNQX and APV evoked GABAA receptor-mediated IPSCs; when pairs of stimuli were delivered 200 ms apart the second IPSC was depressed. Baclofen reversibly depressed IPSCs, and partially occluded paired-pulse depression of IPSCs. The GABAB receptor antagonist CGP 35348 (0.5-1.0 mM) reversed baclofen-induced depression of IPSCs and partially blocked paired-pulse depression. Baclofen-induced and paired-pulse depression of IPSCs were not by affected by THA (0.3 mM). 4. Baclofen reversibly decreased the amplitude and frequency of spontaneous monosynaptic IPSCs (sIPSCs). Depression of sIPSCs by baclofen was unchanged by THA. 5. These results indicate that THA blocks the actions of baclofen and GABA at post- but not presynaptic GABAB receptors. We conclude that post- and presynaptic GABAB receptors in area CA3 of the rat hippocampus couple to different effector mechanisms; postsynaptic GABAB receptors activate THA-sensitive K+ channels, and presynaptic GABAB receptors decrease neurotransmitter release through a THA-insensitive mechanism.

The actions of 3-aminopropanephosphinic acid at GABAB receptors in rat hippocampus.

The actions of 3-aminopropanephosphinic acid (APPA) were examined using whole-cell patch-clamp recording in rat hippocampal slice. In recordings from neurons in subfield CA1 of slices from young (2-4 weeks) and adult (greater than 2 month) rats, APPA (0.5-50 microM) produced membrane hyperpolarization and outward current under voltage-clamp. APPA also inhibited excitatory postsynaptic potentials with an IC50 of 2.3 microM, and reduced inhibitory postsynaptic potentials at concentrations from 0.1 to 1 microM. The hyperpolarizing and synaptic depressant effects of APPA were reduced by 2-OH-saclofen an antagonist at the B-type receptor for the neurotransmitter gamma-aminobutyric acid (GABA). In this preparation APPA exhibited potencies similar to those previously reported for the GABAB receptor agonist baclofen. APPA was much less effective in inhibiting synaptic transmission measured using field potential recordings. The observations made with whole-cell patch-clamp recording indicate that in hippocampus APPA acts as a potent agonist at presynaptic GABAB receptors associated with both excitatory and inhibitory synapses, and also activates postsynaptic GABAB receptors.

Induction of giant depolarizing potentials by zinc in area CA1 of the rat hippocampus does not result from block of GABAB receptors.

The possibility that zinc (Zn2+) induces giant depolarizing potentials (GDPs) by blocking pre- and postsynaptic gamma-aminobutyric acidB (GABAB) receptors in area CA1 of rat hippocampal slices was investigated. Monosynaptic GABAA receptor-mediated fast and GABAB receptor-mediated late inhibitory postsynaptic potentials (IPSPs) were evoked in the presence of the excitatory amino acid (EAA) receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D,L-amino-5-phosphonovalerate (APV). Addition of Zn2+ (0.3 mM) resulted in the appearance of long-lasting GDPs which obscured monosynaptic late IPSPs. The GABAA receptor antagonist bicuculline methiodide (BMI; 30 microM) blocked fast monosynaptic IPSPs and GDPs, revealing a monosynaptic late IPSP that was prolonged in the presence of Zn2+ and blocked by the GABAB receptor antagonist CGP 35,348 (100 microM). The selective GABAB receptor agonist baclofen (10 microM) depressed monosynaptic IPSPs and population excitatory postsynaptic potentials (pEPSPs) by acting at presynaptic GABAB receptors. Depression of synaptic potentials by baclofen was unaffected by Zn2+. These results suggest that induction of GDPs in area CA1 does not result from an action of Zn2+ at GABAB receptors. We suggest instead that Zn2+ induces GDPs by inducing synchronized discharge of GABAergic interneurons.

Hyperpolarizing and depolarizing GABAA receptor-mediated dendritic inhibition in area CA1 of the rat hippocampus.

1. gamma-Aminobutyric acidA (GABAA) receptor-mediated inhibition of pyramidal neuron dendrites was studied in area CA1 of the rat hippocampal slice preparation with the use of intracellular and extracellular recording and one-dimensional current source-density (CSD) analysis. 2. Electrical stimulation of Schaffer collateral/commissural fibers evoked monosynaptic excitatory postsynaptic potentials (EPSPs) and population EPSPs, which were followed by biphasic inhibitory postsynaptic potentials (IPSPs). In the presence of the excitatory amino acid receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D,L-2-amino-5-phosphonovalerate (APV), stimulation in stratum radiatum evoked monosynaptic fast, GABAA and late, GABAB receptor-mediated IPSPs and fast and late positive field potentials recorded in s. radiatum. 3. Fast monosynaptic IPSPs and fast positive field potentials evoked in the presence of DNQX and APV were reversibly abolished by the GABAA receptor antagonist bicuculline methiodide (BMI; 30 microM) and were not changed by the GABAB receptor antagonist P-[3-aminopropyl]-P-diethoxymethylphosphinic acid (CGP 35,348; 0.1-1.0 mM). CGP 35,348 (0.1 mM) reversibly blocked late monosynaptic IPSPs and late positive field potentials. These results suggest that fast field potentials are GABAA receptor-mediated population IPSPs (GABAA, fast pIPSPs) and that late field potentials are GABAB receptor-mediated population IPSPs (GABAB, late pIPSPs). 4. Fast pIPSPs were reversibly abolished when the extracellular Cl- concentration [( Cl-]o) was reduced from 132 to 26 mM in parallel with a depolarizing shift in the reversal potential of fast IPSPs. Paired or repetitive stimulation in s. radiatum reversibly depressed fast pIPSPs and fast IPSPs. Paired-pulse depression of fast pIPSPs was reversibly antagonized by CGP 35,348 (0.4-0.8 mM). 5. Laminar analysis of s. radiatum-evoked fast pIPSPs and one-dimensional CSD analysis revealed active current sources in s. radiatum and passive current sinks in s. oriens and s. lacunosum moleculare. S. radiatum sources were abolished by pressure application of BMI in s. radiatum but not in s. oriens. Stimulation in s. oriens, s. pyramidale, or s. lacunosum moleculare evoked GABAA current sources horizontal to the stimulation site. Changes in the dendritic location of inhibitory current with changes in stimulus location paralleled changes in the distribution of excitatory current. 6. In the presence of 4-aminopyridine (50-100 microM), DNQX and APV long-lasting depolarizing GABAA receptor-mediated responses (LLDs) occurred spontaneously or could be evoked. Current sinks associated with s. radiatum-evoked LLDs were located in the same dendritic area as sources associated with hyperpolarizing fast IPSPs.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholinergic disinhibition in area CA1 of the rat hippocampus is not mediated by receptors located on inhibitory neurons.

We studied the effects of carbamylcholine (carbachol; CCh) on monosynaptic inhibitory postsynaptic potentials (IPSPs) evoked in the presence of the excitatory amino acid receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D,L-2-amino-5-phosphonovalerate (APV). CCh (30 microM) blocked late afterhyperpolarizations but did not depress GABAA receptor-mediated fast monosynaptic IPSPs or GABAB receptor-mediated late monosynaptic IPSPs. In the presence of CCh the GABAB receptor agonist (+/- )-baclofen (2 microM) reversibly hyperpolarized pyramidal neurons and depressed monosynaptic IPSPs as under control conditions. Phorbol-12,13-diacetate (PDAc; 10 microM) increased fast and depressed late monosynaptic IPSPs, and prevented depression of IPSPs by baclofen. These results suggest that cholinergic disinhibition in area CA1 of the hippocampus results from decreased synaptic excitation of inhibitory neurons.

Baclofen-induced disinhibition in area CA1 of rat hippocampus is resistant to extracellular Ba2+.

The mechanism of disinhibition produced by (+/-)-baclofen was studied using intracellular recording in area CA1 of rat hippocampal slices. Baclofen reversibly depressed monosynaptic IPSPs evoked by direct activation of interneurons in the presence of the excitatory amino acid receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D,L-2-amino-5-phosphonovalerate (APV). Ba2+ prevented baclofen-induced hyperpolarization of pyramidal neurons but not depression of monosynaptic IPSPs by baclofen. Baclofen reversibly depressed monosynaptic IPSPs when applied close to the recording site, but was ineffective when applied close to the stimulating site in stratum radiatum. These results suggest that baclofen disinhibits pyramidal neurons in area CA1 of the rat hippocampus by activating receptors on the terminals of inhibitory neurons that are coupled to a Ba(2+)-insensitive effector mechanism.