Thomas S. Otis

Bridging the cleft at GABA synapses in the brain

A fragile balance between excitation and inhibition maintains the normal functioning of the CNS. The dominant inhibitory neurotransmitter of the mammalian brain is GABA, which acts mainly through GABAA and GABAB receptors. Small changes in GABA-mediated inhibition can alter neuronal excitability profoundly and, therefore, a wide range of compounds that clearly modify GABAA-receptor function are used clinically as anesthetics or for the treatment of various nervous system disorders. Recent findings have started to unravel the operation of central GABA synapses where inhibitory events appear to result from the synchronous opening of only tens of GABAA receptors activated by a saturating concentration of GABA. Such properties of GABA synapses impose certain constraints on the physiological and pharmacological modulation of inhibition in the brain.

Zinc-Induced Collapse of Augmented Inhibition by GABA in a Temporal Lobe Epilepsy Model

In the kindling model of temporal lobe epilepsy, several physiological indicators of inhibition by γ-aminobutyric acid (GABA) in the hippocampal dentate gyrus are consistent with an augmented, rather than a diminished, inhibition. In brain slices obtained from epileptic (kindled) rats, the excitatory drive onto inhibitory interneurons was increased and was paralleled by a reduction in the presynaptic autoinhibition of GABA release. This augmented inhibition was sensitive to zinc most likely after a molecular reorganization of GABAA receptor subunits. Consequently, during seizures, inhibition by GABA may be diminished by the zinc released from aberrantly sprouted mossy fiber terminals of granule cells, which are found in many experimental models of epilepsy and in human temporal lobe epilepsy.

Alcohol-induced motor impairment caused by increased extrasynaptic GABAA receptor activity

Neuronal mechanisms underlying alcohol intoxication are unclear. We find that alcohol impairs motor coordination by enhancing tonic inhibition mediated by a specific subtype of extrasynaptic GABAA receptor (GABAR), α6β3δ, expressed exclusively in cerebellar granule cells. In recombinant studies, we characterize a naturally occurring single-nucleotide polymorphism that causes a single amino acid change (R100Q) in α6 (encoded in rats by the Gabra6 gene). We show that this change selectively increases alcohol sensitivity of α6β3δ GABARs. Behavioral and electrophysiological comparisons of Gabra6100R/100R and Gabra6100Q/100Q rats strongly suggest that alcohol impairs motor coordination by enhancing granule cell tonic inhibition. These findings identify extrasynaptic GABARs as critical targets underlying low-dose alcohol intoxication and demonstrate that subtle changes in tonic inhibition in one class of neurons can alter behavior.

Characterization of synaptically elicited GABAB responses using patch-clamp recordings in rat hippocampal slices.

1. Tight‐seal, whole‐cell voltage clamp recording techniques were used to characterize monosynaptically evoked GABAB currents in adult rat brain slices maintained at 34‐35 degrees C. Responses were recorded from granule cells of the dentate gyrus following the blockade of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX)‐, D‐2‐amino‐5‐phosphonovaleric acid (D‐AP5)‐ and picrotoxin‐sensitive fast synaptic transmission, so that the remaining synaptic currents could be studied in isolation. 2. Under these conditions, stimulation in the molecular layer elicited a slow outward current which was blocked by the selective GABAB antagonist CGP 35348 in a concentration‐dependent manner (200‐800 microM). This current was absent in recordings made with pipettes containing 10‐15 mM of the lidocaine derivative QX‐314 or when caesium was substituted for K+. 3. Increasing the [K+]o e‐fold (from 2.5 to 6.8 mM) shifted the reversal potential of the GABAB current from ‐97.9 to ‐73.2 mV, as predicted by the Nernst equation. Peak conductance was constant, but in 6.8 mM [K+]o at voltages hyperpolarized to EK (equilibrium potential for potassium), a small outward rectification was evident. 4. The time course of the current could be described by fourth‐power exponential activation kinetics with double exponential inactivation. At 34‐35 degrees C, the average activation time constant (tau m) was 45.2 ms, while the two inactivation time constants (tau h1 and tau h2) were 110.2 and 516.2 ms, with corresponding weighting factors (wh1 and wh2) of 0.84 and 0.16, respectively. The Q10 (temperature coefficient) values for these time constants were between 1.82 and 2.31. Neither tau m, nor tau h1 and tau h2 were voltage dependent in the range from ‐45 to ‐95 mV. 5. Paired‐pulse depression of the GABAB current was studied by giving identical conditioning and test stimuli over a wide range (50‐5000 ms) of interstimulus intervals (ISIs). The maximal depression (48%) occurred at 200 ms ISI, and the depression lasted for over 5 s. The magnitude of paired‐pulse depression was not dependent on the postsynaptic membrane potential. 6. Application of the competitive antagonist CGP 35348 such that the peak current was diminished by approximately 50% had no effect on the activation or inactivation kinetics of the current. Similarly, during paired‐pulse depression the kinetics of test currents were identical to those of conditioning currents. These findings support the hypothesis that the mechanism responsible for paired‐pulse depression involves a reduction in neurotransmitter release without postsynaptic alterations in K+ channel activation/inactivation kinetics.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuronal Glutamate Transporters Control Activation of Postsynaptic Metabotropic Glutamate Receptors and Influence Cerebellar Long-Term Depression

Neuronal and glial isoforms of glutamate transporters show distinct distributions on membranes surrounding excitatory synapses, but specific roles for transporter subtypes remain unidentified. At parallel fiber (PF) synapses in cerebellum, neuronal glutamate transporters and metabotropic glutamate receptors (mGluRs) have overlapping postsynaptic distributions suggesting that postsynaptic transporters selectively regulate mGluR activation. We examined interactions between transporters and mGluRs by evoking mGluR-mediated excitatory postsynaptic currents (mGluR EPSCs) in slices of rat cerebellum. Selective inhibition of postsynaptic transporters enhanced mGluR EPSCs greater than 3-fold. Moreover, impairing glutamate uptake facilitated mGluR-dependent long-term depression at PF synapses. Our results demonstrate that uniquely positioned glutamate transporters strongly influence mGluR activation at cerebellar PF synapses. Postsynaptic glutamate uptake may serve as a general mechanism for regulating mGluR-initiated synaptic depression.

Perpetual inhibitory activity in mammalian brain slices generated by spontaneous GABA release.

Miniature spontaneous inhibitory postsynaptic currents (sIPSCs) mediated by GABAA receptors were recorded using whole-cell patch clamp recordings in rat brain slices maintained in vitro at 34 +/- 1 degree C. We have found that firing of action potentials by principal neurons or by GABAergic interneurons is not necessary to the generation of sIPSCs since they persist in the presence of 1-5 microM tetrodotoxin (TTX). The average frequency of the discrete sIPSCs exhibits a large cell-to-cell variability and is between 5-15 Hz. The amplitudes of the sIPSCs depend on the difference between the membrane potential and the equilibrium potential for Cl- (ECl). Generally, 70-80 mV away from ECl, sIPSCs have a mean amplitude of 30-80 pA (i.e. peak conductance of 400-1000 pS) with an average decay time constant of 5.8 ms. Accordingly, unitary single sIPSCs arise from the simultaneous activation of no more than 20 GABAA receptor/channels. The perpetual barrage of spontaneous GABAergic activity is very likely to be a critical factor in the regulation of neuronal excitability and the mechanism of action of several neuroactive compounds.

Holographic photolysis of caged neurotransmitters.

Stimulation of light-sensitive chemical probes has become a powerful tool for the study of dynamic signaling processes in living tissue. Classically, this approach has been constrained by limitations of lens-based and point-scanning illumination systems. Here we describe a microscope configuration that incorporates a nematic liquid-crystal spatial light modulator to generate holographic patterns of illumination. This microscope can produce illumination spots of variable size and number, and in patterns shaped to precisely match user-defined elements in a specimen. Using holographic illumination to photolyze caged glutamate in brain slices, we show that shaped excitation on segments of neuronal dendrites and simultaneous, multispot excitation of different dendrites enables precise spatial and rapid temporal control of glutamate receptor activation. By allowing the excitation volume shape to be tailored precisely, the holographic microscope provides an extremely flexible method for activation of various photosensitive proteins and small molecules.

Photochemical control of endogenous ion channels and cellular excitability

Light-activated ion channels provide a precise and noninvasive optical means for controlling action potential firing, but the genes encoding these channels must first be delivered and expressed in target cells. Here we describe a method for bestowing light sensitivity onto endogenous ion channels that does not rely on exogenous gene expression. The method uses a synthetic photoisomerizable small molecule, or photoswitchable affinity label (PAL), that specifically targets K+ channels. PALs contain a reactive electrophile, enabling covalent attachment of the photoswitch to naturally occurring nucleophiles in K+ channels. Ion flow through PAL-modified channels is turned on or off by photoisomerizing PAL with different wavelengths of light. We showed that PAL treatment confers light sensitivity onto endogenous K+ channels in isolated rat neurons and in intact neural structures from rat and leech, allowing rapid optical regulation of excitability without genetic modification.

AMPA receptors with high Ca2+ permeability mediate synaptic transmission in the avian auditory pathway.

1. The permeability of AMPA (alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐ isoxazolepropionate) receptors in the chick cochlear nucleus, the nucleus magnocellularis (nMAG), was examined by measuring the shift in reversal potential (Erev) of current through glutamate or neurotransmitter‐gated channels in solutions of different ionic composition. 2. Outwardly rectifying glutamate‐activated currents in outside‐out membrane patches showed rapid activation and desensitization. The Erev of glutamate‐evoked current in zero sodium solutions was dependent on the extracellular Ca2+ concentration. The relation between Erev and Ca2+ ionic activities could be described by the Goldman‐Hodgkin‐Katz equation with a permeability ratio, PCa/PCs, of 3.3. The PNa/PCs was estimated as 0.66, indicating a PCa/PNa of 5. 3. Evoked excitatory postsynaptic currents (EPSCs) could be recorded during local perfusion of the auditory nerve‐nMAG synapse with isotonic Ca2+. The Erev of the EPSC shifted in the positive direction in high‐Ca2+ solution as predicted from the preceding analysis. The fraction of current carried by Ca2+ during the AMPA receptor EPSC was estimated as 18%.

Molecular basis for the high THIP/gaboxadol sensitivity of extrasynaptic GABAA receptors

Extrasynaptic GABA(A) receptors (eGABARs) allow ambient GABA to tonically regulate neuronal excitability and are implicated as targets for ethanol and anesthetics. These receptors are thought to be heteropentameric proteins made up of two α subunits-either α4 or α6-two β2 or β3 subunits, and one δ subunit. The GABA analog 4,5,6,7-tetrahydroisoxazolo (5,4-c)pyridin-3(-ol) (THIP) has been proposed as a selective ligand for eGABARs. Behavioral and in vitro studies suggest that eGABARs have nanomolar affinity for THIP; however, all published studies on recombinant versions of eGABARs report micromolar affinities. Here, we examine THIP sensitivity of native eGABARs on cerebellar neurons and on reconstituted GABARs in heterologous systems. Concentration-response data for THIP, obtained from cerebellar granule cells and molecular layer interneurons in wild-type and δ subunit knockout slices, confirm that submicromolar THIP sensitivity requires δ subunits. In recombinant experiments, we find that δ subunit coexpression leads to receptors activated by nanomolar THIP concentrations (EC(50) of 30-50 nM for α4β3δ and α6β3δ), a sensitivity almost 1,000-fold higher than receptors formed by α4/6 and β3 subunits. In contrast, γ2 subunit expression significantly reduces THIP sensitivity. Even when δ subunit cDNA or cRNA was supplied in excess, high- and low-sensitivity THIP responses were often apparent, indicative of variable mixtures of low-affinity αβ and high-affinity αβδ receptors. We conclude that δ subunit incorporation into GABARs leads to a dramatic increase in THIP sensitivity, a defining feature that accounts for the unique behavioral and neurophysiological properties of THIP.