Gary L. Westbrook

The physiology of excitatory amino acids in the vertebrate central nervous system

Abbreviations 198

Permeation and block of N-methyl-D-aspartic acid receptor channels by divalent cations in mouse cultured central neurones.

1. Spinal cord and hippocampal neurones in cell culture were voltage clamped using the tight‐seal, whole‐cell recording technique. The concentration of sodium and a series of divalent cations in the extracellular media was varied to study permeation through excitatory amino acid receptor channels activated by the selective agonists N‐methyl‐D‐aspartic acid (NMDA), kainic acid and quisqualic acid. 2. On raising the extracellular calcium concentration, with [Na+]o held constant at 105 mM, the reversal potential of responses to NMDA shifted in the depolarizing direction. This shift was adequately described by the extended constant‐field equation over the range 0.3‐50 mM‐calcium. Using ionic activity coefficients we calculate a value of PCa/PNa = 10.6. Under the same experimental conditions the reversal potential of responses to kainic and quisqualic acids was much less affected by raising the calcium concentration, such that PCa/PNa = 0.15. A depolarizing shift of the NMDA reversal potential was also recorded during application of 20 mM‐barium, strontium or manganese, suggesting permeation of these ions. The permeability sequence was Ca2+ greater than Ba2+ greater than Sr2+ much greater than Mn2+. No depolarizing shift of the NMDA reversal potential occurred during application of 20 mM‐cobalt, magnesium or nickel. 3. In experiments in which the extracellular Na+ concentration was varied the extended constant‐field equation was adequate in predicting shifts of the NMDA reversal potential recorded on varying [Na+]o over the range 50‐150 mM, but failed to accurately predict the reversal potential of responses to NMDA with 10 mM‐[Ca2+]o and only 10 or 20 mM‐[Na+]o. These results imply an apparent increase in PCa/PNa on lowering [Na+]o and may result from interaction of permeant ions within the channel. 4. Barium and to a lesser extent calcium, but not strontium (all 20 mM), reduced the slope conductance of responses to NMDA recorded within +/‐ 15 mV of the reversal potential; over this limited range of membrane potential the current‐voltage relationship remained linear in the presence of each of these ions. In contrast manganese produced a strong, voltage‐dependent block of responses to NMDA, similar to that produced by magnesium, such that even close to the reversal potential the NMDA current‐voltage relationship was highly non‐linear. Thus manganese both permeates and blocks the NMDA receptor channel. 5. Raising the extracellular calcium concentration, from 0.1 to 5 mM, had two effects on the conductance mechanism activated by NMDA.(ABSTRACT TRUNCATED AT 400 WORDS)

Desensitized states prolong GABAA channel responses to brief agonist pulses

We studied the role of desensitization at inhibitory synapses by comparing nonequilibrium GABAA channel gating with inhibitory postsynaptic currents (IPSCs). Currents activated by brief pulses of 1-10 mM GABA to outside-out patches from cultured hippocampal neurons mimicked GABA-mediated IPSCs. Although the average open time of single GABAA channels following brief pulses was less than 10 ms, channels entered long (tau = 38-69 ms) closed states and subsequently reopened. Movement through these states resulted in paired-pulse desensitization. The time required for deactivation after removal of agonist also increased in proportion to the extent of desensitization. These results suggest that visits to desensitized states buffer the channel in bound conformations and underlie the expression of long-lasting components of the IPSC. Reopening after GABAA receptor desensitization may thus enhance inhibitory synaptic transmission by prolonging the response to a brief synaptic GABA transient.

Calcium-induced actin depolymerization reduces NMDA channel activity

Actin filaments are highly concentrated in postsynaptic densities at central excitatory synapses, but their influence on postsynaptic glutamate receptors is unknown. We tested whether actin depolymerization influences NMDA channel activity in whole-cell recording on cultured hippocampal neurons. The ATP- and calcium-dependent rundown of NMDA channels was prevented when actin depolymerization was blocked by phalloidin. Rundown of AMPA/kainate receptors was unaffected by phalloidin. Cytochalasins, which enhance actin-ATP hydrolysis, induced NMDA channel rundown, whereas taxol or colchicine, which stabilize or disrupt microtubule assembly, had no effect. Protease inhibitors also had no effect. Our results suggest that calcium and ATP can influence NMDA channel activity by altering the state of actin polymerization and are consistent with a proposed model in which actin filaments compartmentalize a channel regulatory protein.

The impact of receptor desensitization on fast synaptic transmission.

The role of desensitization of ligand-gated channels at fast chemical synapses has been difficult to establish. Densensitization has been studied traditionally with prolonged agonist exposure, whereas the duration of free neurotransmitter in the synaptic cleft is relatively brief. Studies of acetylcholine-, glutamate- and GABA-gated channels using rapid agonist application now provide a means to assess the effects of densensitization in shaping synaptic responses and in influencing neuronal excitability. These data reveal several strikingly different patterns by which the receptor-specific kinetics of densensitization can determine the size, timecourse and frequency of transmitted signals. Densensitization is thus a surprisingly versatile mechanism for shaping synaptic transmission.

A voltage‐clamp analysis of inward (anomalous) rectification in mouse spinal sensory ganglion neurones.

Mouse embryo dorsal root ganglion neurones were grown in tissue culture and voltage‐clamped with two micro‐electrodes. Hyperpolarizing voltage commands from holding potentials of ‐50 to ‐60 mV evoked slow inward current relaxations which were followed by inward tail currents on repolarization to the holding potential. These relaxations are due to the presence of a time‐ and voltage‐dependent conductance provisionally termed Gh. Gh activates over the membrane potential range ‐60 to ‐120 mV. The presence of Gh causes time‐dependent rectification in the current‐voltage relationship measured between ‐60 and ‐120 mV. Gh does not inactivate within this range and thus generates a steady inward current at hyperpolarized membrane potentials. The current carried by Gh increases when the extracellular K+ concentration is raised, and is greatly reduced in Na+‐free solutions. Current‐voltage plots show considerably less inward rectification in Na+‐free solution; conversely inward rectification is markedly enhanced when the extracellular K+ concentration is raised. The reversal potential of Ih is close to ‐30 mV in media of physiological composition. Tail‐current measurement suggests that Ih is a mixed Na+‐K+ current. Low concentrations of Cs+ reversibly block Ih and produce outward rectification in the steady‐state current‐voltage relationship recorded between membrane potentials of ‐60 and ‐120 mV. Cs+ also reversibly abolishes the sag and depolarizing overshoot that distort hyperpolarizing electrotonic potentials recorded in current‐clamp experiments. Impermeant anion substitutes reversibly block Ih; this block is different from that produced by Cs+ or Na+‐free solutions: Cs+ produces outward rectification in the steady‐state current‐voltage relationship recorded over the Ih activation range; in Na+‐free solutions inward rectification, of reduced amplitude, can still be recorded since Ih is a mixed Na+‐K+ current; in anion‐substituted solutions the current‐voltage relationship becomes approximately linear. It is concluded that in dorsal root ganglion neurones anomalous rectification is generated by the time‐and voltage‐dependent current Ih. The possible function of Ih in sensory neurones is discussed.

Dendrodendritic Inhibition in the Olfactory Bulb Is Driven by NMDA Receptors

At many central excitatory synapses, AMPA receptors relay the electrical signal, whereas activation of NMDA receptors is conditional and serves a modulatory function. We show here quite a different role for NMDA receptors at dendrodendritic synapses between mitral and granule cells in the rat olfactory bulb. In whole-cell patch-clamp recordings in bulb slices, stimulation of mitral cells elicited slowly decaying, GABAA receptor-mediated reciprocal IPSCs that reflected prolonged GABA release from granule cells. Although granule cells had a normal complement of AMPA and NMDA receptors, the IPSC was completely blocked by the NMDA receptor antagonistd,l-AP-5, suggesting that NMDA receptor activation is an absolute requirement for dendrodendritic inhibition. The AMPA receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX) had no effect on IPSCs in the absence of extracellular magnesium but modestly reduced IPSCs in 1 mm magnesium, indicating that the primary effect of the AMPA receptor-mediated depolarization was to facilitate the unblocking of NMDA receptors. Granule cell voltage recordings indicated that effective spike stimulation in granule cells depended on the slow NMDA receptor kinetics. Granule cells also showed a pronounced delay between synaptic stimulation and action potential generation, suggesting that their intrinsic membrane properties underlie the ineffectiveness of brief AMPA receptor-mediated EPSPs. NMDA receptors also seem to have a central role in dendrodendritic inhibition in vivo, because intraperitoneal dizocilpine maleate (MK-801) injection in young adult rats resulted in disinhibition of mitral cells as measured by the generation of c-fos mRNA. The unique dependence of dendrodendritic inhibition on slow EPSPs generated by NMDA receptors suggests that olfactory information processing depends on long-lasting reciprocal and lateral inhibition.

Integrins mediate functional pre- and postsynaptic maturation at a hippocampal synapse

Coordinated signalling between presynaptic terminals and their postsynaptic targets is essential for the development and function of central synapses. In addition to diffusible molecules, this bidirectional flow of information could involve direct interactions through cell-adhesion molecules. Here, we show that one class of cell-adhesion molecule, the integrins, are required for the functional maturation of hippocampal synapses in vitro. At immature synapses, a high probability of glutamate release (Pr) was correlated with the expression of postsynaptic NMDA (N-methyl-D-aspartate) receptors containing the NR2B subunit. The activity-dependent reduction in Pr and a switch in the subunit composition of synaptic NMDA receptors was prevented by chronic blockade with peptides containing the integrin-binding site Arg-Gly-Asp (RGD), or by a functional antibody against the β3 integrin subunit. Active synapses, monitored by the uptake of antibodies against the intraluminal domain of synaptotagmin I, also had β3 subunit immunoreactivity. Our results provide evidence that integrin-mediated signalling is essential for the orchestrated maturation of central excitatory synapses.

Slow excitatory postsynaptic currents mediated by N‐methyl‐D‐aspartate receptors on cultured mouse central neurones.

1. Monosynaptic excitatory postsynaptic potentials (EPSPs) evoked between pairs of cultured neurones from either hippocampus or spinal cord were examined using the tight‐seal whole‐cell recording technique. 2. Using the selective N‐methyl‐D‐aspartate (NMDA)‐receptor antagonist, 2‐amino‐5‐phosphonovaleric acid (APV), two components of the EPSP could be resolved in cultures from both brain regions. The APV‐sensitive (slow) component had the same latency, but a much slower time‐to‐peak and longer duration than the APV‐resistant (fast) component. Other NMDA antagonists such as ketamine also selectively blocked the slow component of the EPSP. 3. In Mg2+‐free medium, the dual‐component EPSP had a duration lasting up to 500 ms, greatly exceeding the membrane time constant of the postsynaptic neurone, suggesting that persistent activation of NMDA receptors was responsible for the long duration of the APV‐sensitive component. 4. Under voltage clamp the excitatory postsynaptic currents (EPSCs) also showed fast and slow components, both of which had a reversal potential near 0 mV in physiological saline. The synaptic current could be fitted with a sum of two exponentials with a decay time constant for the slow EPSC near 80 ms. The slow current contributed approximately 50% of the total charge transfer during the EPSC. 5. In Mg2+‐containing medium, the peak of the fast component was voltage insensitive, whereas the synaptic current measured at a latency of 10‐50 ms was voltage dependent with a region of negative slope conductance at membrane potentials hyperpolarized to ‐30 mV. 6. Raising [Ca2+]o from 1 to 20 mM resulted in a shift of the reversal potential of the APV‐sensitive component from near 0 mV to + 10 mV, but the reversal potential of the fast component remained near 0 mV. This suggests that conductances with different ionic permeability underlie the two components of the EPSC and that the slow component is highly permeable to Ca2+ as well as to monovalent cations. 7. Our results demonstrate that two functionally distinct excitatory amino acid receptor channels are simultaneously activated by transmitter release from a single presynaptic neurone. The conductance mechanism underlying the slow component of the EPSP displays the voltage dependence and Ca2+ permeability expected for NMDA‐receptor channels. We suggest that the available conductance generating the slow EPSP may be sufficient, even at low firing rates, to influence excitability on both a short‐term and more long‐lasting basis.

microRNA-132 regulates dendritic growth and arborization of newborn neurons in the adult hippocampus

Newborn neurons in the dentate gyrus of the adult hippocampus rely upon cAMP response element binding protein (CREB) signaling for their differentiation into mature granule cells and their integration into the dentate network. Among its many targets, the transcription factor CREB activates expression of a gene locus that produces two microRNAs, miR-132 and miR-212. In cultured cortical and hippocampal neurons, miR-132 functions downstream from CREB to mediate activity-dependent dendritic growth and spine formation in response to a variety of signaling pathways. To investigate whether miR-132 and/or miR-212 contribute to the maturation of dendrites in newborn neurons in the adult hippocampus, we inserted LoxP sites surrounding the miR-212/132 locus and specifically targeted its deletion by stereotactically injecting a retrovirus expressing Cre recombinase. Deletion of the miR-212/132 locus caused a dramatic decrease in dendrite length, arborization, and spine density. The miR-212/132 locus may express up to four distinct microRNAs, miR-132 and -212 and their reverse strands miR-132* and -212*. Using ratiometric microRNA sensors, we determined that miR-132 is the predominantly active product in hippocampal neurons. We conclude that miR-132 is required for normal dendrite maturation in newborn neurons in the adult hippocampus and suggest that this microRNA also may participate in other examples of CREB-mediated signaling.