Subunit-specific redox modulation of NMDA receptors expressed in Xenopus oocytes.

We have examined the effects of oxidizing and reducing agents on a number of subtypes of N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus oocytes. Oocytes were injected with cRNA for the zeta 1 subunit from mouse to express homomeric receptors or with zeta 1 in combination with either epsilon 1, epsilon 2, epsilon 3 or epsilon 4 subunits to express heteromeric receptors. All heteromeric combinations resulted in receptors that were affected by the redox reagents, dithiothreitol (DTT) and 5-5-dithio-bis-2-nitrobenzoic acid (DTNB). However, the effects on the small currents from homomeric receptors were quite variable. The zeta 1/epsilon 3 combination showed a greater enhancement by DTT than any of the other combinations. All four receptors expressed showed both a component of persistent potentiation and a slowly reversible component. The reversible component was largest for zeta 1/epsilon 3. Additional experiments were done with S-nitrosocysteine (SNOC), a nitric oxide donor that may affect NMDA receptors by oxidation. SNOC had transient effects on the four heteromeric subunit combinations. The different sensitivities of particular subunit combinations may have pharmacological and clinical significance.

Effects of nitroprusside and redox reagents on NMDA receptors expressed in Xenopus oocytes.

We have examined the effects of oxidizing and reducing agents and sodium nitroprusside (SNP) on currents evoked by NMDA (N-methyl-D-aspartate) using the Xenopus oocyte expression system. Oocytes were injected with RNA prepared from either whole rat brain or from the NMDAR1 clone recently isolated from rat brain. Bath application of 1-1000 microM SNP, which releases nitric oxide and ferrocyanide, caused a rapid inhibition of NMDA-evoked current in both preparations. The inhibitory effect reversed spontaneously within 15 min. Kainate responses were not affected by SNP. Exposure to the reducing agent, dithiothreitol (DTT), enhanced NMDA currents; the oxidant, 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), inhibited NMDA responses, as has been observed in other preparations. The site of action of SNP appeared to be different than the DTT/DTNB redox site for several reasons: SNP and DTNB inhibitions were additive at high doses, DTT did not rapidly reverse SNP effects, and SNP and DTT treatments did not show the same susceptibility to block by the NMDA antagonist, aminophosphonovaleric acid (APV). The results demonstrate that modulation of NMDA receptors by SNP is a property of homomeric channels and is retained when the NMDAR1 subunit is expressed in oocytes.

Protein kinase C-mediated enhancement of NMDA currents by metabotropic glutamate receptors in Xenopus oocytes.

1. N-Methyl-D-aspartate (NMDA) receptors were expressed in Xenopus oocytes injected with rat brain RNA. The modulation of NMDA-induced currents was examined by activating protein kinase C (PKC) either directly (using phorbol esters) or indirectly (via metabotropic glutamate agonists). 2. Bath application of the PKC activator, 4-beta-phorbol-12,13-dibutyrate (PDBu) resulted in a two-fold increase in the NMDA-evoked current at all holding potentials examined (-80 to 0 mV). The inactive (alpha) stereoisomer of phorbol ester was ineffective. 3. The increase was observed under conditions that eliminate the oocyte's endogenous calcium-dependent chloride current, which often contributes to the NMDA response in oocytes. 4. The PDBu effect was specific to the NMDA subclass of glutamate receptors in that no increase was observed in the responses to two other glutamate agonists, kainate and AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid). 5. Stimulation of PKC by activation of metabotropic receptors via either quisqualate or trans-ACPD (trans-1-aminocyclopentane-1,3-dicarboxylic acid) also led to an increase in NMDA currents. 6. Both methods of enhancement induced transient effects. PDBu effects lasted 10-45 min, depending upon both dose and length of application. Quisqualate and trans-ACPD effects were shorter, lasting less than 10 min under these conditions of application. 7. Both methods of enhancement were blocked by the PKC inhibitor, staurosporine. In addition, the phorbol ester-induced enhancement of NMDA responses occluded further enhancement by quisqualate. 8. The results suggest a role for metabotropic glutamate receptors in modulation of NMDA-mediated processes.

Trans-ACPD reduces multiple components of synaptic transmission in the rat hippocampus.

Activation of metabotropic quisqualate receptors by trans-ACPD (trans-1-aminocyclopentane-1,3-dicarboxylic acid) caused a reduction in the amplitude of the synaptic response elicited by stimulation of the Schaffer collateral projection and recorded intracellularly from area CA1 in slices of rat hippocampus. Pharmacological agents were used to isolate components of the response mediated by N-methyl-D-aspartate (NMDA) receptors, non-NMDA receptors, and gamma-aminobutyric acid (GABA) receptors. Each of these components was reduced during the trans-ACPD application. These results indicate that one subtype of glutamate receptor may be able to decrease the synaptic efficacy of other subtypes and may provide an important means for balancing the synaptic enhancement processes often studied in the hippocampus.

Activity-induced decrease in early and late inhibitory synaptic conductances in hippocampus.

The use dependence of inhibitory postsynaptic potentials (IPSPs) and their underlying conductances was studied in area CA1 of the hippocampal brain slice preparation, using a two-pulse paradigm in which paired activation of two separate synaptic inputs resulted in changes in the second, or "primed" response. In intracellular current-clamp recordings, the "primed" response, normally triphasic, exhibited a larger, wider excitatory PSP (EPSP) component and greatly reduced or absent IPSP components. Maximal widening occurred when the interval between synaptic stimuli was between 200 and 250 msec. Hyperpolarization of the postsynaptic cell reversed both the early IPSP and the direction of change of the width of the "primed" EPSP response, suggesting that the changes in the "primed" waveform were not due to the addition of an unidentified inward current(s). Furthermore, the reduction of the IPSPs during the "primed" response could not be accounted for by the fact that the membrane potential of the postsynaptic cell was hyperpolarized and therefore closer to IPSP reversal potential. Using single-electrode voltage-clamp techniques, we found that the early inhibitory conductance generally decreased by approximately 50%, with little if any change in reversal potential. The late inhibitory conductance also showed a priming-induced decrease of approximately 95%. Finally, "primed" four-pulse bursts of stimuli induced a larger depolarization in the postsynaptic cell than did unprimed bursts, also with an optimal interval of about 250 msec. We conclude that activation of certain synaptic pathways in the hippocampus results in a temporal window of 200-300 msec during which inhibitory synaptic activity is depressed and excitatory synaptic transmission is maximally effective, especially if the excitation occurs in short bursts. Such a mechanism would endow the inhibitory synaptic components of the hippocampus with a "gating" function to control long-term synaptic modification at excitatory synapses in the same region.

Intracellular analysis of inherent and synaptic activity in hypothalamic thermosensitive neurones in the rat.

1. Intracellular neuronal activity was recorded in rat preoptic-anterior hypothalamic tissue slices. Thirty neurones were classified as warm sensitive, cold sensitive or temperature insensitive, based on their firing rate response to temperature changes. Seventy-seven per cent of the neurones were temperature insensitive, which included both spontaneously firing and silent neurones. Of all neurones, 10% were warm sensitive and 13% were cold sensitive. 2. Silent temperature-insensitive neurones had lower input resistances (126 +/- 21 M omega) than thermosensitive neurones (179 +/- 24 M omega). Regardless of neuronal type, however, resistance was inversely related to temperature. 3. Warm-sensitive neurones were characterized by a slow, depolarizing pre-potential, whose rate of rise was temperature dependent. This depolarizing potential disappeared during current-induced hyperpolarization, suggesting that intrinsic mechanisms are responsible for neuronal warm sensitivity. 4. Spike activity in cold-sensitive neurones correlated with putative excitatory and inhibitory postsynaptic potentials, whose frequency was thermosensitive. This suggests that cold sensitivity in these neurones depends on synaptic input from nearby neurones. 5. Like cold-sensitive neurones, action potentials of temperature-insensitive neurones often were preceded by short duration (less than 20 ms), rapidly rising pre-potentials, whose rates of rise were not affected by temperature. In some temperature-insensitive neurones, depolarizing current injection increased both firing rate (by 5-8 impulses s-1) and warm sensitivity, with pre-potentials having temperature-dependent rates of rise. We suggest that temperature-insensitive neurones employ two opposing, thermally dependent mechanisms: a voltage-dependent depolarizing conductance and a hyperpolarizing sodium-potassium pump.

Apparent desensitization of NMDA responses in Xenopus oocytes involves calcium-dependent chloride current.

N-Methyl-D-aspartate (NMDA) receptors were expressed and studied in Xenopus oocytes injected with rat brain RNA. NMDA application elicits a rapid inward current that decays in several seconds to a relatively stable level. This decay is reportedly due to desensitization. However, we found the early transient component could be evoked more than once during a single application of NMDA, suggesting that the receptor did not actually desensitize. Removal of external Ca2+, replacement of Ca2+ with Ba2+, or intracellular injection of EGTA abolished the transient component. Furthermore, a variety of Cl- channel blockers nearly eliminated the transient component and inhibited the plateau current as well. We propose that a significant portion of the NMDA current recorded in oocytes is carried by a transient inward Cl- current triggered by Ca2+ influx through the NMDA receptor/channel.

Activity-induced depression of synaptic inhibition during LTP-inducing patterned stimulation.

In the hippocampus, patterns of electrical stimulation that approximate bursting neuronal activity during theta rhythm have been shown to induce a long-term potentiation (LTP) of excitatory synapses. In this study, a single subthreshold stimulus applied to one set of Schaffer/commissural fibers affected the response to a second stimulation delivered 200 ms later to a separate set of Schaffer/commissural fibers in the CA1 field of rat hippocampal slices. The first (priming) stimulus caused a prolongation of the synaptic response elicited by the second (primed) stimulus. In addition, the priming stimulation facilitated the induction of LTP by bursts of stimulation (4 pulses at 100 Hz) of the second afferent pathway. Analysis of the shape of the synaptic responses indicates that the prolongation is due to the removal of an inhibitory component rather than the addition of a novel excitatory component. Blockade of GABAA-ergic transmission with picrotoxin mimicked the priming effect in that it also widened synaptic responses and facilitated burst-induced LTP. We suggest that these patterns of stimulation result in a transient loss of inhibition during the primed stimulation. This, in turn, brings about a prolongation of the synaptic response that allows short bursts of excitatory synaptic activity to depolarize postsynaptic cells sufficiently to trigger LTP.

Hebbian synapses in hippocampus.

A combination of current- and voltage-clamp techniques applied to hippocampal brain slices was used to evaluate the role of postsynaptic electrogenesis in the induction of associative synaptic enhancement. In accordance with Hebb's postulate for learning, repetitive postsynaptic spiking enabled enhancement in just those synapses that were eligible to change by virtue of concurrent presynaptic activity. However, the essential postsynaptic electrogenic event that controlled the enhancement was shown to involve biophysical processes that were unknown when Hebb formulated his neurophysiological postulate. The demonstrated spatiotemporal specificity of this pseudo-Hebbian conjunctive mechanism can account qualitatively for the known neurophysiological properties of associative long-term potentiation in these synapses, which in turn can explain the "cooperativity" requirement for long-term potentiation.

Differential conditioning of associative synaptic enhancement in hippocampal brain slices.

An electrophysiological stimulation paradigm similar to one that produces Pavlovian conditioning was applied to synaptic inputs to pyramidal neurons of hippocampal brain slices. Persistent synaptic enhancement was induced in one of two weak synaptic inputs by pairing high-frequency electrical stimulation of the weak input with stimulation of a third, stronger input to the same region. Forward (temporally overlapping) but not backward (temporally separate) pairings caused this enhancement. Thus hippocampal synapses in vitro can undergo the conditional and selective type of associative modification that could provide the substrate for some of the mnemonic functions in which the hippocampus is thought to participate.

Conductance mechanism responsible for long-term potentiation in monosynaptic and isolated excitatory synaptic inputs to hippocampus.

The biophysical mechanisms underlying long-term potentiation (LTP) were investigated in identifiable and monosynaptic excitatory inputs to hippocampal neurons. The results provide the first insights into the conductance changes that are responsible for the expression of LTP. Both current- and voltage-clamp measurements of the mossy fiber synaptic response in pyramidal neurons of region CA3 were made with a single-electrode-clamp system. The excitatory postsynaptic response was pharmacologically isolated by bathing hippocampal slices in saline containing 10 microM picrotoxin, which blocks the synaptic inhibition that normally accompanies the experimentally evoked mossy fiber response. LTP was induced by tetanically stimulating the mossy fiber input for 1 s at 100 Hz. Before and 20 min to 1 h after inducing LTP, we attempted to measure the mean excitatory postsynaptic potential (EPSP) amplitude, intrasomatic current-voltage relationship to a step (RN) or alpha function (AN) current waveform, membrane time constant (tau m), spike threshold (T50), peak excitatory postsynaptic current amplitude (IP), synaptic conductance increase (delta G), and synaptic reversal potential (VR); but adequate assessments of all eight of these were not always obtained for every cell that was studied. The induction of LTP increased the mean (+/- SE) EPSP amplitude form 10.5 +/- 1.4 mV during the control period to 16.8 +/- 2.4 mV after the induction of LTP (n = 14; P less than 0.05). This change was not accompanied by increases in the mean value of RN (63 +/- 11 M omega before and 61 +/- 11 M omega after induction; n = 8; P greater than 0.05); AN, which approximates the effective synaptic input resistance at the soma (10.0 +/- 1.50 M omega before and 10.5 +/- 1.60 M omega after; n = 10; P greater than 0.05); or tau m (22 +/- 2 ms before and 20 +/- 2 ms after; n = 8; P greater than 0.05). There was no significant change in T50, which was also assessed with an alpha function current waveform (1.48 +/- 0.11 nA before and 1.49 +/- 0.10 nA after; n = 6; P greater than 0.05). The mean value of IP increased from 1.1 +/- 0.2 nA during the control period to 1.8 +/- 0.3 nA after inducing LTP (n = 15; P less than 0.05). Similarly, delta G increased from 30 +/- 4 nS before to 47 +/- 4 nS after induction (n = 10; P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen consumption of hypothalamic tissue slices after varying incubation periods.

In order to quantitate possible time-related changes in the viability of rat hypothalamic tissue slices, tissue oxygen consumption was measured after incubation periods ranging from 0-4 hours. There were no significant differences in mean tissue oxygen consumption between the various incubation periods; nor was there any trend indicating that oxygen consumption gradually decreases over time. Moreover, no regional differences were observed among the various rostral hypothalamic slices. One obvious trend, however, was that during the first two hours of each experiment, tissue oxygen consumption decreased briefly and then returned to normal higher levels. The exact occurrence of this transitory decrease varied from experiment to experiment; but the subsequent recovery in oxygen consumption was always complete by two hours of incubation. This initial transient decrease in tissue oxygen consumption may reflect the initial period of electrophysiological inactivity reported in several in vitro studies.

A slice chamber for intracellular and extracellular recording during continuous perfusion.

The design of a tissue slice perfusion system is described, and examples are given showing the stability of this system for intracellular and extracellular recordings during changes in perfusion media. The stability of this system is attributed to several features. Mini-drips serve to cushion transient changes in flow rate when switching from one medium to another. Solenoid valves are used to quickly switch perfusion media with minimal mechanical movement. A finely-controlled adjustable flow valve provides a uniform flow rate for all media. Constant tissue temperature is maintained by media perfusion through a thermoelectric Peltier assembly. In addition, a filter paper wick insures that the perfusate is constantly removed without movement in the tissue slices. With this design, the slices are supported on a net at the interface between the perfusion medium and a humidified, oxygenated atmosphere. This arrangement appears to be conducive to tissue viability and facilitates the placement of microelectrodes in the slices.

Effect of synaptic blockade on thermosensitive neurons in hypothalamic tissue slices.

To understand the basis of hypothalamic neuronal thermosensitivity, single-unit activity was recorded in vitro, from constantly perfused tissue slices of rat preoptic area and anterior hypothalamus, PO/AH. The firing rate and thermosensitivity of individual PO/AH neurons was determined before, during, and after tissue perfusion with a synaptic blocking medium, containing elevated magnesium and decreased calcium concentrations. During synaptic blockade, thermosensitivity was retained in nearly all of the warm-sensitive neurons, and some temperature-insensitive neurons showed increased warm sensitivity. The thermosensitivity of all cold-sensitive neurons was lost during synaptic blockade. These results support the hypothesis that PO/AH cold-sensitive neurons depend on synapses from nearby warm-sensitive neurons for their temperature sensitivity; whereas warm sensitivity is an independent property of certain PO/AH neurons.

Thermosensitive single-unit activity of in vitro hypothalamic slices.

Single-unit activity was recorded in vitro from tissue slices of rat preoptic area-anterior hypothalamus. The thermosensitivity of 139 units was determined by their changes in firing rate in response to changes in slice temperature. Of these neurons, 30% were warm sensitive, 10% were cold sensitive, and 60% were temperature insensitive. These proportions are similar to results obtained in whole-animal studies, indicating that this is a viable preparation. It also suggests that hypothalamic neuronal thermosensitivity is not dependent on peripheral afferent input. All units had low firing rates (less than 10 imp/s) at 37 degrees C, and 83% of the warm-sensitive units were most thermosensitive above 37 degrees C. This supports the concept that afferent input determines the level of firing rate and range of thermosensitivity of warm-sensitive neurons. The cold-sensitive units also displayed maximal thermosensitivity above 37 degrees C, which would be expected if cold-sensitive neurons received inhibitory synaptic input from nearby warm-sensitive neurons.