NMDA Currents Research Articles

AMPA and NMDA currents show different short-term depression in the dorsal lateral geniculate nucleus of the rat.

Paired-pulse depression was studied at the glutamatergic synapse between retinal afferents and thalamocortical cells in the rat dorsal lateral geniculate nucleus. The main objective of this study was to examine the contributions of the pre- and postsynaptic sites to this depression by comparing AMPA- and NMDA-receptor-mediated responses. Equal depression of the two receptor components would indicate involvement of presynaptic mechanisms, while differences in depression would indicate involvement of postsynaptic mechanisms. Pharmacologically isolated AMPA- and NMDA-receptor-mediated currents were recorded using the whole-cell patch-clamp technique in acute thalamic slices. Both the AMPA and the NMDA components showed pronounced depression when retinal afferents were activated by paired pulses. The depression decayed within 5 s. The AMPA component was more strongly depressed than the NMDA component at paired-pulse intervals ranging from 20 to 200 ms, suggesting the involvement of postsynaptic mechanisms. For intervals of 500 ms and longer, the depression of the two components was identical, suggesting the involvement of purely presynaptic mechanisms. The degree of depression measured without the use of pharmacological tools produced similar results, thus excluding the involvement of presynaptic ionotropic glutamate receptors. Cyclothiazide, a blocker of AMPA-receptor desensitisation, reduced the difference in depression between the two components, suggesting that desensitisation of the AMPA receptors is a postsynaptic mechanism that contributes to the difference in depression between the AMPA and the NMDA components.

α7 Nicotinic Acetylcholine Receptors Occur at Postsynaptic Densities of AMPA Receptor-Positive and -Negative Excitatory Synapses in Rat Sensory Cortex

NMDA receptor (NMDAR) activation requires concurrent membrane depolarization, and glutamatergic synapses lacking AMPA receptors (AMPARs) are often considered "silent" in the absence of another source of membrane depolarization. During the second postnatal week, NMDA currents can be enhanced in rat auditory cortex through activation of the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). Electrophysiological results support a mainly presynaptic role for alpha7nAChR at these synapses. However, immunocytochemical evidence that alpha7nAChR is prevalent at postsynaptic sites of glutamatergic synapses in hippocampus and neocortex, along with emerging electrophysiological evidence for postsynaptic nicotinic currents in neocortex and hippocampus, has prompted speculation that alpha7nAChR allows for activation of NMDAR postsynaptically at synapses lacking AMPAR. Here we used dual immunolabeling and electron microscopy to examine the distribution of alpha7nAChR relative to AMPAR (GluR1, GluR2, and GluR3 subunits combined) at excitatory synapses in somatosensory cortex of adult and 1-week-old rats. alpha7nAChR occurred discretely over most of the thick postsynaptic densities in all cortical layers of both age groups. AMPAR immunoreactivity was also detectable at most synapses; its distribution was independent of that of alpha7nAChR. In both age groups, approximately one-quarter of asymmetrical synapses were alpha7nAChR positive and AMPAR negative. The variability of postsynaptic alpha7nAChR labeling density was greater at postnatal day (PD) 7 than in adulthood, and PD 7 neuropil contained a subset of small AMPA receptor-negative synapses with a high density of alpha7nAChR immunoreactivity. These observations support the idea that acetylcholine receptors can aid in activating glutamatergic synapses and work together with AMPA receptors to mediate postsynaptic excitation throughout life.

Is voltage-dependent synaptic transmission in NMDA receptors a robust mechanism for working memory?

The voltage-dependent synaptic transmission in NMDA receptors had been demonstrated to provide persistence of a stimulated activity pattern independent of any form of synaptic plasticity (Nature Neurosci. 1 (4) (1998) 273). This possible mechanism for working memory, however, relied on some unrealistic conditions. The model requires a dominance of NMDA-mediated over AMPA-mediated currents that is unlikely. Furthermore, as we will show in this paper the mechanism is not robust against realistic amounts of noise. We propose a modified model in which short-time memory of a few hundred milliseconds is maintained by input-driven NMDA currents even in the presence of high levels of noise and AMPA currents.

Pathway-Specific Properties of AMPA and NMDA-Mediated Transmission in CA1 Hippocampal Pyramidal Cells

CA1 pyramidal cells receive glutamatergic input from the entorhinal cortex through the perforant path (PP) and from CA3 through Schaffer collaterals (SC). The PP input terminates in the stratum lacunosum molecular approximately 300 microm from the cell body, whereas SC synapses have a more proximal location in the stratum radiatum. We compared the properties of AMPA- and NMDA-mediated transmission at these two inputs. The AMPA-mediated components have linear voltage dependence in both inputs. The reversal potential in the PP is only slightly more positive than in the SC, indicating that distal membrane voltage could be effectively set. The NMDA-mediated responses in the two pathways, however, are very different. The PP exhibits inward rectification, as evidenced by very low outward currents. The rectification persists in the absence of extracellular Mg2+. It cannot be attributed to clamping problems, because large outward AMPA currents can be observed even when conditions are modified to have the AMPA currents kinetically match the NMDA currents. Thus, it appears that the PP NMDA channels have novel properties. A second difference between the PP and SC pathways is that the PP has a larger NMDA/AMPA charge ratio. This difference could be observed under many conditions, including block of all voltage-dependent conductances and elimination of the negative resistance of NMDA channels by removing extracellular Mg2+. The difference in ratio thus cannot be attributed to regenerative currents. The higher NMDA component of the distal PP synapses could help to make these synapses more powerful under depolarizing conditions.

The effects of serotonin on functionally diverse isolated lamprey spinal cord neurons.

The experiments reported here showed that application of serotonin (5-hydroxytryptamine, 5-HT) (100 microM) did not induce any significant current through the membranes of any of the spinal neurons studied (n = 62). At the same time, the membranes of most motoneurons and interneurons (15 of 18) underwent slight depolarization (2-6 mV) in the presence of 5-HT, which was not accompanied by any change in the input resistance of the cells. Depolarization to 10-20 mV occurred in some cells (3 of 18) of these functional groups, this being accompanied by 20-60% decreases in input resistance. The same concentration of 5-HT induced transient low-amplitude depolarization of most sensory spinal neurons (dorsal sensory cells), this changing smoothly to long-term hyperpolarization by 2-7 mV. The input resistance of the cell membranes in these cases showed no significant change (n = 8). Data were obtained which provided a better understanding of the mechanism by which 5-HT modulates the activity of spinal neurons. Thus, 5-HT facilitates chemoreceptive currents induced by application of NMDA to motoneurons and interneurons, while the NMDA responses of dorsal sensory cells were decreased by 5-HT. 5-HT affected the post-spike afterresponses of neurons. 5-HT significantly decreased the amplitude of afterhyperpolarization arising at the end of the descending phase of action potentials in motoneurons and interneurons and increased the amplitude of afterdepolarization in these types of cells. In sensory spinal neurons, 5-HT had no great effect on post-spike afterresponses. The results obtained here support the suggestion that 5-HT significantly modulates the activity of spinal neurons of different functional types. 5-HT facilitates excitation induced by subthreshold depolarization in motoneurons and some interneurons, facilitating the generation of rhythmic discharges by decreasing afterhyperpolarization. In sensory cells, 5-HT enhances inhibition due to hyperpolarization, suppressing NMDA currents. The differences in the effects of 5-HT on functionally diverse neurons are presumed to be associated with the combination of different types of 5-HT receptors on the membranes of these types of spinal neurons.

Interaction of the hypocretins with neurotransmitters in the nucleus accumbens

The hypocretins (hcrt1 and hcrt2), also known as orexins, are two neuropeptides derived from the same precursor, expressed in a few thousand cells in the lateral hypothalamus. Hypocretin-containing cells project throughout the brain, including ascending projections to the olfactory bulb and cerebral cortex, through the medial septum and the nucleus accumbens. Here, we have studied the interactions of the hypocretins with different neurotransmitters by patch clamp recording of acutely dissociated cells from the nucleus accumbens. Application of hcrt1 or hcrt2 decreased postsynaptic NMDA currents, enhanced GABA currents but did not affect glycine-activated conductances. Our results strongly suggest that the hypocretin peptides may be inhibitory peptides, probably via binding hcrt receptor 2.

Lead inhibition of NMDA channels in native and recombinant receptors.

NMDA channels are key targets for lead (Pb2+) neurotoxicity and Pb2+-induced inhibition of NMDA current is age- and subunit-dependent. In rat cerebellar granule cells maintained in high KCl, glycine affinity as well as sensitivity to ifenprodil change significantly with the days in vitro, indicating a reduction of NR2B subunit expression. Pb2+ blocked NMDA current with IC50 approximately 4 microM and this effect decreased significantly during the second week in vitro. In Xenopus laevis oocytes expressing recombinant NR1-NR2A, NR1-NR2B or NR1-NR2C receptors, Pb2+ inhibited glutamate-activated currents with IC50 of 3.3, 2.5 and 4.7 microM respectively. These data indicate that Pb2+ action is dependent on subunit composition and suggest that down-regulation of the NR2B subunit is correlated to a diminished sensitivity to Pb2+ inhibition.

Selective and biphasic effect of the membrane lipid peroxidation product 4-hydroxy-2,3-nonenal on N-methyl-D-aspartate channels.

Increased oxyradical production and membrane lipid peroxidation occur in neurons under physiological conditions and in neurodegenerative disorders. Lipid peroxidation can alter synaptic plasticity and may increase the vulnerability of neurons to excitotoxicity, but the underlying mechanisms are unknown. We report that 4-hydroxy-2,3-nonenal (4HN), an aldehyde product of lipid peroxidation, exerts a biphasic effect on NMDA-induced current in cultured rat hippocampal neurons with current being increased during the first 2 h and decreased after 6 h. Similarly, 4HN causes an early increase and a delayed decrease in NMDA-induced elevation of intracellular Ca2+ levels. In contrast, 4HN affects neither the ion current nor the Ca2+ response to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). The initial enhancement of NMDA-induced current is associated with increased phosphorylation of the NR1 receptor subunit, whereas the delayed suppression of current is associated with cellular ATP depletion and mitochondrial membrane depolarization. Cell death induced by 4HN is attenuated by an NMDA receptor antagonist, but not by an AMPA receptor antagonist. A secreted form of amyloid precursor protein, previously shown to protect neurons against oxidative and excitotoxic insults, prevented each of the effects of 4HN including the early and late changes in NMDA current, delayed ATP depletion, and cell death. These findings show that the membrane lipid peroxidation product 4HN can modulate NMDA channel activity, suggesting a role for this aldehyde in physiological and pathophysiological responses of neurons to oxidative stress.

Synaptic and neurotransmitter activation of cardiac vagal neurons in the nucleus ambiguus.

Cardiac vagal neurons play a critical role in the control of heart rate and cardiac function. These neurons, which are primarily located in the nucleus ambiguus (NA) and the dorsal motor nucleus of the vagus (DMNX), dominate the neural control of heart rate under normal conditions. Cardiac vagal activity is diminished and unresponsive in many disease states, while restoration of parasympathetic activity to the heart lessens ischemia and arrhythmias and decreases the risk of sudden death. Recent work has demonstrated that cardiac vagal neurons are intrinsically silent and therefore rely on synaptic input to control their firing. To date, three major synaptic inputs to cardiac vagal neurons have been identified. Stimulation of the nucleus tractus solitarius evokes a glutamatergic pathway that activates both NMDA and non-NMDA glutamatergic postsynaptic currents in cardiac vagal neurons. Acetylcholine excites cardiac vagal neurons via three mechanisms, activating a direct ligand-gated postsynaptic nicotinic receptor, enhancing postsynaptic non-NMDA currents, and presynaptically by facilitating transmitter release. This enhancement by nicotine is dependent upon activation of pre- and postsynaptic P-type voltage-gated calcium channels. Additionally, there is a GABAergic innervation of cardiac vagal neurons. The transsynaptic pseudorabies virus that expresses GFP (PRV-GFP) has been used to identify, for subsequent electrophysiologic study, neurons that project to cardiac vagal neurons. Bartha PRV-GFP-labeled neurons retain their normal electrophysiological properties, and the labeled baroreflex pathways that control heart rate are unaltered by the virus.

Balance between Four Types of Synaptic Input for the Integrate-and-Fire Model

We consider the integrate-and-fire model with AMPA, NMDA, GABAAand GABABsynaptic inputs, with model parameters based upon experimental data. An analytical approach is presented to determine when a post-synaptic balance between excitation and inhibition can be achieved. Secondly, we compare the model behaviour subject to these four types of input, with its behaviour subject to conventional point process inputs. We conclude that point processes are not a good approximation, even away from exact presynaptic balance. Thirdly, numerical simulations are presented which demonstrate that we can treat NMDA and GABABas DC currents. Finally, we conclude that a balanced input is plausible neither pre-synaptically nor post-synaptically for the model and parameters we employed.

Activation of type 5 metabotropic glutamate receptors enhances NMDA responses in mice cortical wedges.

1. We measured the effects of agonists and antagonists of metabotropic glutamate (mGlu) receptors (types 1 and 5) on NMDA-induced depolarization of mouse cortical wedges in order to characterize the mGlu receptor type responsible for modulating NMDA responses. We also characterized a number of mGlu receptor agents by measuring [3H]-inositol phosphate (IP) formation in cortical slices and in BHK cells expressing either mGlu 1 or mGlu 5 receptors. 2. (S)-3,5-dihydroxyphenylglycine (DHPG), an agonist of both mGlu 1 and mGlu 5 receptors, at concentrations ranging from 1-10 microM, enhanced up to 105+/-15% the NMDA-induced depolarization. Larger concentrations (100-300 microM) of the compound were inactive in this test. When evaluated on [3H]-IP synthesis in cortical slices or in cells expressing either mGlu 1 or mGlu 5 receptors, DHPG responses (1-300 microM) increased in a concentration-dependent manner. 3. (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) and (S:)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG), had partial agonist activity on mGlu 5 receptors, with maximal effects reaching approximately 50% that of the full agonists. These compounds, however, enhanced NMDA-evoked currents with maximal effects not different from those induced by DHPG. Thus the enhancement of [3H]-IP synthesis and the potentiation of NMDA currents were not directly related. 4. 2-methyl-6-(phenylethynyl)-pyridine (MPEP, 1-10 microM), a selective mGlu 5 receptor antagonist, reduced DHPG effects on NMDA currents. 7-(hydroxyimino)cyclopropan[b]-chromen-1a-carboxylic acid ethylester (CPCCOEt, 30 microM), a preferential mGlu 1 receptor antagonist, did not reduce NMDA currents. 5. These results show that mGlu 5 receptor agonists enhance while mGlu 5 receptor antagonists reduce NMDA currents. Thus the use of mGlu 5 receptor agents may be suggested in a number of pathologies related to altered NMDA receptor function.

Exploration of the role of reactive oxygen species in glutamate neurotoxicity in rat hippocampal neurones in culture.

1. Exposure of hippocampal neurones to glutamate at toxic levels is associated with a profound collapse of mitochondrial potential and deregulation of calcium homeostasis. We have explored the contributions of reactive oxygen species (ROS) to these events, considered to represent the first steps in the progression to cell death. 2. Digital imaging techniques were used to monitor changes in cytosolic Ca2+ concentration ([Ca2+]c; fura-2FF) and mitochondrial potential (Deltapsim; rhodamine 123); rates of ROS generation were assessed using hydroethidium (HEt); and membrane currents were measured with the whole-cell configuration of the patch clamp technique. 3. Inhibitors of lipid peroxidation (trolox plus ascorbate) and scavengers of superoxide or hydrogen peroxide (manganese(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP) and TEMPO plus catalase), had only minimal impact on the mitochondrial depolarisation and the sustained increase in [Ca2+]c during and following a 10 min exposure to glutamate. 4. The antioxidants completely suppressed ROS generated by xanthine with xanthine oxidase. No significant increase in ROS production was detected with HEt during a 10 min glutamate exposure. 5. A combination of antioxidants (TEMPO, catalase, trolox and ascorbate) delayed but did not prevent the glutamate-induced mitochondrial depolarisation and the secondary [Ca2+]c rise. However, this was attributable to a transient inhibition of the NMDA current by the antioxidants. 6. Despite their inability to attenuate the glutamate-induced collapse of Deltapsim and destabilisation of [Ca2+]c homeostasis, the antioxidants conferred significant protection in assays of cell viability at 24 h after a 10 min excitotoxic challenge. The data obtained suggest that antioxidants exert their protective effect against glutamate-induced neuronal death through steps downstream of a sustained increase in [Ca2+]c associated with the collapse of Deltapsi(m).

Maturation of glutamatergic neurotransmission in dentate gyrus granule cells

We studied the development of glutamatergic neurotransmission in dentate gyrus granule cells (GCs) in hippocampal slices from 5 to 12-day-old rats. The active postnatal neuronogenesis in dentate permits GCs with staggered birthdates to be studied in situ in a single preparation. We recorded evoked responses to medial perforant path stimulation using visually-guided whole-cell patch clamping to select immature GCs, and biocytin filling to correlate electrophysiologic responses with maturational stage. Even within this immature cell population we found four distinct electrophysiologic patterns. Type 1 cells had no glutamatergic current; Type 2 cells had only N-methyl- d-aspartate receptor (NMDA) current; Type 3 cells had both NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) current although the NMDA component could be isolated at low stimulus intensity (NMDA threshold<AMPA threshold); Type 4 cells had both AMPA and NMDA currents with NMDA threshold≥AMPA threshold. Type 1 cells were least mature, and Type 4 cells most mature as assessed by cell properties, dendritic arborization, and penetration of dendrites into the molecular layer. Thus NMDA-mediated currents predominate early in GC development as is consistent with their role in processes that determine dentate architecture — neuronal migration, dendritic outgrowth and regression, and synapse stabilization. By analogy with ‘silent synapses’ (i.e. synapses that contain only NMDA receptors), Type 2 cells are candidate ‘silent cells’ that may undergo activity-dependent acquisition of functional fast-conducting AMPA receptors with maturation.

Imidazoline-induced neuroprotective effects result from blockade of NMDA receptor channels in neuronal cultures

Imidazolines have been shown to be neuroprotective in focal and global ischemia in the rat. However, their mechanism of action is still unclear. We have studied the neuroprotective effects of imidazolines against NMDA-induced neuronal death and hypoxic insult in cerebellar and striatal neuronal cultures. All of the imidazolines tested decreased the NMDA-mediated neurotoxicity in a non-competitive manner. Antazoline was the most effective (IC 50 of 5 μM, maximal neuroprotection reaching 90% at 100 μM). The neuroprotective effects were still present when the imidazolines were applied during the post-insult period. Antazoline, idazoxan and guanabenz also showed neuroprotective effects against hypoxia-induced neuronal death (neuroprotection reaching 95% for antazoline at 100 μM). Antazoline was still active if applied during the reoxygenation period (15% neuroprotection). To determine the mechanism of the neuroprotective effects, the possible interaction of imidazolines with NMDA receptors was studied. Imidazolines dose-dependently and non-competitively inhibited NMDA currents. As found for the neuroprotective effects, antazoline was the most effective imidazoline, with an IC 50 of 4 μM and a maximal inhibition of 90% at 100 μM. This blockade was rapid, reversible and voltage-dependent. We compared these effects to those of the classical non-competitive antagonist of NMDA channels, MK-801. In contrast to imidazolines, blockade of the NMDA current by MK-801 was voltage-independent and reversible only at positive potentials. When co-applied with MK-801, antazoline prevented the long lasting blockade of the NMDA current by MK-801. These results are consistent with the existence of overlapping binding sites for these drugs on the NMDA receptor channel. They indicate that imidazolines exert a strong neuroprotective effect against excitotoxicity and hypoxia in cerebellar and striatal primary neuronal cultures by inhibiting NMDA receptors. Since these effects were non-competitive, imidazolines appear to be interesting new drugs with therapeutic potential.

Enhancement of NMDA-induced current by the putative NR2B selective antagonist ifenprodil.

Ifenprodil has been widely used as an antagonist selective for NMDA receptors containing the NR2B subunit. Evidence suggests, however, that ifenprodil also increases NMDA receptor affinity. Using rat brain slices, we found that ifenprodil enhanced NMDA-induced current in both cortical and subcortical areas examined. To test whether the effect is due to an increase in NMDA receptor affinity, we compared the effect of ifenprodil on currents induced by different concentrations of NMDA. Consistent with the hypothesis, the enhancing effect (percent increase) was relatively constant at low NMDA concentrations. As NMDA concentration increased, however, the effect decreased. To test whether the effect is blocked when NMDA binding sites are saturated with NMDA, high concentrations of NMDA were applied. To partially block Ca(2+) influx and prevent cells from deteriorating, the experiments were performed in the presence of either MK801 or kynurenate, two noncompetitive antagonists. Under such conditions, ifenprodil not only failed to potentiate NMDA currents, but consistently suppressed the current. When the same concentration of NMDA was applied in the presence of the competitive antagonist CGP37849, ifenprodil regained its ability to potentiate NMDA currents. Furthermore, the higher the concentration of CGP37849 the more the NMDA current was potentiated by ifenprodil. These results, combined with previous studies, suggest that the enhancing effect is due to an increase in NMDA receptor affinity and is specific for responses induced by low NMDA concentrations. As NMDA concentration increases, the affinity-enhancing effect decreases. Consequently, the channel-suppressing effect becomes more prominent.

The postsynaptic density protein PSD-95 differentially regulates insulin- and Src-mediated current modulation of mouse NMDA receptors expressed in Xenopus oocytes.

The NMDA subtype of glutamate receptor is physically associated with the postsynaptic density protein PSD-95 at glutamatergic synapses. The channel activity of NMDA receptors is regulated by different signaling molecules, including protein tyrosine kinases. Because previous results have suggested a role for protein kinase C (PKC) in insulin potentiation of NMDA currents in oocytes, the effects of coexpression of PSD-95 on insulin and PKC potentiation of NMDA currents from these receptors were compared. Another primary objective was to determine if PSD-95 could enable Src to potentiate currents from NR2A/NR1 and NR2B/NR1 receptors expressed in Xenopus oocytes. The results show opposite effects of PSD-95 coexpression on Src and insulin modulation of NR2A/NR1 receptor currents. Src potentiation of mouse NR2A/NR1 currents required PSD-95 coexpression. In contrast, PSD-95 coexpression eliminated insulin-mediated potentiation of NR2A/NR1 receptor currents. PSD-95 coexpression also eliminated PKC potentiation of NR2A/NR1 receptor currents. PSD-95 may therefore play a key role in controlling kinase modulation of NR2A/NR1 receptor currents at glutamatergic synapses.

C-Terminal Truncation of NR2A Subunits Impairs Synaptic But Not Extrasynaptic Localization of NMDA Receptors

NMDA receptors interact via the extended intracellular C-terminal domain of the NR2 subunits with constituents of the postsynaptic density for purposes of retention, clustering, and functional regulation at central excitatory synapses. To examine the role of the C-terminal domain of NR2A in the synaptic localization and function of NR2A-containing NMDA receptors in hippocampal Schaffer collateral-CA1 pyramidal cell synapses, we analyzed mice which express NR2A only in its C-terminally truncated form. In CA1 cell somata, the levels, activation, and deactivation kinetics of extrasynaptic NMDA receptor channels were comparable in wild-type and mutant NR2A(Delta)(C/)(Delta)(C) mice. At CA1 cell synapses, however, the truncated receptors were less concentrated than their full-length counterparts, as indicated by immunodetection in cultured neurons, synaptosomes, and postsynaptic densities. In the mutant, the NMDA component of evoked EPSCs was reduced in a developmentally progressing manner and was even more reduced in miniature EPSCs (mEPSCs) elicited by spontaneous glutamate release. Moreover, pharmacologically isolated NMDA currents evoked by synaptic stimulation had longer latencies and displayed slower rise and decay times, even in the presence of an NR2B-specific antagonist. These data strongly suggest that the C-terminal domain of NR2A subunits is important for the precise synaptic arrangement of NMDA receptors.

Activity coregulates quantal AMPA and NMDA currents at neocortical synapses.

AMPA and NMDA receptors are coexpressed at many central synapses, but the factors that control the ratio of these two receptors are not well understood. We recorded mixed miniature or evoked synaptic currents arising from coactivation of AMPA and NMDA receptors and found that long-lasting changes in activity scaled both currents up and down proportionally through changes in the number of postsynaptic receptors. The ratio of NMDA to AMPA current was similar at different synapses onto the same neuron, and this relationship was preserved following activity-dependent synaptic scaling. These data show that AMPA and NMDA receptors are tightly coregulated by activity at synapses at which they are both expressed and suggest that a mechanism exists to actively maintain a constant receptor ratio across a neuron's synapses.

Protein kinase C potentiation of currents from mouse ζ1/ε2 NMDA receptors expressed in Xenopus oocytes depends on f-actin/g-actin cycling

NMDA currents from Xenopus oocytes expressing recombinant ζ1/ε2 NMDA receptors can be potentiated by activation of protein kinase-C (PKC) and also demonstrate time-dependent rundown. In order to determine whether cytoskeletal proteins are involved in either of these phenomena, experiments were performed using the f-actin stabilizer phalloidin, the f-actin de-stabilizer cytochalasin-D, and the microtubule stabilizer taxol. Phalloidin treatment both prevented rundown and inhibited PKC-potentiation of whole-cell currents but did not affect baseline current amplitudes. Treatment with cytochalasin-D also prevented rundown and inhibited PKC-potentiation of whole-cell currents, but baseline currents from cytochalasin treated cells were only 50% as large as those from control cells. Taxol had no effect on either rundown or PKC potentiation of NMDA currents. The results indicate that both spontaneous rundown and PKC potentiation of currents from heterologously expressed ζ1/ε2 NMDA receptors depend on dynamic actin polymerization/depolymerization but do not involve changes in microtubules.

Pituitary Adenylate Cyclase Activating Peptide Phase Shifts Circadian Rhythms in a Manner Similar to Light

The endogenous circadian pacemaker in mammals is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Various cues can reset circadian rhythm phase, thereby entraining the internal rhythm to the environmental cycle, and these effects can be investigated using an in vitro method to measure phase shifts of the SCN. Although pituitary adenylate cyclase activating peptide (PACAP) is localized in retinal inputs to the SCN, it has been reported to alter clock phase only during the subjective day (Hannibal et al., 1997), whereas light alters phase only in the subjective night. In this study we show that PACAP can reset the clock in the photic pattern during the subjective night when applied in 10 pM to 1 nM doses. This appears to be mediated via a glutamatergic mechanism, possibly by potentiation of NMDA currents as is seen at 10-100 pM. Given at higher doses (>10 nM), PACAP shifts in the subjective day, apparently via activation of adenylate cyclase and increased intracellular cAMP. These results indicate dose and phase specificity of the effects of PACAP, and a new role as a transmitter in the retinohypothalamic tract.